European Rodents Research Articles

Beyond the Mediterranean peninsulas: evidence of central European glacial refugia for a temperate forest mammal species, the bank vole (Clethrionomys glareolus)

This study details the phylogeographic pattern of the bank vole, Clethrionomys glareolus, a European rodent species strongly associated with forest habitat. We used sequences of 1011 base pairs of the mitochondrial DNA cytochrome b gene from 207 bank voles collected in 62 localities spread throughout its distribution area. Our results reveal the presence of three Mediterranean (Spanish, Italian and Balkan) and three continental (western, eastern and 'Ural') phylogroups. The endemic Mediterranean phylogroups did not contribute to the post-glacial recolonization of much of the Palaearctic range of species. Instead, the major part of this region was apparently recolonized by bank voles that survived in glacial refugia in central Europe. Moreover, our phylogeographic analyses also reveal differentiated populations of bank voles in the Ural mountains and elsewhere, which carry the mitochondrial DNA of another related vole species, the ruddy vole (Clethrionomys rutilus). In conclusion, this study demonstrates a complex phylogeographic history for a forest species in Europe which is sufficiently adaptable that, facing climate change, survives in relict southern and northern habitats. The high level of genetic diversity characterizing vole populations from parts of central Europe also highlights the importance of such regions as a source of intraspecific genetic biodiversity.

The collapse of cycles in the dynamics of North American grouse populations

AbstractCyclic dynamics of bird and mammal populations are commonly reported in northern latitudes throughout the world, and recent European observations on rodents and grouse suggest that cycle periods decline towards southern latitudes. To investigate latitudinal patterns of cyclic dynamics in North America, we assembled 27 long‐term data sets collected between 1939 and 2001 for three grouse species. By fitting the data with autoregressive models to measure direct and delayed density dependence, we show that, in contrast to European studies, North American grouse exhibit period increases from north to south, with cycles collapsing via period lengthening. This occurs because delayed density dependence decreases in southern latitudes, whereas direct density dependence increases. These results show that cyclic dynamics can dissipate by period lengthening as well as the period shortening postulated for European grouse and rodents.

Eastern European rodent (Rodentia, mammalia) faunas from the Early–Middle Pleistocene transition

The rodent faunas from nine sites on the Russian Plain and Taman Peninsula corresponding to the Early–Middle Pleistocene transition have been analyzed. These localities are situated in the Dnieper, Don, Dniester and Danube drainage basins and in the northern regions of the Black and Azov Seas. Seven of the sites are associated with the upper part of Matuyama reversed polarity Chron and located between the beginning of Jaramillo event and the Brunhes/Matuyama boundary. One of the sites was recovered from the Chauda marine deposits of the Black Sea and referred to the very beginning of Brunhes Epoch. The species composition of the rodent fauna sequence has been established. The faunas referred to the Jaramillo event (∼1.1–1.0 Ma) contain the advanced Mimomys, steppe lemmings Prolagurus pannonicus and Lagurodon arankae, and Allophaiomys pliocaenicus. Faunas were recovered from Korotoyak (Ostrogozhsk suite), Roksolany, Zapadnye Kairy and Ushkalka localities. The more advanced rodent faunas of the Morozovian assemblage (Port-Katon site) include also the first representatives of Terricola. Karai–Dubina and Shamin localities have been found below the Brunhes/Matuyama boundary (0.85 Ma), including the first representatives of Microtus ( Microtus ex gr. oeconomus and Microtus arvalinus). The “Pitymys” like voles— Microtus ( Stenocranius) hintoni, steppe lemming P. pannonicus and yellow steppe lemming Eolagurus argyropuloi compose the core of these faunas. The Litvin locality, located in the deposits of the Chauda transgression of the Black Sea and corresponding to the very beginning of the Middle Pleistocene, contains a rodent fauna closely resembling the Shamin one. It yielded the remains of Mimomys savini, advanced A. pliocaenicus, P. pannonicus, Microtus ( Stenocranius) hintoni, Microtus ex gr. oeconomus and Mcirotus arvalinus. Thus, numerous data permit elucidation of the history of rodent faunas of the Russian Plain during Early–Middle Pleistocene transitions and reveal the definitive stages in their evolution.

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia.

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Association of Borrelia afzelii with rodents in Europe.

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested low land locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochactes than voles. However, the infectivity of soles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

Seasonal Dynamics ofAnaplasmaphagocytophilain a Rodent-Tick (Ixodes trianguliceps) System, United Kingdom

We investigated the reservoir role of European wild rodents for Anaplasma phagocytophila using polymerase chain reaction (PCR) analysis of blood collected from individually tagged rodents captured monthly over 2 years. The only tick species observed in the woodland study site was Ixodes trianguliceps, and ruminant reservoir hosts were not known to occur. A. phagocytophila infections were detected in both bank voles and wood mice but were restricted to periods of peak nymphal and adult tick activity. Most PCR-positive rodents were positive only once, suggesting that rodent infections are generally short-lived and that ticks rather than rodents may maintain the infection over winter. Bank voles were more likely to be PCR positive than wood mice, possibly because detectable infections are longer lived in bank voles. This study confirms that woodland rodents can maintain A. phagocytophila in Great Britain in the absence of other reservoir hosts and suggests that I. trianguliceps is a competent vector.

Mating behaviour of bank voles (Clethrionomys glareolus) modified by hormonal and social factors

Summary The bank vole is a common European rodent. In a natural population the adult females are territorial, and during the breeding season each female's home range overlaps with the home range of several males. The aim of the present study was to investigate the influence of sexual experiences of females and males on mating behaviour. The results indicate that the activity of the female was modified by her sexual experience and by the experience of the partner. Females were more active in presence of sexually non-experienced than experienced males. Males also showed higher activity in the presence of virgin than in the presence of experienced females. Females in postpartum estrus were more active than virgins and injection of gonadotropins stimulated virgin females. Males tested with gonadotropin-treated virgin females or postpartum estrous females showed similar sexual activity. Sexual experience increased the activity of males; they approached receptive females frequently and exhibited a higher number of mountings than did non-experienced males. The higher activity of an experienced partner can prevent copulation with another male during the same estrus, so that only one male becomes the father of the offspring. The present results indicate that in sexually experienced males bulbectomy but not vomeronasalectomy decreased the sexual activity and only 2 out of 6 lesioned males copulated with receptive females.

Diet of Puma (Puma concolor, Carnivora: Felidae) in Coastal and Andean Ranges of Southern Chile

Between 1985 and 1999 we collected a total of 62 puma (Puma concolor) feces in two reserves located on the coastal range and on three national parks located in the Andean range, all in the Lake Region of southern Chile. The identification of 79 prey items showed that the puma diet in the southern rainforests was made up mainly of European hares (Lepus europaeus ; an exotic species), ungulates (chiefly the pudu deer; Pudu puda), rodents, birds and marsupials, in that order of importance. Except for the lowland site San Martín, the trophic ecology of pumas did not differ substantially among sites. At San Martín, in addition to hares, pumas hunted Black-necked swans (Cygnus melancoryphus) and coypu (Myocastor coypus). Although pumas preyed upon the largest prey available, the mean prey weight was of only 7.7 kg, much smaller than on other previous studies. European hares due to their high abundance, have replaced pudus as the main prey of pumas in the forests of the Lake Region. In order to maintain viable populations of pumas in the biologically most interesting coastal forests, it will be necessary to protect larger native forests that provide enough food and habitat for pumas.

Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils.

In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence.

Species richness and rarity in European rodents

The presence or absence of 77 rodent species was scored within 427 squares of 100×100 miles. The grid encompassed the entirety of Europe west of the Urals and north of the Caucasus, but excluded all offshore islands. Rodent distributional ranges varied between 26 000 and 22 670 000 km2. Very large ranges were uncommon and 53% of species had ranges covering <20% of the study area. Range size showed no correlation with body size, nor did it depend on taxonomic provenance. However, saltatorial rodents appeared to have the largest median ranges and fossorial species the smallest. Species densities varied between 4 and 26 species/grid square, with the highest densities coinciding broadly with the steppe–deciduous forest ecotone. The degree of endemism showed a different geographic pattern from that of species density, with the highest values at lower latitudes. Forty‐nine squares (11% of Europe's surface) identified as species richness and endemism “hot spots” contained >80% of genera and species, their largest contiguous coincidence being in the southern Balkans, the Carpathian Basin, southern Ukraine and cis‐Caucasia.

ALTERNATIVE SEASONAL REPRODUCTIVE STRATEGIES IN WILD RODENT POPULATIONS

Many temperate rodents show a break in reproductive activity to conserve energy during harsh winter conditions. However, even in species that use photoperiodic changes to adjust reproductive functions, winter breeding has been recorded frequently and most likely is caused by different responses to changes in photoperiod. Furthermore, these reproductive phenotypes show clear physiologic and morphologic differences. We investigated winter reproduction in natural populations of 2 European rodents, the yellow-necked mouse (Apodemus flavicollis) and the bank vole (Clethrionomys glareolus). Analyses of our data indicate that winter breeding was due to nonresponsive phenotypes in both sexes. A broad range of gonadal changes was found, particularly in males, and individual differences in testis size were linked to differences in body weight. Population genetic analyses provided no hint of assortative mating between the 2 phenotypes.

Relationship between El Niño /South Oscillation (ENSO) and population outbreaks of some lemmings and voles in Europe

Ecologists have been puzzled by population cycles of lemmings and voles for the over 70 years. At present, our understanding and explanation to this phenomenon remain controversial. Recently, El Nino/South Oscillation has attracted attention of ecologists on its links with population outbreaks of terrestrial animals. This paper aims to investigate the statistical relationship between outbreaks of microtine rodents and ENSO events by scanning available literature. During 1862–1894, outbreaks of Norway lemmings in Norway tended to occur in the Southern Oscillation Index (SOI) peak years or 1 year after the SOI peak years with an approximate significance level (p = 0.057). During 1885–1931, outbreaks of voles in France tended to occur 1 year before the SOI peak years (p = 0.01). During 1946–1993, outbreaks of lemmings and voles in North Finland tended to occur 1 year before the SOI peak years with a significant level (p = 0.022); the peaks of population abundance corresponded well to the SOI trough years (equal to 1 year before the SOI peak years). Outbreaks of common voles in Poland during 1946–1975 tended to occur in the SOI peak years or 1 year before the SOI peak years (p = 0.011), and also tended to occur 1 year before the SOI peak years (p = 0.030). It was also found that the rodent outbreaks in Norway and France, rodent outbreaks in Finland and Poland synchronized well. It was suggested that the ENSO-related climate or food were key factors in causing outbreaks of microtine rodents in Europe.

Molecular Phylogeny of European Muroid Rodents Based on Complete Cytochrome b Sequences

Phylogenetic relationships among 18 species of mainly European muroid rodents that belong to three subfamilies were estimated using complete sequences of the mitochondrial cytochrome b gene. The inferred monophyly of the subfamilies Murinae (mice and rats) and Arvicolinae (voles, lemmings, and muskrats) is in agreement with previous studies. Within the Murinae, the morphology-based division of the genus Apodemus into three subgenera is supported by these DNA sequence data. The relationships among the different genera of the Murinae were generally poorly resolved, and the relationships of Micromys and Acomys to the other murine genera remained unresolved. Within the subfamily Arvicolinae, the relations of the genera Arvicola, Clethrionomys, and Microtus remained tentative with our data. However, within the Microtus group, there is a good molecular support for the phylogenetic relationships. These findings suggest that the origin of the different murine and arvicoline lineages was rapid, indicating an adaptive radiation with fast speciation.

A longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents

Cowpox is an orthopoxvirus infection endemic in European wild rodents, but with a wide host range including human beings. In this longitudinal study we examined cowpox in two wild rodent species, bank voles Clethrionomys glareolus and wood mice Apodemus sylvaticus, to investigate the dynamics of a virus in its wild reservoir host. Trapping was carried out at 4-weekly intervals over 3 years and each animal caught was uniquely identified, blood sampled and tested for antibodies to cowpox. Antibody prevalence was higher in bank voles than in wood mice and seroconversion varied seasonally, with peaks in autumn. Infection was most common in males of both species but no clear association with age was demonstrated. This study provides a model for studying other zoonotic infections that derive from wild mammals since other approaches, such as one-off samples, will fail to detect the variation in infection and thus, risk to human health, demonstrated here.

Gnaw marks on Eocene seeds: evidence for early rodent behaviour

Gnawed holes in fossil seeds of the freshwater aquatic floating plant Stratiotes, collected from the Solent Group of Late Eocene (Priabonian) age at two localities in the Hampshire Basin, UK, are recognised as the oldest direct evidence of seed predation by rodents. The type of gnawing is similar to that made by Wood Mice, Apodemus sylvaticus, today and a more reliable means of differentiating the resultant pattern from those of other gnawing types is described. By using the size relationship between modern gnaw marks and the rodent incisors that made them, isolated fossil incisors from the same strata at sites near to those where the seeds were found are matched closely to the fossil gnaw marks. By comparison with incisors in situ in jaws from the Continental European Palaeogene, and by eliminating other contenders amongst the known Solent Group rodent fauna, it is concluded that the fossil Stratiotes seeds were predated by the extinct glirids Glamys priscus and Glamys devoogdi. Different patterns of gnaw marks associated with different hole sizes are interpreted as different stages in the opening of the seeds and as demonstrating learning behaviour on the part of the predators. Similarity of fossil astragali (ankle bones) from the Solent Group, identified as Glamys devoogdi, to the recent glirid Eliomys quercinus suggests a scansorial rather than arboreal locomotor mode for the former, in keeping with a ground-level foraging strategy as indicated by the habitat of the Stratiotes plant. The direct evidence of feeding from the fossil seeds and the clear link to the specific predators are an important indication of ancient dietary adaptation independent of that deduced from tooth morphology. It supports the change in dominant frugivorous feeding type amongst European rodents from soft fruit-eating pseudoparamyine paramyids and pseudosciurids in the Middle Eocene to hard seed-eating glirids in the Late Eocene, coincident with the global climatic deterioration shown by oxygen isotopes. One new ichnogenus with one new ichnospecies is erected for one type of gnawing in fossil seeds: Glirotremmorpha entectus igen. et isp. nov., from the English Late Eocene.

Granulocytic Ehrlichia infection in ixodid ticks and mammals in woodlands and uplands of the U.K.

The prevalence of infection with Ehrlichiae of the Ehrlichia phagocytophila genogroup (the granulocytic Ehrlichiae), in questing Ixodes ricinus ticks of U.K. upland and woodland habitats, was investigated by PCR. The prevalence of infection in the three feeding stages of I. ricinus indicated that granulocytic Ehrlichiae are transmitted transstadially with no, or inefficient, transovarial transmission. The presence of infected ticks in both habitats indicates that endemic cycles of granulocytic Ehrlichia (GE) infection are maintained by both domesticated sheep and by wild reservoirs, and coexist with endemic cycles of Borrelia burgdorferi infection. Moreover, demonstration, for the first time, of GE infection in engorged Ixodes trianguliceps ticks and blood collected from wild rodents, suggests that European wild rodents are competent reservoirs. GE infection prevalence in nymphal and adult I. ricinus was significantly greater in uplands than woodlands, which is consistent with ticks of all three feeding stages feeding on reservoir-competent sheep in uplands. In one woodland studied, pheasants are important hosts for nymphal I. ricinus but are incompetent or inefficient reservoirs, so reducing GE infection prevalence in I. ricinus ticks in this habitat. 16S rRNA sequences of GE from ticks of these U.K. habitats, showed a high degree of homology with those of granulocytic Ehrlichiae isolated from humans, but also showed some evidence of genetic diversity of granulocytic ehrlichiae in the U.K. The implications of these findings, for the taxonomy of granulocytic ehrlichiae and the potential for human infections to occur in the U.K., is discussed.

DETERMINANTS OF GEOGRAPHIC VARIATION IN GROWTH AND REPRODUCTIVE TRAITS IN THE ROOT VOLE

I studied growth rate and reproduction in two geographically distinct strains of root voles, Microtus oeconomus, in the laboratory to unravel the mechanisms underlying the observed increase in body size from south to north among European microtine rodents. In the field, adult northern root voles are 51% heavier than southern. Many proximate and ultimate mechanisms underlying this geographic pattern have been suggested, but none has been adequately tested. I measured offspring growth rates in one northern and one southern strain over three consecutive laboratory generations, employing two separate experimental protocols (cross-mating and cross-fostering) to control for differential maternal investment. Paternal investment in the young was evaluated in separate tests. Northern root voles were larger at birth and grew faster than southern voles. Litter size and litter mass at birth were largest in the southern strain, when maternal body mass was adjusted for, indicating a larger prenatal reproductive expenditure in southern females. Cross-fostering and cross-breeding experiments demonstrated that the north-south difference in body mass is of genetic origin. Postnatal growth rate of cross-bred litters was intermediate between the two purebred strains, thus being consistent with an additive genetic mechanism. In contrast, neonatal mass of offspring from cross-bred pairs was similar to that of the southern strain, consistent with southern genetic dominance. Cross-fostering between the two strains showed that parental investment had no discernible effects on postnatal growth rate, although southern fathers showed more parental care than did northern fathers. Together, the cross-fostering and the cross-breeding experiments suggest that the growth rate of southern root voles was con- strained by their own genotype and not by the characteristics of their mothers. Low en- vironmental predictability in southern habitats and high productivity of northern habitats probably result in different selection pressures on growth rates and reproductive effort.

Magnetostratigraphy and paleontology of Aït Kandoula basin (High Atlas, Morocco) and the African-European late Miocene terrestrial fauna exchanges

A magnetostratigraphic study has been carried out on a middle Miocene to upper Pliocene lacustrine sedimentary deposit in the central part of the Aït Kandoula basin, which contains micromammal faunas and is situated in the southern High Atlas (Morocco). In total, 113 samples were subjected to paleomagnetic analysis: 60 out of the 113 studied samples representing 52 different stratigraphic levels yielded a paleomagnetic direction and at least the polarity could be recognized in 42 specimens. Eleven specimens were submitted to AF demagnetization. The mean direction for normal-polarity samples was D = 349.4, I = 50.7 (N = 36, α 95 = 4.5, k = 27) and for reversed polarity samples D = 191.2, I = −53.4 (N = 16, α 95 = 12.32, k = 9). These results yielded a polarity sequence which we interpret as spanning from Chron C5n.2n to the beginning of Chron C3n.4n. This interpretation relies on biostratigraphic data previously proposed for a part of the continental fauna found in the basin. This result is in agreement with the 40Ar/ 39Ar dating previously carried out on a volcanic ash layer, which provided an age of 5.9 ± 0.5 Ma [1] and which is shown here to be reversely magnetized. This layer is correlated here with the reverse polarity zone corresponding to Chron C3r. Biostratigraphic studies on the same section have shown that the micromammal levels extend here only from middle Vallesian to upper Turolian (upper Miocene). Four localities have yielded western European species of micromammals, indicating trans-Mediterranean terrestrial faunal exchanges between these two continents during the late Miocene. The European murid rodent Occitanomys is recorded for the first time in North Africa in level 8 of the Afoud section, an age younger than 5.32 Ma being assigned to this level by the present study. Level 1 of the same section yields the lagomorph Prolagus cf. michauxi, with an age of 6.1 Ma. The magnetostratigraphic data suggest therefore that the beginning of terrestrial faunal exchanges between North Africa and Western Europe took place in subchron C3An.1n, at about 6.1 Ma, some 0.4 Myr before the beginning of the Messinian salinity crisis.

Morphological trends and rates of evolution in arvicolids (arvicolidae, rodentia): Towards a punctuated equilibria/disequilibria model

This paper seeks to test morphological trends and rates of evolution using both paleontology and biology. Two major morphological trends are observed in the fossil record: morphological stasis and phyletic gradualism. The ‘how often?’ statistical test of the European arvicolid rodent radiation shows (1) that over 5 Ma, 52 lineages appear (FAD) by allopatric speciation, of which 34 die out (LAD) in the same time span; (2) that cumulative phyletic gradualism (diachrons) accounts for around 63 % of evolutionary trends, whereas stasis accounts for only 37%; (3) that morphological changes occur either by phyletic gradualism through successive distinct diachrons (73% of morphological changes) or during punctuations (27% of morphological changes). Thus it is clear that, from a theoretical point of view, the punctuated equilibria model must be complemented by a disequilibria component which includes phyletic gradualism. As phyletic gradualism is partly serial, the punctuated equilibria/disequilibria model can be described by a linear and periodic equation established on the basis of a Plio-Pleistocene Mimomys lineage.

Sacaresia moyaeponsi nov. gen. nov. sp., rongeur Thryonomyidé (Mammalia) dans le Paléogène de Majorque (Baléares, Espagne)

Isolated teeth of a new rodent found in a South Western Majorcan fauna of European affinities, completely differ from all the other European Oligocene rodents and can be better compared with African Thryonomyids. Detailed study of the dental features makes it possible to refer these teeth to the family Thryonomyidae. Sacaresia leads one to suppose that transmediterranean crossing opportunities could occur during the Oligocene.