ABSTRACT
Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626–0.1732, whereas the distances within each subcluster were 0.003–0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010–0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.
Leptotrombidium (Acari: Trombiculidae) mites are important from a public health perspective because of their role as carriers of Orientia tsutsugamushi, a bacterial pathogen causing scrub typhus in humans in the regions of the Pacific Rim such as East and Southeast Asian countries including Japan, Pacific islands and northern Australia, and the parts of the Indian Ocean Rim including India and Pakistan, which is an area referred as “tsutsugamushi triangle” (Tamura et al., 1995). For the mites, O. tsutsugamushi is a maternally inherited endosymbiont but can cause diseases (scrub typhus in humans) when transmitted to mammals upon feeding (Kawamura et al., 1995). The mites feed on mammals (wild rodents or other animals, including humans) only once during the larval stage (their earliest developmental stage, also called chiggers), whereas in their later stages, they feed on the arthropod eggs in the soil (Kawamura et al., 1995). By feeding on mammals heavily infected with O. tsutsugamushi, uninfected larval mites can acquire O. tsutsugamushi, which, however, are not transmitted to their progeny (Takahashi et al., 1988).
Several serotypes of O. tsutsugamushi were reported in Japan (Kawamura et al., 1995) with varying degrees of pathogenicity against mice; Kato, Karp, and Gilliam serotypes were virulent in mice, whereas Kuroki, Kawasaki, and Shimokoshi were less virulent or avirulent (Nagano et al., 1996). Notably, it has been reported that the serotypes of O. tsutsugamushi depend on the mite species; Leptotrombidium akamushi is the reservoir for the Karp serotype, Leptotrombidium pallidum for Gilliam and Karp serotypes (Takahashi et al., 1990, 2004a), and Leptotrombidium scutellare for Kuroki and Kawasaki serotypes (Yamashita et al., 1994; Kawamura et al., 1995). Recently, Leptotrombidium palpale was proposed as the reservoir for the Shimokoshi type (Seto et al., 2013). Although several species of Leptotrombidium are reported, the number of carrier species of O. tsutsugamushi is limited. Therefore, the classification of larval mites is vital for the epidemiological survey of mites, especially for the surveillance of scrub typhus vectors.
Within the reservoir species, carrier rates of O. tsutsugamushi remain at a few percent (Kawamura et al., 1995). Because maternally transmitted endosymbionts should have linkage disequilibrium with mitochondria (Perlman et al., 2015), we hypothesized that the mitochondrial haplotype variation can help explain the presence or absence of O. tsutsugamushi in these mites. For this, we utilized deoxyribonucleic acid (DNA) barcoding targeting mitochondrial gene sequences widely used for taxonomic identification and classification of eukaryotes (Beebe, 2018; Ahmed et al., 2022). The universal primer set LCO1490 and HCO2198, designed initially against highly conserved regions of mitochondrial cytochrome c oxidase subunit I (COI) genes across several taxa, were commonly used for DNA barcoding of invertebrate species (Folmer et al., 1994). However, it was reported that the universal primers should be redesigned for some taxonomic groups (Sharma and Kobayashi, 2014).
The objective of our study was to redesign a COI primer set for DNA barcoding using consensus sequences derived from different Leptotrombidium species, which are significant carriers of O. tsutsugamushi, and then apply this improved DNA barcoding for the classification of Leptotrombidium to evaluate the genetic variation of major species carrying O. tsutsugamushi in Japan.
MATERIALS AND METHODS
A sampling of mites and DNA extraction
Unfed larval mites inhabiting the soil were collected using the modified “black cloth method” (Uchikawa et al., 1994; Takada et al., 2001), and engorged larval mites were collected from wild rodents as described previously (Takahashi et al., 1988, 2004b) from 7 geographically separated regions of Japan (Fig. 1). Species of the mites for sequencing a full-length COI gene (Fig. 2) were carefully identified by observing detailed morphological features under a stereomicroscope. In contrast, the mites used for DNA barcoding were briefly identified by following only a few major morphological characteristics (Table I). For DNA extraction, the mites were cut or punctured with a 21-gauge needle on a clean glass slide placed under a stereomicroscope, transferred into PrepMan™ Ultra sample preparation reagent (Thermo Fisher Scientific K.K., Tokyo, Japan) in a microtube, and then heated at 96 C for 5 min. The samples were directly used for polymerase chain reaction (PCR). All the obtained sequences were deposited in the DNA data bank Japan (DDBJ) of National Institute of Genetics (NIG), Japan.
DNA sequencing of full-length COI genes
For sequencing of a full-length COI gene, PCR was performed with the primer set COX1fullF (5′–TTCTTTAAATTTGCAATTTAATWTC–3′) and COX1fullR (5′–AAAATAGATTGTCAGATTGGCA–3′) using EX Taq HS polymerase (Takara Bio Inc., Shiga, Japan). These primers were designed on the basis of the consensus sequences from the references of L. akamushi (NC_007601), L. pallidum (NC_007177), and Leptotrombidium delicense (NC_007600), which were obtained from the database of National Center for Biotechnology Information (NCBI, Bethesda, Maryland). DNA extracted from L. scutellare, Leptotrombidium fuji, L. palpale, and 2 L. pallidum mites, morphologically identified under a stereomicroscope, were used for PCR. Thermal conditions of PCR were as follows: denaturation at 94 C for 1 min followed by 30 cycles of amplification (denaturation at 94 C for 30 sec, annealing at 55 C for 30 sec, and extension at 68 C for 1 min) and a final extension at 68 C for 10 min. DNA sequencing was determined by dye terminator using ABI 3500 Genetic Analyzer for Resequencing & Fragment Analysis (Thermo Fisher Scientific K.K.) following the manufacturer's protocol.
Phylogenetic analysis
COI sequences aligned using ClustalW implemented in MEGA6 software (Tamura et al., 1995) were subjected to phylogenetic tree reconstruction using the neighbor-joining method with bootstrap analysis of 1,000 pseudoreplicates by MEGA6. On the basis of a previous report, a bootstrap value ≥70% was considered the threshold for reasonable confidence (Hillis and Bull, 1993). In the analysis, sequences of Walchia hayashii (NC_010595.1), obtained from the database of NCBI along with those of Leptotrombidium, were used. These sequences were either obtained in this study or from the database. Mean genetic distances (p-distance) within or between clusters and subclusters were estimated by MEGA6.
To perform network analysis, COI sequences were aligned using ClustalW implemented in MEGA6, and ambiguous sites containing degenerate nucleotides were manually removed. The cleaned sequence was subjected to draw a maximum parsimony TCS network (Clement et al., 2000) using the software PopART (Leigh and Bryant, 2015).
DNA barcoding of Leptotrombidium
We improved the universal primer set LCO1490 and HCO2198 for DNA barcoding on the basis of the COI gene using the consensus sequence of significant Leptotrombidium, which are carefully and definitively identified by morphological classification using a stereomicroscope (Fig. 2). DNA barcoding was performed by PCR using the improved degenerate primer set LCO1490_Ltromb (5′–TTTCHACWAAYCCYAARGAYATTGG–3′) and HCO2186_Ltromb (5′–GWCCRAARAAYCARAAHARATGTTG–3′), as well as a conventional universal primer, set LCO1490 (5′–GGTCAACAAATCATAAAGATATTGG–3′) and HCO2198 (5′–TAAACTTCAGGGTGACCAAAAAATCA–3′; Folmer et al., 1994). For DNA barcoding, DNA extracted from 213 L. scutellare, 32 L. pallidum, 16 L. fuji, and 14 Walchia masoni (suspected) mites roughly morphologically identified using a stereomicroscope as well as from 16 unidentified mites were used. PCR was performed under the following cycling conditions: initial denaturation at 94 C for 1 min followed by 30 cycles of amplification (denaturation at 94 C for 5 sec, annealing at 55 C for 1 sec, and extension at 68 C for 40 sec) and final extension at 68 C for 1 min. The expected PCR product size is 697 base pairs (bp) with the improved primer set and 709 bp with the conventional primer set. DNA sequencing was performed using the same improved PCR primers as described above. All the obtained sequences were deposited in DDBJ of NIG, Japan.
RESULTS
DNA sequences and phylogenetic analysis of full-length COI genes
We obtained full-length sequences of COI genes (1,533 bp) from L. scutellare (LC681948), L. fuji (LC681944), L. palpale (LC681945), and the 2 L. pallidum mites (LC681946 and LC6819476). Phylogenetic analysis showed that sequences of L. pallidum formed a single clade clustered with L. fuji (Fig. 2), whereas L. scutellare formed a clade with L. akamushi/L. deliense. However, the position of L. palpale was unclear.
PCR detection using the improved primer set for DNA barcoding
We compared the full-length sequences of COI genes and found that the corresponding sequences to the original universal primers, LCO1490 (forward) and HCO2198 (reverse), differed among the strains (Fig. 3). Therefore, we designed degenerate primers, LCO1490_Ltromb (forward) and HCO2186_Ltromb (reverse), to obtain a suitable consensus sequence. HCO2186_Ltromb is located at the position 12 bp shifted from HCO2198 toward the 5′ sides. PCR with the new primer set led to an increase in identification rates of L. scutellare COI (approximately 17% increase), whereas the identification rates of COI of other mites remained almost the same (Table I).
DNA barcoding with the improved primer set
We performed DNA barcoding by sequencing the 215 samples that showed intense bands by PCR. These include 137 L. scutellare, 42 L. pallidum, 8 L. fuji, 3 Leptotrombidium intermedium, 1 L. palpale, and 24 mites belonging to other genera such as Walchia (LC682866 to LC6803080) including the 5 sequences of full-length COI genes determined in this study. Phylogenetic analysis showed that all species of Leptotrombidium were clustered together and separated from similar mite species of other genera (Fig. 4). The genus Leptotrombidium was further divided into 4 clusters, Lpt-1, Lpt-2, Lpt-3, and Lpt-4. It should be noted that the phylogenetic relationship between the 4 clusters is unclear, and Lpt-3 does not represent a well-supported clade, which may be further separated into multiple clades by further sampling. Lpt-1 and Lpt-2 are expanded in Figure 5.
The first cluster, Lpt-1 (L. scutellare), was divided into 3 subclusters, Lsc-1, Lsc-2, and Lsc-3. Included in the subcluster Lsc-1 was an L. scutellare individual for which full-length COI was determined in this study (LC681948, marked by a star in Fig. 5). Lsc-1 consists of samples derived from the 6 regions (Gumma, Saitama, Kanagawa, Ishikawa, Fukui, and Kagoshima) in Japan, whereas Lsc-2 and Lsc-3 are comprised of representatives from the limited areas (Lsc-2 from Kanagawa and Kagoshima; Lsc-3 from Kanagawa and Fukui; Figs. 5, 6). Circular structure in the haplotype network of Lsc-1 shows the presence of homoplasy in the COI sequence, which may imply natural selection acting on COI (Fig. 6). Uneven presence of samples derived from different regions may reflect a limited gene flow due to geographic distance, particularly between Kagoshima and other regions (Fig. 6).
The second cluster, Lpt-2, can be separated into 4 small clusters: L. pallidum, L. intermedium, L. fuji, and a cluster that does not contain known species (Fig. 5). The cluster of L. pallidum was further separated into 2 clear subclusters, Lpd-1, and Lpd-2. The subcluster of Lpd-1 includes 2 L. pallidum individuals for which full-length COI were determined in this study (LC681946 and LC6819476, marked by a star in Fig. 5). Lpd-1 and Lpd-2 contained the samples from the same 2 regions (Saitama and Nagano; Figs. 5, 6).
Mean genetic distances between or within L. scutellare and L. pallidum were estimated. The mean genetic distance between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044. Within L. scutellare, the mean genetic distances between Lsc-1 and Lsc-2, Lsc-1 and Lsc-3, and Lsc-2 and Lsc-3 (the subclusters) were 0.1626, 0.1675, and 0.1732 respectively, whereas the distances within Lsc-1, Lsc-2, and Lsc-3 were 0.017, 0.003, and 0.017 respectively. Within L. pallidum, the mean genetic distance between Lpd-1 and Lpd-2 (the subclusters) was 0.1029, whereas the distances within Lpd-1 and Lpd-2 were 0.010 and 0.013 respectively.
The cluster L. fuji included an L. fuji individual for which full-length COI was determined in this study (LC681944, marked by a star in Fig. 5). The third cluster Lpt-3 consisted of L. deliense, L. akamushi, and miscellaneous species and the fourth cluster Lpt-4 consisted of L. palpale including an L. palpale individual for which full-length COI was determined in this study (LC681945, marked by a star in Fig. 5; Fig. 4).
Comparison between morphological and molecular classification
The molecular classification of the tested mites by DNA barcoding was compared with morphological classification (Table II). In each species, the accuracy of morphological classification is evaluated by the percentage of samples identified by molecular classification/number of samples classified by morphological inspection. The accuracy of morphological classification was 97.8% (133/136) for L. scutellare, 100% (35/35) for L. pallidum, and 66.7% (8/12) for L. fuji. Nine of Walchia masoni were classified into Walchia spp. since further classification was not done in this study. By molecular classification, 3 samples morphologically misclassified as L. scutellare were found to be L. pallidum, L. palpale, and other non-Leptorombidium mites. By molecular classification, 4 samples were morphologically misclassified as L. fuji and identified as L. intermidium and other non-Leptorombidium mites.
DISCUSSION
In this study, we found that COI genes of Leptotrombidium mite were very different from some other similar mites and varied even among the species in Japan (Fig. 4). According to the consensus sequence of the full-length COI genes (Fig. 3), we redesigned the primer set for DNA barcoding of Leptotrombidium, which drastically increased the sensitivity (Table I), especially against L. scutellare, one of the significant mite species in Japan that transmits O. tsutsugamushi, the etiological agent of scrub typhus (Yamashita et al., 1994; Kawamura et al., 1995).
The DNA barcoding performed for 291 mites using the new primer set revealed that they were classified into 5 species of the genus Leptotrombidium: L. scutellare, L. pallidum, L. fuji, L. intermedium, and L. palpale; the genus Walchia; and other genera (Fig. 5). Specifically, the sequences of both L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi, were further divided into some subclusters (Fig. 5). However, the morphological inspection did not allow further classification beyond individual species level (Table I). Both major subclusters, Lsc-1 of L. scutellare and Lpd-1 of L. pallidum, included the sequences of full-length COI genes of each species determined in this study using the mite sample morphologically classified with detailed features. This suggests that Lsc-1 and Lpd-1 constitute a significant group of each mite species, and the other subclusters Lsc-2, Lsc-3, and Lpd-2 are subgenotypes of each mite species. Detailed morphological classification should be done for the mites classified into subclusters (Lsc-2, Lsc-3, and Lpd-2), even though these mite samples had the same major morphological features. Furthermore, nuclear gene sequences of the mites would be required to confirm the existence of subgenotypes.
Microscopical classification is difficult owing to the minuscule size of the larval mites (approximately 0.2 mm); morphological identification requires considerable skills and taxonomic expertise. The accuracy of morphological classification was high for L. scutellare and L. pallidum, whereas not very high for L. fuji (Table II). Most of the misclassified samples of L. fuji were genetically classified into L. intermedium; however, the cluster of L. intermedium was not well divided from that of L. fuji. These results suggest that L. fuji and L. intermedium should be classified into a single species. These results indicate the advantage of DNA barcoding, which showed a more precise classification of Leptotrombidium even within the species (Figs. 4, 5).
Recent studies from our laboratory showed that certain species of Leptotrombidium were carriers of several novel symbiotic bacteria including Wolbachia spp., Rickettsiella spp., and Rickettsia spp. other than O. tsutsugamushi (Ogawa et al., 2020). Symbiotic bacteria, including O. tsutsugamushi, are transmitted through a mite life cycle by transstadial transmission and to their eggs and progeny by transovarial transmission (Takahashi et al., 1988; Urakami et al., 1994). This suggests that both the host mites and their symbiotic bacteria can coevolve. Only a few percent of mites carry O. tsutsugamushi (Kawamura et al., 1995) in some areas. However, Wolbachia, a nonpathogenic symbiotic genus of bacteria, was distributed in a wide area of Japan. DNA barcoding in this study may make it possible to clarify the relationship between DNA haplotypes of mites and their bacterial symbionts, such as O. tsutsugamushi. The host DNA haplotypes may allow distinction between harmful mites and nonharmful ones to humans.
This study showed that the improved DNA barcoding with the redesigned primers had higher detection sensitivity than universal primers; we also obtained a higher classification accuracy than morphological classification methods. Finally, the DNA barcoding uncovered the broad genetic diversity in Leptotrombidium mites, especially in L. scutellare and L. pallidum, the primary species that carry O. tsutsugamushi.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant number: 60K21321, R2-Chousenteki houga kenkyu].