Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 1 April 2021 J-STAGE Advance Published Date: 12 April 2021

©2021 The Japanese Society of Veterinary Science Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Parasitology

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3 Note

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5 Title

6 Raccoon dogs (Nyctereutes procyonoides) are the new natural definitive hosts of Metagonimus

7 hakubaensis

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9 Authors

10 Jun HAKOZAKI1, Sho KUZUKAMI1, Asako HARAGUCHI1, Kazuhiko NAKAYAMA1,

11 Kodai KUSAKISAKO1, Noboru KUDO1, and Hiromi IKADAI1*

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13 Institution

14 1)Laboratory of Veterinary Parasitology, School of Veterinary Medicine, Kitasato University,

15 Towada, Aomori 034–8628, Japan

16 *Correspondence to: Ikadai, H., Laboratory of Veterinary Parasitology, School of Veterinary

17 Medicine, Kitasato University, Towada, Aomori 034–8628, Japan.

18 e-mail: [email protected]

19

20 Running head

21 NEW DEFINITIVE HOST OF M. HAKUBAENSIS

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23 ABSTRACT

24 The definitive hosts of Metagonimus hakubaensis are reported to be hamsters, rats,

25 mice, dogs, cats, chickens, and quails in experimental infection and Japanese water shrews in

26 natural infection. Here we report that raccoon dogs are new natural definitive hosts of M.

27 hakubaensis, based on morphological and molecular analyses of Metagonimus flukes

28 collected from the host species from Aomori Prefecture, Japan. Moreover, M. hakubaensis

29 recovered from raccoon dogs showed higher fecundity than those recovered from Japanese

30 water shrews. Therefore, raccoon dogs were considered as a more suitable natural definitive

31 host of M. hakubaensis than Japanese water shrews.

32

33 KEY WORDS

34 Japanese water shrew, Metagonimus hakubaensis, natural definitive host, raccoon dog,

35 suitable definitive host

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45 Metagonimus hakubaensis was initially described in laboratory rats orally inoculated

46 with metacercariae from sand lampreys (Lethenteron reissneri) that were collected in Nagano

47 Prefecture, Japan [10]. Previously, only Japanese water shrews (Chimarrogale platycephala)

48 have been reported as the natural definitive hosts of M. hakubaensis [4]. In contrast, hamsters,

49 rats, mice, dogs, cats, chickens, and quails have been reported as the experimental definitive

50 hosts of M. hakubaensis [3].

51 Raccoon dogs (Nyctereutes procyonoides) belong to the family Canidae and have

52 been reported to be natural definitive hosts of Metagonimus yokogawai, Metagonimus

53 takahashii, and Metagonimus miyatai, which are closely related to M. hakubaensis [8, 9, 14].

54 This study was aimed at clarifying whether raccoon dogs are natural definitive hosts of M.

55 hakubaensis based on morphological and molecular identification of Metagonimus flukes

56 collected from the host animals. The host suitability of M. hakubaensis has been evaluated

57 based on the number of intrauterine eggs in the flukes. The flukes from dogs and hamsters had

58 more intrauterine eggs compared with those from other animal species; hence, dogs and

59 hamsters were considered more suitable definitive hosts of M. hakubaensis [3]. This study

60 compared the number of intrauterine eggs of M. hakubaensis in raccoon dogs and Japanese

61 water shrews, which are the only natural definitive hosts of M. hakubaensis, and evaluated the

62 suitability of these hosts for the flukes.

63 Eight raccoon dogs, designated N1–N8 and seven Japanese water shrews, C1–C7,

64 were collected in Aomori Prefecture, Japan, during 1989–2000, and 1994–1996, respectively.

65 In total, 2,648 Metagonimus flukes were recovered from the intestines of 8 raccoon dogs,

66 ranging from 1 to 1,686 per animal (Table 1). In contrast, 5,416 Metagonimus flukes were

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67 recovered from the intestines of 7 Japanese water shrews (Table 1).

68 Of the Metagonimus flukes, 50 from raccoon dogs and 271 from Japanese water

69 shrews were pressed between a glass slide and a coverslip, fixed in 70% ethanol, stained with

70 alum carmine, and observed for morphological characteristics, such as the oral and ventral

71 sucker size ratio, position of the testes and the uterus containing the eggs, and distribution of

72 the vitelline follicles, which are the key morphological characters distinguishing Metagonimus

73 spp. in Japan [2, 6, 7, 10–12]. The remaining flukes were preserved in 70% alcohol without

74 being pressed and stained.

75 Of the 50 stained flukes from raccoon dogs, 16 had oral suckers slightly smaller than

76 ventral suckers, and their uteri, containing the eggs, were not extended to the posterior

77 extremity between both testes; therefore, they were identified as M. hakubaensis (Fig.1 and

78 Table 1). The remaining 34 flukes had ventral suckers significantly larger than oral suckers,

79 and had uteri extended to the posterior extremity between both testes; therefore, they were

80 identified as M. miyatai (Table 1). In contrast, of the 271 stained flukes from Japanese water

81 shrews, 219 were identified as M. hakubaensis, 1 as M. miyatai, and 51 as Metagonimus sp.

82 (Table 1).

83 Of the unstained specimens, 2,340 flukes from raccoon dogs and 1,618 from

84 Japanese water shrews were identified morphologically because M. hakubaensis, as only M.

85 hakubaensis has a uterus that is not extended to the posterior extremity between both testes

86 among Metagonimus spp. [10].

87 The mitochondrial cytochrome c oxidase subunit 1 (mtCOX1) DNA sequence of M.

88 hakubaensis was analyzed using 17 and 12 unstained flukes from raccoon dogs and Japanese

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89 water shrews, respectively. Genomic DNA from each fluke was extracted using Quick-

90 gDNATM Miniprep Kit (Zymo Research, Irvine, CA, U.S.A.) according to the manufacturer’s

91 protocol. A 399-bp fragment of the mtCOX1 gene was amplified by polymerase chain reaction

92 (PCR) using primers JB3 and JB4.5 [1]. The PCR amplification consisted of one cycle of

93 denaturation at 98°C for 3 min, followed by 40 cycles of denaturation at 98°C for 30 sec,

94 annealing at 60°C for 1 min, and extension at 72°C for 1 min. Finally, both strands of the PCR

95 products were sequenced directly by Fasmac sequencing service (FASMAC, Atsugi, Japan)

96 using either JB3 or JB4.5. Phylogenetic trees were constructed from mtCOX1 sequences using

97 maximum likelihood as implemented in MEGA X (https://www.megasoftware.net). The

98 Tamura 3-parameter model was selected for the best-fit model of maximum likelihood [13].

99 A bootstrap method, performed in 1,000 replicates, was used to assess the stability of the

100 phylogenetic trees.

101 Six and four mtCOX1sequence variants were detected in M. hakubaensis flukes from

102 raccoon dogs and Japanese water shrews, respectively; these were registered in GenBank as

103 accession numbers LC576452–LC576461. These variants were classified into the same clade

104 as the reference sequences of M. hakubaensis (KM061415, KM061416, and KM061417) in

105 the phylogenetic tree [5] (Fig. 2). These molecular findings strongly supported that the flukes

106 from raccoon dogs and Japanese water shrews were M. hakubaensis.

107 The fecundity of M. hakubaensis recovered from raccoon dogs and Japanese water

108 shrews were compared to determine which host species was more suitable as the natural

109 definitive host based on the number of intrauterine eggs. The number of intrauterine eggs in

110 a single M. hakubaensis specimen was counted and categorized according to Kudo et al. [3].

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111 The number of intrauterine eggs was counted for 16 and 219 stained M. hakubaensis flukes

112 from raccoon dogs and Japanese water shrews, respectively (Table 2). Metagonimus

113 hakubaensis specimens from raccoon dogs had more intrauterine eggs compared with those

114 from Japanese water shrews. Kudo et al. [3] reported that M. hakubaensis adults possess many

115 eggs in highly suitable host animals. Accordingly, raccoon dogs would be more suitable

116 definitive hosts of M. hakubaensis than Japanese water shrews.

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118 POTENTIAL CONFLICTS OF INTEREST

119 The authors have nothing to disclose.

120

121 ACKNOWLEDGMENTS

122 We would like to thank the two anonymous reviewers for their suggestions and

123 comments, which considerably helped us to improve our manuscript. We express our thanks

124 to Dr. Mizuki Sasaki, Department of Parasitology, Asahikawa Medical University, for her

125 valuable comments on this manuscript.

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179 13. Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are

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189 Thailand. Exp. Parasitol. 146: 64–70.

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199 FIGURE LEGENDS

200 Fig 1. Metagonimus hakubaensis specimen recovered from a raccoon dog.

201 Bar = 100 µm.

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203 Fig 2. The phylogenetic tree of Metagonimus spp. based on the dataset of mtCOX1 gene

204 sequences. The published sequences of mitochondrial cytochrome c oxidase subunit 1

205 (mtCOX1) in M. hakubaensis (GenBank accession numbers KM061415, KM061416, and

206 KM061417), M. yokogawai (KM061412, KM061413, and KM061414), M. takahashii

207 (KM061406, KM061407, and KM061408), M. miyatai (KM061409, KM061410, and

208 KM061411), M. otsurui (KM061421, KM061422, and KM061423), and M. katsuradai

209 (KM061418, KM061419, and KM061420) were used [5]. A partial sequence of Fasciola

210 gigantica mtCOX1 gene (KF687896) was used as a potential outgroup [15]. Branch lengths

211 are drawn to scale, with the scale bar indicating the number of nucleotide substitutions.

212 *M. hakubaensis (LC576452–LC576461) recovered from raccoon dogs (N1 and N2) and

213 Japanese water shrews (C1–C3).

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Fig. 1 Hakozaki et al. Fig. 2 Hakozaki et al. 76 M. otsurui KM061423 99 M. otsurui KM061421 83 M. otsurui KM061422 M. katsuradai KM061420 56 M. katsuradai KM061419 99 M. katsuradai KM061418 M. miyatai KM061410 M. miyatai KM061411 32 99 M. miyatai KM061409 76 M. yokogawai KM061413 99 M. yokogawai KM061412 M. yokogawai KM061414 50 M. takahashii KM061406 M. takahashii KM061408 99 M. takahashii KM061407 M. hakubaensis LC576461 ・・・・・・*C3 M. hakubaensis LC576455 ・・・・・・*N1 M. hakubaensis LC576457 ・・・・・・*N2 99 M. hakubaensis LC576458 ・・・・・・*C3 M. hakubaensis LC576459 ・・・・・・*C2 50 M. hakubaensis LC576456 ・・・・・・*N1 55 M. hakubaensis LC576453 ・・・・・・*N1 M. hakubaensis LC576452 ・・・・・・*N1 M. hakubaensis LC576454 ・・・・・・*N1,N2 M. hakubaensis KM061416

28 M. hakubaensis LC576460 ・・・・・・*C1 16 M. hakubaensis KM061417 65 M. hakubaensis KM061415 Fasciola gigantica KF687896

0.050 Table 1. The number of Metagonimus flukes recovered from racoon dogs and Japanese water shrews

Number of stained specimens Number of unstained specimens Host Host animal code M. hakubaensis M. miyatai Metagonimus sp. M. hakubaensis Metagonimus sp.

N1 3 0 0 1683 0 N2 11 7 0 595 35 Raccoon dog N3 1 13 0 0 45 N4 1 4 0 0 0 (N. procyonoides ) N5 0 9 0 0 0 N6 0 0 0 0 178 N7 0 1 0 0 0 N8 0 0 0 62 0 Total 16 34 0 Total 2340 258 C1 48 1 12 127 590 C2 6 0 14 228 658 C3 0 0 0 443 134 Japanese water shrew C4 0 0 0 307 0 (C. platycephala ) C5 35 0 19 171 422 C6 49 0 5 244 1634 C7 81 0 1 98 89 Total 219 1 51 Total 1618 3527 Table 2. The different intrauterine egg count in Metagonimus hakubaensis recovered from raccoon dogs (Nyctereutes procyonoides) and Japanese water shrews (Chimarrogale platycephala )

Host The number of flukes with Host animal code 1–100 eggs/fluke 101–200 eggs/fluke 201–300 eggs/flukes N1 0 2 0 Raccoon dog N2 0 3 8 (N. procyonoides ) N3 0 2 0 N4 0 0 1 Total 0 7 9 C1 46 2 0 C2 6 0 0 Japanese water shrew C5 32 3 0 (C. platycephala ) C6 43 6 0 C7 69 12 0 Total 196 23 0