Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses

Abstract

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

Figure 1

Physical map of the 16,322 bp circular genome of mitochondrion (MT) of Peromyscus leucopus LL stock of Peromyscus Genetic Stock Center. The map is based on GenBank accession number MG674647. Transcription direction is indicated by arrowhead. Ribosomal RNAs 12S and 16S and transfer RNAs (T) coding sequences are indicated by red; associated amino acids for tRNAs are given by single letter code. Protein coding sequences for cytochrome B (CYB) and numbered subunits of NADH dehydrogenase (ND), cytochrome c oxidase (CO), and ATP synthase F0 (ATP) are indicated by yellow. The D-loop region is indicated by gray.

Figure 2

Comparison of mitochondrial genomes of Peromyscus species and selected other rodents. (A) Neighbor-joining distance phylogram of aligned complete mitochondrial genomes of Peromyscus leucopus (White-footed Deermouse), P. maniculatus (North American Deermouse), other Peromyscus species, cricetines Cricetulus griseus (Chinese hamster) and Mesocricetus auratus (golden hamster), and murids Mus musculus (house mouse) and Rattus norvegicus (brown rat). The correspondingd accession numbers are given in Table S1 of Supplementary Materials. The scale for distance by criterion of observed differences is indicated. Percent bootstrap (100 iterations) support values of ≥90% at a node are shown. (B) Sliding window plot of nucleotide diversity (π) per site of five aligned mitochondrion genome sequences of P. leucopus.

Figure 3

Threshold dot plots of alignments of two fragments (panels A and B) of nuclear mitochondrial pseudogenes paired with the corresponding sequences of the mitochondrion genome (mtDNA) in the reference P. leucopus PGSC animal. The positions in the mtDNA (accession number MG674647) are shown on the x-axis, and positions in chromosome and other scaffolds are shown on the y-axis. The approximate locations of the coding sequences or their pseudogenes in each of the pairs are shown on the diagonal. The coding sequence abbreviations are defined in the Fig. 1 legend. Computation was carried out with dotmatcher of EMBOSS (http://www.bioinformatics.nl/cgi-bin/emboss/dotmatcher).

Figure 4

RNA-seq analysis of RNA abundances along the length of the mitochondrion genome in 5 P. leucopus infected with Borrelia hermsii and 3 uninfected controls. Blood was obtained 4 days after injection of 10³ bacteria or buffer control. Libraries of cDNA from the RNA were subjected to 100 cycles of paired-end Illumina sequencing. Reads matching to each of 100 nt length windows of the mitochondrion genome were counted and normalized as TPM values. (A) Box plots for each of the 100 nt windows for animals grouped by infection state. (B) Plots of the negative log₁₀ of the p-value of 2-tailed t-tests of log-transformed TPM values for each of the windows.

Figure 5

Aerial photograph of mixed hardwood forest field site at Lake Gaillard, CT in June 2018. Numbers indicate the locations of trapping sites 1–3 for capture-and-release of P. leucopus. The GPS coordinates for center of the lake are 41.347 N 72.783 W. Eye altitude is approximately 10,000 m. Photograph obtained with Google Earth Pro.

Figure 6

Neighbor-joining distance phylogram of 881 positions of aligned D-loop sequences of mitochondria of P. leucopus and P. maniculatus. Haplotype 1 is that of the index P. leucopus and other animals of the LL stock colony of the PGSC. Haplotypes 40 and 41 are from P. leucopus captured in the north-central U.S. The other numbered haplotypes are of animals captured at the Lake Gaillard site. P. maniculatus (Pman) representatives are from subspecies bairdii (BW, BW2, BZ1, and SH1) and gracilis (ON1, TA1, and MN1). Accession numbers are given in Table S1 or in Methods. The scale for distance by criterion of observed differences is indicated. Percent bootstrap (1000 iterations) support values of ≥90% at a node are shown.