Comparison of Chiggers (Acari: Trombiculidae, Leeuwenhoekiidae) on Two Sibling Mouse Species, Apodemus draco and A. ilex (Rodentia: Muridae), in Southwest China

Simple Summary Chigger mites (Acari) are common ectoparasites and the exclusive vector of scrub typhus. South China field mouse (Apodemus draco) and Lantsang field mouse (A. ilex) are two sibling rodent species. The investigation in southwest China (2001–2015) showed that chigger infestations on two mouse species were quite different, including different species composition, overall infestation, community parameters and dominant chigger species. There were 36 chigger species found on A. draco and 11 on A. ilex, and the overall mean intensity of chiggers on A. draco (MI = 4.26) was higher than that on A. ilex (MI = 3.91, p < 0.05). Dominant chiggers were unevenly distributed among different individuals of mouse hosts, and chigger infestation showed sex bias on different mouse sexes. The species abundance of the chigger community on A. draco was revealed as a log-normal distribution pattern. Abstract Chigger mites (Acari) are common ectoparasites on rodents, and they are the exclusive vector of scrub typhus. South China field mouse (Apodemus draco) and Lantsang field mouse (A. ilex) are two sibling rodent species. Based on field investigations in southwest China (2001–2015), this paper compared the infestation of these two mouse species with chiggers. Of 42 chigger species identified from two mouse species, 36 were found on A. draco, 11 on A. ilex and 5 common species on both mice. Jaccard similarity index (J = 0.12, J < 0.25) showed a very different species composition of chiggers on two mouse species, and some parameters of the chigger community were also different. The overall mean intensity of chiggers on A. draco (MI = 4.26) was higher than that on A. ilex (MI = 3.91, p < 0.05). The dominant chigger species on A. draco were Trombiculindus yunnanus, Leptotrombidium scutellare (a major vector species in China) and L. sinicum with a total constituent ratio Cr = 42.9% (106/247). Leptorombidium sinicum and L. scutellare independently occurred on A. draco with an association coefficient V = 0.09 (V ≈ 0). The dominant chigger species on A. ilex were L. rusticum, L. densipunctatum and L. gongshanense, with a total Cr = 58.14% (25/43). Leptorombidium rusticum and L. densipunctatum on A. ilex had a slight positive association (V = 0.49, 0.5 < V < 1). All dominant chigger species were unevenly distributed among different individuals of two mouse species. Chigger infestation showed sex bias on different sexes of two mouse species. The species abundance of the chigger community on A. draco was revealed as a log-normal distribution pattern.


Introduction
Chigger mites belong to the families Trombiculidae and Leeuwenhoekiidae in the subclass Acari [1,2]. It is estimated that about 3013 species of chigger mites have been

Field Survey Sites
The original data came from the field investigations in 91 survey sites of five provincial regions in southwest China from 2001 to 2015 [29,30]. Among the 91 sites investigated, there are 8 sites where A. draco was captured, and 3 sites where A. ilex was captured, totaling 11 sites (Figure 1).

Collection and Identification of Chiggers and Their Animal Hosts
Rodents and other small mammals (animal hosts) were captured with mousetraps, and chiggers on their body surfaces were routinely collected and fixed. Each host was identified into species according to its morphological appearance (body size, shape and coat color), various measurements (body length, body weight, tail length, ear height, hind foot length, etc.) and other morphological features [31,32]. The collected chiggers were mounted with Hoyer's medium and made into slide specimens. After dehydration, drying and transparency, each chigger was identified into species under an optical microscope (Olympus Corporation, Tokyo, Japan) [1,3,[33][34][35]. Based on the identification results of chiggers and their animal hosts, A. draco and A. ilex, together with their chiggers, were chosen as the target of present study. The capture and use of animals were officially approved by the local wildlife affairs authority and the Animal Ethics Committee of Dali University, and the ethics approval number is DLDXLL2020-1104.

Infestation Statistics Analysis
The species and numbers of all chiggers on the body surface of each A. draco and A. ilex were counted, respectively. The constituent ratio (Cr), infestation prevalence (PM), mean abundance (MA) and mean intensity (MI) were adopted to calculate the infestation of mice with chiggers [7,34,36].

= × 100%
(1) In the above formulae, Ni = the number of a certain chigger species (species i) on a certain species of host, N = the total number of all the chigger species, H = the total number of hosts captured, Hi = the number of hosts infested with chiggers.

Collection and Identification of Chiggers and Their Animal Hosts
Rodents and other small mammals (animal hosts) were captured with mousetraps, and chiggers on their body surfaces were routinely collected and fixed. Each host was identified into species according to its morphological appearance (body size, shape and coat color), various measurements (body length, body weight, tail length, ear height, hind foot length, etc.) and other morphological features [31,32]. The collected chiggers were mounted with Hoyer's medium and made into slide specimens. After dehydration, drying and transparency, each chigger was identified into species under an optical microscope (Olympus Corporation, Tokyo, Japan) [1,3,[33][34][35]. Based on the identification results of chiggers and their animal hosts, A. draco and A. ilex, together with their chiggers, were chosen as the target of present study. The capture and use of animals were officially approved by the local wildlife affairs authority and the Animal Ethics Committee of Dali University, and the ethics approval number is DLDXLL2020-1104.

Infestation Statistics Analysis
The species and numbers of all chiggers on the body surface of each A. draco and A. ilex were counted, respectively. The constituent ratio (C r ), infestation prevalence (P M ), mean abundance (MA) and mean intensity (MI) were adopted to calculate the infestation of mice with chiggers [7,34,36].
In the above formulae, N i = the number of a certain chigger species (species i) on a certain species of host, N = the total number of all the chigger species, H = the total number of hosts captured, H i = the number of hosts infested with chiggers.

Basic Community Structure Statistics
Species richness (S), Shannon-Wiener diversity index (H), Simpson dominance index (D) and Pielou evenness index (E) were used for chigger community statistics. Jaccard similarity coefficient (J) was used to analyze the similarity of community species composition [36,37].
In the above formulae, N = the total number of all the chigger species, N i = the number of a certain chigger species (species i) on a certain species of host, S = species richness (the number of species), G = the number of chigger species in community A, F = the number of chigger species in community B, M = the number of common species existed in both community A and B. 0.00 < J < 0.25 means extremely dissimilar, 0.25 ≤ J < 0.50 means moderately dissimilar, 0.50 ≤ J < 0.75 means moderately similar, and 0.75 ≤ J < 1.00 means extremely similar.

Measurement of Spatial Distribution Patterns
The spatial distribution of dominant chigger species among different individuals of mouse hosts was determined by the indexes of diffusion (C), mean crowding (m*) and clumping index (I). The calculation formulae and judgment criteria are listed in Table 1 [38,39]. Table 1. Index formulae and judgment criteria for spatial distribution patterns.

Name
Formula Aggregation Distribution

Random Distribution
Uniform Distribution Annotation : σ 2 = variance, m = mean and n = the total number of host samples.

Measurement of Interspecific Association
The association coefficient (V) was used to analyze the interspecific relationship between any two dominant chigger species on A. draco and A. ilex, and Chi-square test was used to test the statistical significance of V [5,40].
In the above formula, V = association coefficient between any two chigger species, X and Y, on a certain host species; a = host individuals on which both chigger species X and Y concurrently appear; b = host individuals on which chigger species Y appears, but chigger species X does not appear; c = host individuals on which chigger species X appears, but chigger species Y does not appear; and d = host individuals on which neither chigger species X nor Y appears. When 0 < V ≤ 1 and p < 0.05, the interspecific relationship between chigger species X and Y is determined as positive association, and when −1 ≤ V < 0 and p < 0.05, negative association. When V = 0 or V ≈ 0, it can be considered that chigger species X and Y independently occur on the host.

Species Abundance Distribution
X-axis (indicating individuals of chiggers) was labeled with log intervals based on log 3 M, and Y-axis (representing the number of chigger species) was marked with arithmetic scales. Based on following formulae, Preston's lognormal model was used to fit the theoretical curve of species abundance distribution of chigger community with the calculation of fitting goodness, R 2 [34,[41][42][43].
(e = 2.71828 . . .) Preston slognormal model (10) In the above formulas, S(R) = the theoretical number of chigger species at the R-th log interval, S 0 = the number of chigger species at the R 0 log interval, m = the number of log intervals, R 0 = the mode log interval, S'(R) = the actual chigger species at R-th log interval and − S(R) = the average chigger species for each log interval. The value of α was determined according to the best-fitting goodness, R 2 . Among the 11 survey sites shown in Figure 1, 567 A. draco were captured in 8 sites, and 154 A. ilex were captured in 3 sites, with a total of 721 hosts (567 + 154). From the body surface of 721 hosts, 313 chiggers were collected. Of the 313 chiggers collected, 290 ones were identified as 42 species and 7 genera in 3 subfamilies under 2 families, and 23 mites were unidentified because of broken bodies, dirt-covered bodies, blurred structures or suspected new species. The 23 unidentified chiggers were not included in the statistical analysis of this study. Among the 42 chigger species identified, there are 36 species on A. draco, 11 species on A. ilex, and 5 common species on both Apodemus ( Table 2). Jaccard similarity index (J) shows that the species composition of chiggers on two mouse species of Apodemus is very different, with J = 0.12 (J < 0.25, extremely dissimilar). There are some differences in the community parameters of chiggers on two mouse species. The species richness (S = 36) and Shannon-Wiener diversity index (H = 2.89) of chigger community on A. draco are higher than those on A. ilex (S = 11, H = 2.10), but Pielou evenness index and Simpson dominance index (E = 0.81, D = 0.09) on A. draco are lower than those on A. ilex (E = 0.88, D = 0.15).  Annotation: "+"= The host surface contains this chigger mite. "-"= The host body surface does not contain this chigger mite.

Overall Infestation and Dominant Species of Chiggers on Two Mouse Species
There were consistent differences in the overall infestation of chiggers in the two mouse species. Of all A. draco mouse hosts, 58 mice of them were infested with chiggers with 10.23% of overall infestation (P M = 10.23%, 58/567), 0.44 mites/per mouse of mean abundance (MA = 0.44) and 4.26 mites/per mouse of mean intensity (MI = 4.26), which were higher than the corresponding indices on A. ilex (P M = 7.14%, MA = 0.28 mites/mouse, MI = 3.91 mites/mouse). The difference in overall mean intensity (MI) of chiggers on two mouse species was statistically significant (p < 0.05), but there was no statistical significance in the differences in overall prevalence (P M ) and overall mean abundance (MA) of the mites on the mice (p > 0.05). The dominant chigger species on the two mouse species were also different. The dominant chigger species on A. draco are Trombiculindus yunnanus Wang and Yu, 1965, L. scutellare and L. sinicum Yu, Yang and Gong, 1981 (total C r = 42.9%, 106/247). The dominant chigger species on A. ilex, however, are L. rusticum Yu, Yang and Gong, 1986, L. densipunctatum Yu, Yang and Gong, 1982 and L. gongshanense Yu, Yang and Gong, 1981 (total C r = 58.14%, 25/43) ( Table 3). The diagnostic characteristics of the six dominant chigger species on two mouse species are listed in Table 4, and the corresponding abbreviations refer to the relevant taxonomic literature [1,2]. The photos of the representative dominant mite species are shown in Figures 2-5.

Names of Dominant Chigger Species Diagnostic Characteristics
A. draco        The calculation of the Jaccard similarity index showed that the species compositio of chiggers on different sexes (males and females) of two mouse species was quite differ ent (moderately dissimilar), J < 0.5 (A. draco: J = 0.27, A. ilex: J = 0.45). The infestation in dexes (PM, MA, MI) of chiggers were also different between the two sexes of the mice, bu the differences are of no statistical significance (Table 5).   The calculation of the Jaccard similarity index showed that the species composition of chiggers on different sexes (males and females) of two mouse species was quite differ ent (moderately dissimilar), J < 0.5 (A. draco: J = 0.27, A. ilex: J = 0.45). The infestation in dexes (PM, MA, MI) of chiggers were also different between the two sexes of the mice, bu the differences are of no statistical significance (Table 5). The calculation of the Jaccard similarity index showed that the species composition of chiggers on different sexes (males and females) of two mouse species was quite different (moderately dissimilar), J < 0.5 (A. draco: J = 0.27, A. ilex: J = 0.45). The infestation indexes (P M , MA, MI) of chiggers were also different between the two sexes of the mice, but the differences are of no statistical significance (Table 5). Annotation: There were five individuals (n = 5) of A. draco without sex records, and these five mice were not included in the calculation of the above table.
The association coefficient (V) between L. sinicum and L. scutellare on A. draco was close to 0 (V = 0.09, V ≈ 0), and that between L. rusticum and L. densipunctatum on A. ilex was close to 0.5 (V = 0.49) (Tables 6 and 7).   All the calculated indexes of dominant chigger species on two mouse species for spatial distribution patterns were higher than the border values (C > 1, m* > m, I > 1) of determining aggregated distribution (Tables 1 and 8).

Species Abundance Distribution of Chigger Community
Of the 36 species and 247 identified chiggers on A. draco, the number of chigger individuals at Log interval 4 was the highest, but the number of species was minimal. At Log interval 0 (R 0 = 0), there was only one individual chigger, but the number of chigger species was the largest (S 0 = 13). The species abundance distribution of the chigger community on A. draco was successfully fitted by Preston's lognormal model with α = 0.36 and R 2 = 0.86. The theoretical curve equation was (R) = 13e −[0.36(R−0)] 2 (S 0 = 13, R 0 = 0) (Table 9, Figure 6).

Discussion
Previously A. draco and A. ilex were regarded as the same species, and A. ilex was once considered a subspecies of A. draco [18,44,45]. Recent studies have proved that A. draco and A. ilex are two independent species of rodents, and they are two sibling species. Although A. draco and A. ilex are quite similar in morphology, they still have some differences in morphology, molecular characteristics and geographical distribution areas

Discussion
Previously A. draco and A. ilex were regarded as the same species, and A. ilex was once considered a subspecies of A. draco [18,44,45]. Recent studies have proved that A. draco and A. ilex are two independent species of rodents, and they are two sibling species. Although A. draco and A. ilex are quite similar in morphology, they still have some differences in morphology, molecular characteristics and geographical distribution areas [16,17,28]. Apodemus draco is mainly distributed in countries and regions such as China, north Myanmar and northeast India. In China, A. draco is mainly distributed in Yunnan, Tibet and Fujian provincial regions. The distribution range of A. ilex is narrow and mainly distributed south of the Yangtze River in south China and west of the Lantsang River in southwest China [16,17,28]. The present study revealed that A. draco and A. ilex were distributed in southwest China, but they were not the dominant rodent species in this area (only 567 A. draco and 154 A. ilex were captured). The distribution range of two mouse species in southwest China was obviously different. Apodemus draco was mainly captured east of the Jinsha River, and A. ilex was mainly captured west of the Lantsang River (Figure 1). This result is consistent with some previous research reports [16,28].
The results of this study showed that A. draco and A. ilex were not only obviously different in distribution areas but also different in chigger infestations, including species composition, community structure, infestation status and dominant chigger species. The Jaccard similarity index (J) used in this study is an index reflecting the similarity of species composition of any two communities. When 0.00 < J < 0.25, the species composition of two communities is extremely dissimilar, 0.25 ≤ J < 0.50 means moderately dissimilar, 0.50 ≤ J < 0.75 means moderately similar, and 0.75 ≤ J < 1.00 means extremely similar [46]. The results reveal that the species composition of the chigger community on two sibling species of Apodemus are very different with J = 0.12 (J < 0.25), and the community parameters (S, H, E and D) are also different. The overall infestation prevalence (P M ), mean abundance (MA) and mean intensity (MI) of chiggers on A. draco are higher than those on A. ilex. The dominant chigger species on A. draco are T. yunnanus, L. scutellare and L. sinicum, which are obviously different from the dominant mite species on A. ilex (L. rusticum, L. densipunctatum and L. gongshanense) ( Table 3). Previous studies have shown that different small mammal species have different susceptibilities to the infestation of ectoparasites, including chiggers, which leads to differences in species composition, infestation status and dominant parasite species on different species of animal hosts [1,47]. Chevrier's field mouse (A. chevrieri Miline-Edwards, 1868) is a mouse species in the same genus (Apodemus) as A. draco and A. ilex, and it is one of the dominant rodent species in southwest China [30]. A special study on chiggers of A. chevrieri in southwest China showed that its overall infestation prevalence (P M = 31.95%), mean abundance (MA = 6.32 mites/mouse) and mean intensity (MI = 19.77 mites/mouse) with chiggers were significantly higher than the corresponding infestation indexes on the two mouse species of Apodemus in this study. The dominant chigger species on A. chevrieri are L. scutellare, L. densipunctatum and L. cricethrionis Wen, Sun and Sun, 1984, which are also obviously different from A. draco and A. ilex in this study [30]. The above differences reflect the different susceptibility of different mouse host species to chigger infestation. In this study, there are a series of differences in species composition, infestation status and dominant species composition of chiggers between two mouse species of Apodemus, which further verify the different susceptibility of different hosts to chiggers and the different preference of chiggers to different hosts. From the aspect of ectoparasites, the differences in species composition, infestation status and dominant species composition of chiggers between two sibling mouse species also support that A. draco and A. ilex belong to two independent species [16,28]. As one of the dominant chigger species of A. draco, L. scutellare is not only the second major vector of scrub typhus in China but also a potential vector of hemorrhagic fever with renal syndrome [9,48]. The occurrence of L. scutellare on the body surface of A. draco may increase the potential risk of spreading the pathogen of scrub typhus, Orientia tsutsugamushi, from rodents to humans.
The calculation results of the Jaccard similarity index (J) showed that the species composition of chiggers on different sexes of two mouse species, A. draco and A. ilex, were quite different (J < 0.5, moderately dissimilar). Besides, the infestation indexes (P M , MA, MI) of chigger on different sexes of two mouse species were also different ( Table 5). These results indicate that there is sex bias in chigger infestation between male and female hosts [29,36].
Sex bias is prevalent in parasite infections (including ectoparasite infestations), and many parasites are more likely to choose to parasitize in male hosts [4,49,50], but the preference of chiggers to male hosts is not obvious in this paper (Table 5), which may need further studies.
In this study, the interspecific relationship between the dominant chigger species on two mouse species was measured by association coefficient (V). The results showed that the association coefficient (V) between L. sinicum and L. scutellare on A. draco was close to 0 (V = 0.09, V ≈ 0). The V between L. rusticum and L. densipunctatum on A. ilex was close to 0.5 (V = 0.49, V ≈ 0.5). The association coefficient (V) used in this paper is one of the simple and practicable methods to judge the interspecific relationship between any two species in a certain community. The range of V is from 0 to positive and negative 1, that is, [1, ±1]. When V approaches 0, the distribution of the two species is independent of each other. When V is positive and close to 1, it means that two species have a tendency to coexist in a certain environment or on a certain species of host for parasites. When the V is negative and close to -1, it means that there is a mutually exclusive relationship between the two species [5,40]. This study implies that the distribution of L. sinicum and L. scutellare on A. draco seems independent of each other without obvious interspecific dependency. A low degree of interdependence, however, exists between L. rusticum and L. densipunctatum on A. ilex, and these two chigger species tend to choose the same individuals of A. ilex at the same time, but the degree of interdependence is still relatively low (V < 0.5).
The results showed that all the distribution indexes calculated were larger than the critical value (C > 1, m* > m, I > 1) of determining the aggregated distribution ( Table 1,  Table 8), and this indicates that the dominant chigger species are of aggregated distribution among different individuals of their corresponding mouse host, A. draco and A. ilex. This aggregated distribution further indicates that the distribution of dominant chigger species among different individuals of their hosts is very uneven. Some host individuals may harbor a large number of chiggers on their body surface, while some other hosts may have no or only a few chiggers. The aggregated distribution of ectoparasites, including chiggers, suggests that there is an intraspecific relationship of mutual attraction and interdependence between different individuals of the same parasite species. This mutual attraction and interdependence within a certain species are conducive to the survival, mating and reproduction of the population [5,51].
The species abundance distribution aims to reveal the relationship between the number of species and the number of individuals in a community, which reflects the proportion structure of common and rare species in the community [33,42,43]. In the present study, the species abundance distribution of the chigger community on A. draco was successfully fitted by Preston's lognormal model, which shows that most chigger species are rare species with few individuals, while few mite species are dominant species with abundant individuals. With the increase of chigger individuals, the number of chigger species gradually decreased (Table 9, Figure 6). The result is consistent with some previous reports on the species abundance of chiggers [36,43]. Due to the small number of individuals and species of chiggers and mice collected, however, Preston's lognormal model is not applicable to the chigger community on A. ilex. In ecological practice, if the species abundance distribution of a specific community is successfully fitted by Preston's log-normal distribution model, the number of expected total species in the community can be roughly estimated, but this estimation must be based on a large number of samples [52]. Due to the small number of host samples (only 567 A. draco and 154 A. ilex captured) and a small number of chiggers (total 290), the total number of chigger species was not estimated in this paper, which remains to be conducted in further studies.
It must be pointed out that the present study is just a preliminary comparison of chiggers on two sibling mouse species due to the small number of host samples, especially A. ilex, and some results may still be unstable. With the expansion of survey areas and the increase of host samples in future research, some results may fluctuate and change to some extent.

Conclusions
In southwest China, the susceptibility of two sibling mouse species (A. draco and A. ilex) to chigger infestation is quite different, with different species composition and community structure, different infestation status and different dominant chigger species. The results support that A. draco and A. ilex are two independent rodent species from the aspect of chigger infestation.  Institutional Review Board Statement: The use of animals (including animal euthanasia) for our research was officially approved by the Animals' Ethics Committee of Dali University, approval code: DLDXLL2020-1104, approval date: 4 November 2020.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The experimental data used to support the findings of this study are available from the corresponding author upon request.