Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia

ABSTRACT

We developed microsatellite markers for Appasus japonicus (Heteroptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula and mainland China) and often listed as an endangered species in the Red List or the Red Data Book at the national and local level in Japan. Here, we describe twenty novel polymorphic microsatellite loci developed for A. japonicus, and marker suitability was evaluated using 56 individuals from four A. japonicus populations (Nagano, Hiroshima and Yamaguchi prefectures, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged from 1 to 12 (mean = 2.5), and the average observed and expected heterozygosity and fixation index per locus were 0.270, 0.323 and 0.153, respectively. In addition, a population structure analysis was conducted using the software STRUCTURE, and its result suggested that the 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which should contribute to population genetics studies of this species.

MAIN

Freshwater biodiversity, including that of aquatic invertebrates, is the overriding conservation priority of the International “Water for Life” Decade for Action (Dudgeon et al., 2006; Doi et al., 2017). Recently, studies of phylogenetic evolution of freshwater invertebrates, which have weak dispersal ability, have been attracting much attention, because they establish the freshwater invertebrates’ strong associations with geohistory and provide substantial information on the creation of genetic and species diversity (e.g., Tojo et al., 2017; Saito et al., 2018; Sekiné and Tojo, 2019; Takenaka and Tojo, 2019; Takenaka et al., 2019; Yano et al., 2019). The freshwater giant water bug, Appasus japonicus, which we focus on in this study, is likewise known to have an interesting evolutionary history (Suzuki et al., 2013, 2014).

Appasus japonicus is an aquatic insect that is distributed throughout the Japanese Archipelago, the Korean Peninsula and mainland China. This species is often listed as endangered in the Red List or the Red Data Book at the national and local level in Japan (Ministry of the Environment, 2006). Its evolutionary history has been revealed by our previous research using the mtDNA COI and 16S rRNA regions. In previous studies we identified three largely divided genetic lineages within this species, and their evolution is strongly associated with geohistory (Suzuki et al., 2013, 2014). Furthermore, “back dispersal” of A. japonicus, i.e., dispersal from the Japanese Archipelago to the Eurasian continent, was suggested from that work (Suzuki et al., 2014). Such a back dispersal phenomenon is an extremely rare and interesting case, since almost all biogeographic studies of Japanese organisms have been focused on when and/or how they reached the Japanese Archipelago. Detailed studies of such dispersal patterns are very important in understanding the biodiversity creation process of the Japanese Archipelago, and further fine-scale biogeographical analyses are needed to elucidate it. However, fine-scale analyses using population genetic approaches have not yet been conducted for this species. Microsatellite markers are one of the most useful tools for detecting fine-scale population structure (e.g., Phillipsen and Lytle, 2013; Phillipsen et al., 2015; Hirao et al., 2017; Komaki et al., 2017; Yamazaki et al., 2020). Furthermore, information on population genetic structure obtained from microsatellite markers is very important for conservation. Such information is expected even to detect gene flow between populations with high sensitivity. Therefore, in this study, we developed 20 microsatellite markers for A. japonicus and evaluated their suitability for population genetic analyses.

Whole-genome shotgun sequencing was performed using the Ion PGM system (Life Technologies). Library preparation and PGM sequencing were conducted at the Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Japan. Total genomic DNA was extracted from ethanol-preserved tissue of a specimen collected at Matsumoto, Nagano, and purified using a DNeasy Blood & Tissue Kit (QIAGEN) in accordance with the manufacturer’s instructions. The concentration of genomic DNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies), and 13.6 ng/μl of DNA was used for the following processes. The genomic DNA was sheared to approximately 350–450 bp using Ion Shear Plus Reagents (Life Technologies), and adapter ligation, nick-repair and purification of the ligated DNA were conducted using an Ion Plus Fragment Library Kit (Life Technologies). After size selection (target insert sizes 300–400 bp) was performed using an E-Gel Agarose Gel Electrophoresis System (Life Technologies), library amplification was conducted using an Ion Plus Fragment Library Kit (Life Technologies). The library was assessed and quantified using a Bioanalyzer (Agilent Technologies), and then diluted to 8 pM for template preparation using an Ion PGM Template OT2 400 kit (Life Technologies) and enriched. Sequencing was performed using an Ion PGM Sequencing 400 kit (Life Technologies) with 850 flows on the Ion 314 Chip V2 (Life Technologies) in accordance with the manufacturer’s protocol. After sequencing, signal processing and base-calling were performed using Torrent Suite 3.6 (Life Technologies), and a library-specific FASTQ file was generated. The data sets were collated and applied to the QDD bioinformatics pipeline (Meglécz et al., 2010) to filter sequences containing microsatellites with appropriate flanking sequences to define PCR primers. QDD detected 10,760 loci, each containing a microsatellite consisting of at least five repeats. We selected primer pairs according to the following criteria: amplification products within the size range of 90–200 bp; optimal melting temperature range 58–62 ℃. A total of 50 primer pairs were obtained for screening. Twenty primer pairs showing clear peak patterns were selected after an initial primer screening using eight samples from the Matsumoto, Nagano, population, and eight samples from the Shimonoseki, Hiroshima, population (Table 1). We selected primer pairs that not only showed clear peak patterns in both populations but also were missing in one of the two populations, because we want to use these primer pairs for wide-scale to fine-scale population genetic structure analyses.

Table 1. Characteristics of 20 microsatellite primers developed for Appasus japonicus

Locus	Primer sequences (5′-3′)	Ta (℃)	BStag	Repeat motif	Size range (bp)	DDBJ accession no.
AJP01	F: CCCTGTAACAGTTGAGGATTTACA	58	F9GAC-FAM	(TA)7	119–127	LC548110
	R: AAACCTAATGTGTTCCGATATTCA
AJP02	F: CTGACACCAATCGGAGGAGT	58	F9TAC-NED	(AT)6 (AC)4	95–105	LC548111
	R: GATCTCATGCCCGTTGAGAG
AJP04	F: TGAAACTCACGAGATTGTTATTCA	58	F9GTC-HEX	(CT)7	105–133	LC548112
	R: GGAGTCGATGAGTGAGCCAG
AJP07	F: GTTCGTAACCGATCATGCG	58	F9GAC-FAM	(GT)16	129–154	LC548113
	R: ACCCAAGTCATACTCGGAGG
AJP08	F: GACGTGGAATGAATTGTGTAAGT	58	F9GCC-PET	(AT)7	151–155	LC548114
	R: TTTACAAGCTCAATAACAAGCTGA
AJP09	F: ACAGGGACTGCTTTGATCGT	58	F9GTC-HEX	(CT)5	120–124	LC548115
	R: CCCTCTCCTGTGGAAGAGAA
AJP10	F: CGAAGGGACAGACAGAAATGA	58	F9GTC-HEX	(AT)9	95–113	LC548116
	R: CGCATAATAAGCTTCCAGGC
AJP11	F: AAATGGGCTGTAGTGCCA	58	F9TAC-NED	(ATA)7	129–147	LC548117
	R: TTGCAACGAGTTGTTGATCG
AJP12	F: TCACGCGGATATAAATTGCC	58	F9TAC-NED	(AT)7	118–122	LC548118
	R: CGGAAATTAATGTGAGTCCAGG
AJP20	F: TTCCAGTCTGTGGGTTCCAT	58	F9GTC-HEX	(AT)7	180–190	LC548119
	R: CAGAGGTCAAACCTCAAACACA
AJP21	F: CGGAACTCCATCCCAGTAGT	58	F9GCC-PET	(TA)7	119–128	LC548120
	R: CTGTCGCCCACATTTAGGTT
AJP24	F: TCAGGTACGCAGAGGTCTCTAA	58	F9GCC-PET	(AG)11	136–150	LC548121
	R: TGAGAGCCCGATTAATTCCC
AJP28	F: TTTGGAGTTTGTTCAAGTCATGT	58	F9GAC-FAM	(TTA)7	182–191	LC548122
	R: TGCAGGCGTCATTCTCTAAA
AJP31	F: TGTTTCGGATTAAACCACTCG	58	F9GAC-FAM	(AAC)7	154–160	LC548123
	R: CCACGCCCAGTAATAATCAA
AJP34	F: AACGAAATTGGCACGTGTTAC	58	F9GTC-HEX	(CT)7	154–156	LC548124
	R: CAAAGCAATATGTTTGTCTGTTATGC
AJP36	F: ACGGGTATCGACATGCTGAC	58	F9TAC-NED	(AT)8	149–155	LC548125
	R: AATTAGAGCCCAACAATGCG
AJP38	F: TCGTTAATACACGGGACAGAAA	58	F9GAC-FAM	(AG)7	111–119	LC548126
	R: GACCCACTGCTCTTCTTCCA
AJP39	F: ATCTGAGTTCACCCACGTCA	58	F9GCC-PET	(GT)9	120–126	LC548127
	R: GCAGGGCACGAAGTTAGGTA
AJP43	F: GCGCAGAACGCATAATTTGT	58	F9TAC-NED	(TG)9	191–195	LC548128
	R: AAACCGGTCTTTCTCACGAC
AJP47	F: TGAAACGACCACTCGGGTA	58	F9GCC-PET	(GA)7	112–116	LC548129
	R: CAAAGTTGAACTGTTCCGCA

T_a = annealing temperature.

To test the genetic variation of the 20 selected microsatellite loci, 20 samples from Matsumoto, Nagano, 10 samples from Mihara, Hiroshima, 10 samples from Shimonoseki, Yamaguchi, and 16 samples from Daechi, Chungcheongnam-do, were used. PCR amplification with fluorescent dye-labeled primers was performed using a protocol described by Shimizu and Yano (2011). PCR amplification was done in 10 μl reactions with KOD FX Neo DNA polymerase (TOYOBO). Each reaction contained the following components: 1 μl of total genomic DNA, 4.8 μl of 2 × buffer, 1.6 μl of 2.0 mM dNTP mix, 0.05 μl of forward primer, 0.2 μl of reverse primer, 0.05 μl of fluorescent dye-labeled primer and 2.3 μl of SQ. The PCR protocol was: 94 ℃ for 2 min; 30 cycles of 98 ℃ for 10 s, 58 ℃ for 10 s and 68 ℃ for 30 s; and 68 ℃ for 5 min. We labeled BStag primers with the following fluorescent dyes: F9GAC-FAM (5′-CTAGTATCAGGACGAC-3′), F9GTC-HEX (5′-CTAGTATGAGGACGTC-3′), F9TAC-NED (5′-CTAGTATCAGGACTAC-3′), F9GCC-PET (5′-CTAGTATTAGGACGCC-3′) and F9CCG-FAM (5′-CTAGTATTAGGACCCG-3′). Product sizes were determined using an ABI 3130xl Genetic Analyzer and GeneMapper software (Applied Biosystems) with GeneScan 500 LIZ dye Size Standard v2.0 (Applied Biosystems). We calculated observed heterozygosity (H_O), expected heterozygosity (H_E), inbreeding coefficients (F_IS) and pairwise population genetic subdivision (F_ST) values between populations using GenAlEx 6.5 (Peakall and Smouse, 2012). We tested whether the F_IS values of each of the four populations were significantly different from zero by χ²-test. We also tested deviation from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium among the polymorphic loci using GENEPOP 4.7 (Rousset, 2008), and checked for the presence of null alleles using MICRO-CHECKER 2.2.3 (Van Oosterhout et al., 2004). We used STRUCTURE v2.3.4 (Pritchard et al., 2000) to determine the number of distinct genetic clusters. STRUCTURE simultaneously identifies potential populations (clusters) and probabilistically assigns individuals to each of the K populations based on the sample genotypes. We did not use loci that significantly deviated from HWE and detected the null alleles; these loci did not show clear peak patterns and were treated as missing data. Finally, 15 loci were used for STRUCTURE analysis (Table 2). STRUCTURE runs were performed using the admixture model and correlated allele frequencies, with 100,000 iterations of the Markov Chain Monte Carlo (MCMC) used as “burn-in” that were followed by 1,000,000 MCMC iterations; the probability to observe the data [Ln P(D)] was calculated for values of K ranging from 1 to 10, with 10 iterations for each K-value. The best estimate of K was taken to be the maximum value observed before the plateau of the curve Ln P(D) against K (Pritchard et al., 2000). STRUCTURE HARVESTER v0.6.94 (Earl and vonHoldt, 2012) was also used to identify the most pronounced level of population structure using the method of Evanno et al. (2005). CLUMPP v1.1 (Jakobsson and Rosenberg, 2007) was used to find the optimal alignment from replicate STRUCTURE runs, with the summary of results generated using DISTRUCT v1.1 (Rosenberg, 2004).

Table 2. Genetic variation of the 20 microsatellite loci for four populations of Appasus japonicus

Locus	Matsumoto, Nagano (n = 20)				Mihara, Hiroshima (n = 10)				Shimonoseki, Yamaguchi (n = 10)				Daechi, Chungcheongnam-do (n = 16)
	A	HO	HE	FIS	A	HO	HE	FIS	A	HO	HE	FIS	A	HO	HE	FIS
AJP01	2	0.600	0.500	−0.200	3	0.333	0.568	0.413	3	0.700	0.595	−0.176	7	0.750	0.805	0.068
AJP02	4	0.400	0.336	−0.190	−	−	−	−	−	−	−	−	5	0.438	0.564	0.225
AJP04	2	0.150	0.139	−0.081	2	0.100	0.095	−0.053	3	0.400	0.515	0.223	−	−	−	−
AJP07	3	0.600	0.609	0.014	2	0.100	0.095	−0.053	3	0.500	0.545	0.083	12	0.750	0.900	0.167
AJP08	3	0.100	0.096	−0.039	1	0.000	0.000	NA	1	0.000	0.000	NA	3	0.438	0.461	0.051
AJP09	3	0.600	0.586	−0.023	1	0.000	0.000	NA	1	0.000	0.000	NA	2	0.250	0.219	−0.143
AJP10	5	0.450	0.451	0.003	3	0.600	0.445	−0.348	3	0.300	0.515	0.417	4	0.200	0.296	0.323
AJP11	−	−	−	−	3	0.500	0.555	0.099	4	0.500	0.645	0.225	−	−	−	−
AJP12	3	0.450	0.436	−0.032	1	0.000	0.000	NA	1	0.000	0.000	NA	4	0.000	0.711	1.000
AJP20	3	0.250	0.509	0.509	−	−	−	−	−	−	−	−	5	0.438	0.418	−0.047
AJP21	3	0.600	0.514	−0.168	−	−	−	−	−	−	−	−	−	−	−	−
AJP24	4	0.300	0.341	0.121	5	0.800	0.685	−0.168	4	0.700	0.700	0.000	−	−	−	−
AJP28	2	0.050	0.049	−0.026	1	0.000	0.000	NA	2	0.200	0.420	0.524	−	−	−	−
AJP31	3	0.250	0.386	0.353	2	0.400	0.320	−0.250	2	0.500	0.495	−0.010	2	0.313	0.404	0.227
AJP34	−	−	−	−	2	0.200	0.480	0.583	2	0.100	0.255	0.608	−	−	−	−
AJP36	3	0.600	0.531	−0.129	2	0.700	0.495	−0.414	3	0.700	0.505	−0.386	4	0.563	0.451	−0.247
AJP38	3	0.100	0.096	−0.039	−	−	−	−	−	−	−	−	4	0.375	0.525	0.286
AJP39	−	−	−	−	2	0.111	0.105	−0.059	4	0.500	0.415	−0.205	−	−	−	−
AJP43	2	0.000	0.260	1.000	2	0.100	0.455	0.780	3	0.222	0.370	0.400	−	−	−	−
AJP47	3	0.700	0.601	−0.164	−	−	−	−	−	−	−	−	−	−	−	−

n, number of individuals analyzed; A, number of alleles; H_O, observed heterozygosity (in bold numbers, if values deviate significantly from HWE); H_E, expected heterozygosity; F_IS, fixation index.

As a result, all 20 microsatellite markers identified in this study had meaningful polymorphism. Seventeen loci were stably amplified and genotyped in the Matsumoto, Nagano population, 15 loci were stably amplified and genotyped in the Mihara, Hiroshima and Shimonoseki, Yamaguchi populations, and 11 loci were stably amplified and genotyped in the Daechi, Chungcheongnam-do population (Table 2). The number of genotyped loci of A. japonicus collected from Daechi (Chungcheongnam-do, Korean Peninsula) was lower than that from the Japanese populations. Our previous study revealed that there has been no gene flow between the Japanese and continental populations since the formation of the Tsushima Strait (ca. 1.55 Ma; Suzuki et al., 2014). Therefore, it is considered that the individuals of the continental populations may have some nucleotide mutations within their PCR primer region(s).

The number of alleles per locus across the four populations was between 1 and 12 (mean = 2.5). Four and three loci were not polymorphic in the Hiroshima and Yamaguchi populations, respectively (Table 2). The ranges of H_O, H_E and F_IS per locus were 0.000–0.800 (mean = 0.270), 0.000–0.900 (mean = 0.323) and −0.414–1.000 (mean = 0.153), respectively (Table 2). No significant differences from zero for the F_IS values were observed in any of the four populations (P > 0.05). Some loci significantly deviated from HWE in each population (Table 2; P < 0.05), and loci that were positive for linkage disequilibrium were not detected. The result of the MICRO-CHECKER 2.2.3 analysis suggested null alleles in AJP20, AJP31 and AJP43 in the Matsumoto, Nagano population, and AJP12 in the Daechi, Chungcheongnam-do population (estimated null allele frequency: 0.16, 0.12, 0.22 and 0.42, respectively). An examination of the likelihood probabilities from the 10 replicate runs across K-values ranging from 1 to 10 indicated that the optimal K-value was 4 (i.e., K = 4), suggesting that four genetic groups exist within our dataset (Fig. 1, Supplementary Fig. S1). In the result where K = 4, the four populations that were used for STRUCTURE analysis were clearly divided into distinct genetic clusters (Fig. 1). The result of pairwise comparisons of F_ST values also suggested clear genetic differentiation between each population (Table 3). Therefore, we consider that these microsatellite markers, which were newly developed in this study, are useful for discussing finer-scale phylogeography of A. japonicus than can be achieved by mtDNA markers. We will therefore conduct population genetic analyses based on nDNA (SSRs) using more samples and populations, in order to confirm the validity of this in a future study.

Fig. 1.

Clustering analysis of the multilocus microsatellite data of Appasus japonicus performed using STRUCTURE (K = 2–4), and sampling localities of A. japonicus used in this study. The photo on the right shows an egg-carrying male of A. japonicus.

Table 3. Pairwise F_ST values between four populations of Appasus japonicus

	1	2	3	4
1. Matsumoto, Nagano	0.000
2. Mihara, Hiroshima	0.416	0.000
3. Shimonoseki, Yamaguchi	0.446	0.472	0.000
4. Daechi, Chungcheongnam-do	0.543	0.627	0.585	0.000

In addition, the result in the case of K = 2 revealed that the Shimonoseki (Yamaguchi Prefecture) and Korean populations of A. japonicus were within the same cluster (Fig. 1). It was also shown in our previous study based on their mtDNA sequences that these two populations of A. japonicus (i.e., populations of western Honshu and the Korean Peninsula) constitute a monophyletic group (Suzuki et al., 2014). Therefore, the result of the population structure analysis where K = 2 based on the SSR (nDNA) markers appears to support strongly the results of our previous phylogenetic analyses based on mtDNA.

Furthermore, in the result where K = 3, the Mihara (Hiroshima Prefecture) and Shimonoseki (Yamaguchi Prefecture) populations were identified as being within the same cluster (Fig. 1). Similarly, in our previous study based on the mtDNA sequences, it was also suggested that this geographic area corresponded to a secondary contact zone of the two Japanese clades (Suzuki et al., 2014). We consider that it would be very interesting to conduct finer-scale population genetic analyses using SSR markers in this area, and we will work to do so in a future study.

In conclusion, we sequenced A. japonicus genomic DNA using Ion PGM and found microsatellite regions. Based on these data, we developed 20 polymorphic microsatellite markers for this species. These polymorphic markers are the first developed for A. japonicus. Appasus japonicus has high potential as a model organism for the study of arthropod evolution (e.g., speciation and evolution of a paternal care system). These microsatellite markers are useful for elucidating broad- and fine-scale population genetic structure and evolution of the unique paternal care mating system in A. japonicus, as well as for conservation genetics research (c.f. Tomita et al., 2020).

ACKNOWLEDGMENTS

We are sincerely thankful to Professor Emeritus K. Suzuki of Shinshu University for arranging the study environment. We also express our thanks to Dr. M. Konishi (Shinshu University) and Dr. S. Komaki (Iwate Medical University) for their advice and assistance with experiments, Dr. S. Ohba (Nagasaki University) for his many valuable comments on this study, Mr. M. Shimono (Iwakuni City) and Ms. K. Momose (Shinshu University) for their cooperation with the field research and collection of specimens, and Dr. H. Matsumura (Shinshu University) for providing facilities for genetic analysis. This study was supported by JSPS KAKENHI Grant Numbers JP20687005, JP23657046, JP16K14807 (K. T.), 285211031 (K. T.), and JP26891010, JP19K16209 (T. S.), the River Fund of The River Foundation, Japan [27-1215-013 (K. T.), and 27-1263-012 (T. S.)], and the Sasakawa Scientific Research Grant (26-529; T. S.).

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