Isolation and characterization of microsatellite markers in Calomys musculinus (Muridae,Sigmodontinae, Phyllotini), the natural reservoir of Junin virus

Calomys musculinus, one of the most abundant rodent species in central Argentina, is the reservoir of Junin virus, the aetiological agent of Argentine hemorrhagic fever. We isolated six polymorphic microsatellite loci for microgeographical studies of population structure in this species. Amplification of these loci in 36 individuals from three natural populations revealed five to 14 alleles per locus and expected heterozygosities from 0.426 to 0.868. Cross‐species amplifications suggest that primers designed for these loci may be useful in other closely related species of the tribe Phyllotini, but not in species of other more distantly related tribes of the subfamily Sigmodontinae.     

Haplotype diversity of the mitochondrial DNA D-loop region in Calomys musculinus (Rodentia,Muridae) detected by PCR-RFLP

In order to contribute to knowledge of colonization patterns in the rodent Calomys musculinus, a natural reservoir of the virus producing Argentine hemorrhagic fever (AHF), we studied the haplotype diversity of the mitochondrial DNA D-loop region in five natural populations from central Argentina. Digestion with eight restriction enzymes (RsaI, MseI, Tsp509I, AluI, AciI, HaeIII, NlaIII, and AseI) revealed polymorphism in the 1300 bp fragment amplified by PCR. Twenty different composite haplotypes were detected. Hierarchical analyses indicated that almost all variation (94%) is contained within local populations. Haplotypes 1 and 2, shared by all populations, were the most frequent. Nonsignificant genetic differentiation was found among populations of the endemic and nonendemic areas of AHF: All locations sampled presented exclusive haplotypes in spite of their geographic proximity, which would support previous observations indicating restricted gene flow among C. musculinus populations.     

Inheritance of random amplified polymorphic DNA (RAPD-PCR) markers and their use in population genetic studies of Calomys musculinus (Rodentia,Muridae), the reservoir of Argentine hemorrhagic fever

In order to assess the reliability of RAPD markers in the estimation of the genetic structure of natural populations of the murid rodent Calomys musculinus (reservoir of Junin virus, ethiological agent of Argentine Hemorrhagic Fever), we have analyzed the heritability of RAPD bands in 10 parents and their offspring (33 individuals). Fourteen out of a total of 119 bands obtained were absent in the parental patterns, but consistently amplified in offspring from some families. These bands can be eliminated from analyses. Overall degree of band sharing between individuals, including non-parental bands, correctly grouped members of a family in the same cluster in a UPGMA tree, with a high bootstrap percentage. Results support the usefulness of RAPDs as hereditable markers. One hundred polymorphic RAPD loci were identified in three natural populations of C. musculinus. Mean expected heterozygosity in three natural populations ranged from 0.206 to 0.220. Allele frequency based and phenotype based measures of genetic differentiation among natural populations of C. musculinus gave similar results (Weir and Cockerham's theta = 0.133; Excoffier et al.'s phi = 0.127). These values were considerably higher than those found previously using allozymes as genetic markers, and are compatible with moderate to low levels of gene flow among populations.     

Enzyme polymorphism in a population of Calomys musculinus (Rodentia,Cricetidae)

NAD-linked lactate, malate, glycerophosphate, alcohol and nonspecific dehydrogenases, aspartate aminotransferases, and soluble esterases from extracts of tissues of individuals from a wild population of Calomys musculinus (Rodentia, Cricetidae) have been analyzed by means of starch gel electrophoresis and specific staining. Allelic frequencies and heterozygosity have been determined. Mendelian inheritance of some of the variants detected was confirmed by breeding experiments. Ten out of fifteen (66.6%) of the genetic loci investigated presented polymorphism. Mean heterozygosity per locus was very high (H=0.2014, se 0.046).This work has been supported, in part, by grants from the Secretaria de Ciencia y Tecnología de la Nación (National Program for Endemic Diseases) and from the Fundación Emilio Ocampo. C. N. G. is a Fellow and A. B. a Career Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.     

Gene Flow Among Calomys Musculinus (Rodentia, Muridae) Populations in Argentina

Calomys musculinus is a Sigmodontinae rodent inhabiting periodically disturbed habitats in the central eastern plains of Argentina. It is the natural reservoir host of Junin virus, the etiological agent of Argentine Hemorrhagic Fever (AHF). In order to analyze the levels of gene flow among populations of this species, allozymic variability at 26 loci was studied in 291 individuals from the endemic zone of AHF and localities outside it. All populations showed high levels of polymorphism (H _e between 0.107 and 0.144; P_95% between 38 and 54%). Individual loci f values were in most cases negative, although not significantly different from zero. Mean genetic differentiation among populations was low, but statistically significant (=0.020; P<0.01). There was no correlation between genetic and geographic distances between pairs of populations, and scatter of the pairwise points suggests that, at the regional scale, genetic drift is more influential than gene flow. This result can be interpreted as indicative of a relatively recent expansion of C. musculinus habitat and restricted on-going gene flow, which would be compatible with a relatively slow expansion rate of AHF.     

Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae)

Small South American rodents of the genus Calomys have been used extensively for virology and ecological research. Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced. The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization. Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction. The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated. In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail. The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro. This is the first report of capacitation and in vitro fertilization for Calomys sp. These results provide opportunities to use C. musculinus and C. laucha as new laboratory animals for research into reproductive biology.     

Co-amplification of mitochondrial pseudogenes in Calomys musculinus (Rodentia, Cricetidae): a source of error in phylogeographic studies.

In a previous phylogeographic study of the rodent Calomys musculinus, 24 haplotypes of the mitochondrial DNA D-loop region were detected using the restriction fragment length polymorphism technique (PCR-RFLP). Seven percent of the individuals showed patterns in which the sum of the sizes of the restriction fragments exceeded the size of the original PCR product. In the present paper we analyze possible causes of these atypical haplotypes. PCR products were cloned, and two or three different clones from a single individual were detected by their RFLP patterns. Nine clones with different restriction patterns were selected for sequence analyses. A maximum parsimony phylogenetic analysis revealed two well-supported paraphyletic groups. One group comprised sequences showing low nucleotide divergence compared with the most common haplotypes detected in the phylogeographic study. The other group was basal to the three species of Calomys other than C. musculinus included in the study; the mutations in the short portion of the cytochrome b gene amplified corresponded to 12 amino acid substitutions. The results suggest that two independent insertions of mtDNA sequences into the nucleus occurred; these sequences would co-amplify in the PCR procedure. Identification of pseudogenes is crucial to obtain reliable reconstruction of the intraspecific genealogy in phylogeographic studies.     

Oligoryzomys flavescens (Rodentia, Muridae): gene flow among populations from central-eastern Argentina

In species acting as hosts of infectious agents, the extent of gene flow between populations is of particular interest because the expansion of different infectious diseases is usually related to the dispersal of the host. We have estimated levels of gene flow among populations of the sigmodontine rodent Oligoryzomys flavescens, in which high titers of antibodies have been detected for a Hantavirus in Argentina that produces a severe pulmonary syndrome. Enzyme polymorphism was studied by means of starch gel electrophoresis in 10 populations from the area where human cases of Hantavirus have occurred. Genetic differentiation between populations was calculated from F_ST values with the equation Nm = [(1/F_ST−1]/4. To assess the relative importance of current gene flow and historical associations between populations, the relationship of population pairwise log Nm and log geographic distance was examined. Low F_ST (mean = 0.038) and high Nm (15.27) values suggest high levels of gene flow among populations. The lack of an isolation by distance pattern would indicate that this species has recently colonized the area. The northernmost population, located on the margin of a great river, shows very high levels of gene flow with the downstream populations despite the large geographic distances. Passive transport of animals down the river by floating plants would promote unidirectional gene flow. This fact and the highest mean heterozygosity of that northernmost population suggest it is a center of dispersal within the species' range. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bibliography of the genus Apodemus (Rodentia, Muridae)

A complete bibliography of the genus Apodemus for the period 1970–1980 is presented, with a selection of the more important papers from earlier years, and a few references for 1981. A coded index to species and subjects is provided. All titles have been translated into English.     

Seasonal Changes in Microhabitat Use and Niche Overlap between Akodon azarae and Calomys laucha (Rodentia,Muridae) in Agroecosystems of Central Argentina

We examined seasonal changes in microhabitat use by the murid rodents Akodon azarae and Calomys laucha in agroecosystems of the former pampa region of Argentina. In summer, trap data showed that both species had low densities and were almost equally distributed between the mature cropfields and their surrounding weedy borders. Analysis of vegetation at the trap sites revealed no selection for microhabitats at the borders, whereas in the cropfields both species shared preferences for covered microhabitats. In contrast, winter data revealed a sharp habitat segregation, being Calomys numerically dominant in post-harvest cropfields and Akodon more abundant in borders. Moreover, there were clear differences in microhabitat selection at the borders, A. azarae occupying the more covered microhabitats and C. laucha the less covered. Breadth and overlap of niches were calculated using principal component analysis, in order to recognize interspecific competition and its influence on community niche space. Available evidence indicates that the structure of this rodent community is strongly influenced by seasonal changes in habitat stucture and rodent abundance. The socially dominant A. azarae retains the best part of the available niche space, a fact becoming more evident under the harsh winter conditions.     

Isolation and characterization of microsatellite markers in Oligoryzomys longicaudatus (Muridae, Sigmodontinae, Oryzomini), the natural reservoir of genotype Andes hantavirus

The rodent Oligoryzomys longicaudatus or long-tailed pygmy rice rat is the reservoir of the aetiological agent of the hantavirus pulmonary syndrome in southern Argentina and Chile. We characterize 11 polymorphic microsatellite loci which would be useful for studies on microgeographical population structure in the species. Amplification of these loci in 42 individuals from four natural populations revealed four to 21 alleles per locus, and values of observed heterozygosities ranging from 0.371 to 0.896. Cross-species amplifications showed that some of the primers designed may be useful for other species of the genus Oligoryzomys.     

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae)

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.     

Reproductive characteristics of free-livingCalomys venustus (Rodentia, Muridae)

Calomys venustus showed a cyclic seasonal pattern of reproductive activity characterized by a period of repose and a variable length of the period of sexual activity between August—September to May—June. The study was done from 1983 to 1999. During this time the population density increased from October—November to the end of autumn and sharply decreased in winter. Three peaks in rate of pregnancy were recognized in the breeding period: spring, summer and late summer. The average litter size was different among the three peaks in rate of pregnancy. The reproductive pattern had a peak at the beginning of the spring produced by overwintering animals, and another peak in summer-autumn caused by the reproductive activity of young born in the same period in which they breed. The spring cohort exhibited the greatest breeding contribution suggesting that this cohort is principally responsible for the yearly peak in abundance.     

Gene flow among native bush rat,Rattus fuscipes (Rodentia: Muridae), populations in the fragmented subtropical forests of south‐east Queensland

Abstract Many natural populations in areas of continuous habitat exhibit some form of local genetic structure. Anthropogenic habitat fragmentation can also strongly influence the dynamics of gene flow between populations. We used eight microsatellite markers to investigate the population genetic structure of an abundant forest species, the Australian bush rat (Rattus fuscipes), in the subtropical forests of south‐east Queensland. Five sites were sampled, allowing pairwise comparisons within continuous habitat and across clearings. Weak, but significant population differentiation and a significant pattern of isolation by distance was detected over the small scale (<10 km) of this study. Fine‐scale analysis at a single site (<1 km) showed a significant correlation between individual female genetic distance and geographical distance, but no similar pattern among male individuals. There was no evidence of increased population differentiation across clearings relative to comparisons within continuous forest. This was attributed to dispersal within corridors of remnant and revegetated habitat between the forested areas. We concluded that an inherently restricted dispersal ability, female philopatry and natural habitat heterogeneity play an important part in the development of genetic structure among populations of R. fuscipes. It is important to understand the relationship between landscape features and the pattern of gene flow among continuous populations, as this allows us to predict the impact of fragmentation on natural populations.     

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS((AGY)) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem-loop structure and control region has several conserved sequence elements.     

Breeding season, litter size and nestling mortality of the Harvest mouse, Micromys minutus (Rodentia: Muridae), in Britain

Data are presented on the breeding biology of Micromys minutus in Britain. It is shown that although litters are produced over a protracted period (May-December), 74% of the litters are born in August and September. Mean litter size is 5·40±0·16( n = 62), which is a significant reduction on the mean litter size of 6·75±0·40( n = 16) calculated from pre-1917 literature. Litter size was found to be constant throughout the breeding season, and there was no difference in mean litter size between two samples collected close to the northern and southern limits of the known British range. By comparing the mean litter size of samples 1–5, 6–10 and 11+ days old, no significant loss of individual mice within litters could be detected, although 12·3% of all litters died prior to weaning. This loss was predominantly of autumnal litters, cold and wet being important climatic factors that terminate the breeding season. Comparisons are made with data from other parts of the animal's range.