Chapter 1. General Introduction

Marine fishes are frequently infected with nematodes. However, information on them, such as taxonomy and biology, are generally limited. In this study, I focused on two nematode families, namely Philometridae and Anisakidae considering their importance. Philometrid nematodes are known for their economic impact on fish production, especially those species infecting the gonads and body muscles of their respective host fishes. Proper species identification is important, not only in the re-evaluation of the taxonomical classification of these poorly studied group, but also in understanding the biology and pathology on their host fishes. Taxonomical and biological studies were carried out on six different gonad-infecting Philometra species and one muscle-infecting Philometroides species, thus adding some basic but very relevant information on this group of nematode.

On the other hand, anisakid nematodes are known for their zoonotic contribution to human anisakiasis and as source of allergens. With this, studying this group of nematode is very important from the viewpoint of food safety. Previous identification of Anisakis larvae are based on two types, Anisakis Type I and Anisakis Type II, wherein A. simplex, A. ziphidarum, A. pegreffii and A. typica were on the former type, whereas A. paggiae, A. brevispiculata and A. physeteris were on the latter type. At present, little is known about the taxonomy and infection, especially those of the two widely reported sibling species of Anisakis simplex complex, namely A. simplex (sensu stricto)(s.s.) (Rudolphi, 1809) and A. pegreffii Campana-Rouget et Biocca, 1955. Taxonomical studies and experimental infection studies were carried out on Anisakis species in Japanese waters, with emphasis on these two sibling species.

Chapter 2. Studies on philometrid nematodes

1. Taxonomical studies

In philometrid nematodes, males are generally variant in morphology and crucial to their taxonomy. Due to the difficulty in finding tiny males, description of many philometrids were based mainly on large females. In this study, males of six philometrid species were successfully obtained and used for their taxonomy. Six gonad-infecting Philometra species and one muscle-infecting Philometroides species in Japanese waters were taxonomically examined by morphological and molecular analyses. Males of Philometra lateolabracis (Yamaguti, 1935), females of which have been reported from various fish species, were discovered for the first time in the gonads of its type host, Japanese seaperch, Lateolabrax japonicus. Morphological comparisons were carried out by light microscopy and scanning electron microscopy between P. lateolabracis collected from Japanese seaperch and other philometrid nematodes previously reported as P. lateolabracis from chicken grunt, Parapristipoma trilineatum, and red sea bream, Pagrus major. Results revealed that the latter two represent new species, Philometra isaki Quiazon, Yoshinaga et Ogawa, 2008 and Philometra madai Quiazon, Yoshinaga et Ogawa, 2008, respectively. Also, new Philometra species, P. sawara Quiazon, Yoshinaga et Ogawa, 2008, was described based on male and female specimens collected from the gonads of Japanese Spanish mackerel, Scomberomorus niphonius. Three additional species, Philometra nemipteri Luo, 2001, Philometra sciaenae Yamaguti, 1941 and Philometroides seriolae (Ishii, 1931) were confirmed as valid species and were redescribed based on specimens collected from the gonads of golden threadfin bream, Nemipterus virgatus and silver croaker, Pennahia argentata, and from the body muscle of Japanese amberjack, Seriola quinqueradiata, respectively. Male P. nemipteri were first reported and described in this study, whereas male Philometroides seriolae were unsuccessfully collected. Molecular results of the ITS2 rDNA sequences supported the morphological findings that all seven philometrid species examined are independent from each other and from other species, the sequence of which were already reported. These findings indicate that philometrids have strong host specificity and the identity of a species reported from several host species should be re-evaluated.

2. Biological studies

In this study, the infection of six gonad-infecting Philometra species (P. sciaenae, P. sawara, P. isaki, P. madai, P. lateolabracis and P. nemipteri) and one muscle-infecting Philometroides species were examined. Results of examination revealed that both live and dead Philometra species were mainly found in the ovarian lumen and some were also found in the oviduct. In male host fishes, only P. nemipteri were found mainly in the spermatic duct, whereas majority of the other five Philometra species, live and dead, were found in the seminiferous tubules. The developmental stages and prevalence of gonad-infecting Philometra species were found synchronized with the spawning season of their host fishes. Hence, it is clear that presence of fully gravid female Philometra in the gonads of their hosts possibly facilitate the release of first-stage larvae, as host gametes were released during host spawning. On the other hand, Philometroides seriolae were mainly found in the body muscle and only very few of them were situated under the skin of the fish. For those found under the skin, only few (8.7% of fully gravid females) were found protruding their anterior end outside the fish skin, possibly to release first-stage larvae into the environment as suggested in a previous report. As all developmental stages, particularly fully gravid females, were observed throughout the year, no seasonal fluctuation in the prevalence of Philometroides seriolae was observed. Up to date, its mode of releasing hatched larvae remains largely unknown. However, the fully gravid females under the skin possibly contribute to the continuous existence of this parasite in the marine environment. In the infection with Philometroides seriolae, a layer of inflammatory tissue was formed in the muscle surrounding worms, where numerous leukocytes infiltrated. In gonad-infecting Philometra species, in general, leukocyte infiltrations were mostly observed in live worms and fibrous tissue layers were frequently observed around remnant dead worms.

Chapter 3. Studies on anisakid nematodes

1. Taxonomical studies

Studies on anisakid nematodes were carried out in aspects mainly focusing on A. simplex (s.s.) and A. pegreffii:(1) morphological differences between A. simplex (s.s.) and A. pegrefii,(2) morphological and molecular approaches for multi-species identification of Anisakis species in Theragra chalcogramma, (3) comparisons of A. pegreffii from the Far East and Mediterranean, and (4) distribution of different Anisakis species in Japanese waters.

Morphological differences in larvae and adults between A. simplex (s.s.) and A. pegreffii Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. The foremost problem in the identification of Anisakis larvae in fishes is that L3 larvae cannot be easily differentiated morphologically, especially between A. simplex (s.s.) and A. pegreffii. Instead, molecular means such as allozyme, mitochondrial DNA (mtDNA) cox2 region and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses had been successfully used. However, morphological distinction is important in facilitating studies especially on these two species. In this study, morphological differences of L3 larvae collected from fishes and in vitro-cultured L4 larvae and adults between A. simplex (s.s.) and A. pegreffii were evaluated. L3 larvae were collected from seven different host fishes within Japan. L4 larvae and adults were obtained by culturing L3 larvae in vitro. After morphological examination, each parasite was subjected to PCR-RFLP analysis of the ITS region (ITS1-5.8S-ITS2) of rDNA for species identification. The identification was confirmed by mtDNA cox2 gene sequencing of representative specimens. Results revealed that L3, L4 and adult stages of A. simplex (s.s.) and A. pegreffii are morphologically distinguishable based on ventriculus length, wherein the former has longer ventriculus (0.90-1.50 mm) than the latter (0.50-0.78 mm). Also, adult male A. simplex (s.s.) and A. pegreffii were found to be distinguishable by differences in the distribution pattern of the caudal papillae, particularly the 3rd pair of distal papillae.

Multi-species identification of Anisakis in Theragra chalcogramma Morphological and molecular approaches were conducted in order to precisely identify different Anisakis species infecting Alaska pollock, T. chalcogramma, in northern Japan. Morphologically, both Anisakis Type I and Anisakis Type II larvae were found. Anisakis identification using PCR-RFLP analysis generated four different fragment patterns. Analysis of the nucleotide and amino acid sequences of the ITS and mtDNA cox2 regions, respectively, indicated that the four fragment patterns corresponded to A. simplex (s.s.), A. pegreffii, A. brevispiculata and A. paggiae. Among these four species, the predominant species infecting T. chalcogramma was A. simplex (s.s.) (91.0%), followed by A. pegreffii (5.2%), A. paggiae (2.4%), and A. brevispiculata (1.4%). This is the first evidence of the occurrence of A. brevispiculata and A. paggiae in the north-west Pacific region.

Comparison of A. pegreffii from the Far East and Mediterranean Anisakis pegreffii has been widely reported, not only in the Mediterranean and Atlantic but also in the Far East region. Recently, two base difference was reported in the sequence of 5.8S rDNA between A. pegreffii from the Mediterranean and that from the Far East region. Based on this difference, the latter, which was originated from China, was tentatively designated as Anisakis pegreffii JP (Japan) in a previous report. In this study, A. pegreffii from the Mediterranean and from the Far East were morphologically and molecularly compared to confirm the validity of A. pegreffii JP and to confirm the two base difference in the 5.8S rDNA region. Morphologically, little difference was detected among the specimens. In PCR-RFLP and DNA sequences of ITS rDNA, identical results were obtained from all the present and previously reported specimens of A. pegreffii except that the two base difference was detected previously in only one sequence reported from the Mediterranean (GenBank Acc. No. AY826720), in which an unclear base was sandwiched between the different base positions, suggesting possible error in sequencing. In mtDNA cox2 region, no geographical difference was detected. These results indicate that A. pegreffii JP is identical morphologically and molecularly with A. pegreffii from Europe.

Anisakis distribution in Japanese waters Combining data obtained in the present studies (1) and (2), other host fishes presently examined and data obtained from available literature, distributions of Anisakis species in Japanese waters were described. It was revealed that A. simplex (s.s.) are mainly present in all host fishes examined in northern part of Japan in Hokkaido and in the Pacific side, whereas A. pegreffii are mainly present in all host fishes examined in the Sea of Japan and the East China Sea. In addition, low prevalence and intensity of three Anisakis species belonging to Anisakis Type II (A. physeteris, A. brevispiculata and A. paggiae) and hybrid genotypes were found. The former species was reported from Kyushu, whereas the latter two species were found in north-west Pacific side near Iwate Prefecture, whereas hybrid genotypes were found in northern and southern part of Japan. Interestingly, the current data indicate difference in distribution pattern between A. simplex (s.s.) and A. pegreffii. It is also very interesting to know the exact bounderies of such distribution between the Pacific, the Sea of Japan and East China Sea sides, from the viewpoint of the distribution of host populations including fishes and cetaceans.

2. Experimental infection studies

Anisakis Type I larvae are commonly found in many marine fish species. In spite of its presence in the body muscles of some host fishes, little information is available regarding the tissue specificity of A. simplex (s.s.) and A. pegreffii. In this study, experimental infection of rainbow trout, Oncorhynchus mykiss, and Japanese flounder, Paralichthys olivaceus with L3 larvae of A. simplex (s.s.) and A. pegreffii was conducted independently on both parasites. Fishes were orally challenged with L3 larvae. Sites of infection of the parasites were monitored 3, 7, 14, 21, 28 and 35 days postinfection (dpi). In rainbow trout, all A. simplex (s.s.) and A. pegreffii were recovered at 3dpi in the gastrointestinal lumen and body cavity. Migration of A. simplex (s.s.) in the body muscle was observed 7 dpi onwards, whereas A. pegreffii remained freely moving in the body cavity and finally disappeared at 35 dpi. In Japanese flounder, only 78% and 53% of A. simplex (s.s.) and A. pegreffii, respectively, were recovered at 3 dpi in the body cavity only. Migration in the body muscles was only obserevd in A. simplex (s.s.) from 7 dpi onwards, whereas A. pegreffii was found encycted in the body cavity in lumps. The results demonstrate that the migration and survival of both parasites differ depending on host species.

多くの海産魚には線虫が寄生するが、これらの線虫に関する分類や生物学特性に関する情報は大きく不足しているのが現状である。そこで本研究では、海産魚の線虫のなかでも、宿主の生殖巣や筋肉組織に寄生して宿主の生殖能や商品価値に影響を与えているフィロメトラ科線虫ならびにヒトに健康被害を起こすことが知られているアニサキス科アニサキス属線虫を研究対象として、こられについて、分類ならびに生物学的知見の集積を目的に研究を進めた。

1.フィロメトラ科線虫に関する研究

(1)分類学的研究

様々な海産魚の生殖巣から得たPhilometra属6種ならびにブリの筋肉から得たPhilometroids seriolaeについて分類学的研究を行った。一般にフィロメトラ科線虫では形態学的に種間変異に富んでいる雄虫を分類に用いるべきであると考えられている。しかし、実際には雄虫は非常に小型で採集が困難なため、これまで多くの分類が種間変異の小さい雌虫を用いて行われている。本研究では、Philometra属6種について雌虫に合わせて雄虫も得ることができたので、これらの虫体の光学顕微鏡観察と走査型電子顕微鏡観察によって形態学的分類を行った。特に、これまでに様々な海産魚類から報告され、種特異性が低いと考えられてきたP. lateolabracisの雄虫と雌虫を模式宿主であるスズキから初めて採集し、再記載した。この種と、従来P. lateolabracisと同定されていたイサキ寄生種とマダイ寄生種の形態を比較し、この2種は本種とは異なる新種(P. isaki およびP. madai)であることを明らかにした。併せて、サワラ寄生種についても新種(P. sawara)であることを明らかにした。また、シログチ寄生種 (P. sciaenae)ならびにイトヨリダイ寄生種(P. nemipteri) を再記載した。Philometroides seriolaeの雄虫を得ることはできなかったが、雌虫について再記載を行った。以上のように、調べたフィロメトラ科線虫7種はすべて独立した種であり、その結果はITS2領域の塩基配列からも支持された。フィロメトラ科線虫は宿主特異性が高く、これまで複数の宿主種から報告されている種は、その分類について再検討が必要であることが示された。

(2)生物学的特性に関する研究

前述のPhilometra属6種は、生虫あるいは死虫の両方が宿主の生殖巣に見出された。これらの生殖巣に寄生する種では、虫体の発達ならびに寄生率は宿主の産卵期と同調しており、この同調性により、成虫は産み出した1期幼虫を容易に宿主体外に放出できることが示唆された。一方、P. seriolaeはそのほとんどが筋肉内に寄生しており、虫体の一部を宿主体外に突き出して1期幼虫の放出をしていると考えられている皮下に寄生する成熟雌虫は、成熟虫体の一部(8.7%)にとどまった。組織学的には、Philometra属の生虫には宿主白血球の浸潤が観察され、死虫はその周囲に繊維細胞層の形成が認められた。P. seriolaeの周囲の筋肉内には炎症組織の層が形成されていた。

2.アニサキス科線虫

(1)分類学的研究

ヒトへの健康被害が問題となるアニサキス科線虫のうち、同胞種であり形態学的識別は困難とされていたAnisakis simplex (s.s.)とA. pegreffiiに着目し、分類学的研究を行った。

日本近海の海産魚から得たA. simplex (s.s.)とA. pegreffiiの3期幼虫のITS領域をPCR-RFLP法で解析して分子生物学的に同定し、さらに、ITS領域とmt cox2遺伝子の塩基配列を決定することにより、行った同定の妥当性を確認した。そのうえで、これらの3期幼虫ならびにin vitro培養法で得た成虫を光学顕微鏡観察と走査電子顕微鏡観察で形態学的に比較した。その結果、この2種は胃長(ventriculus length)で識別可能であること、雄成虫では尾乳頭の配列に違いがあることがわかった。

日本北部で漁獲されたスケトウダラからアニサキス属線虫を採集し、そのITS領域のPCR-RFLP解析, ITS領域とmt cox2遺伝子の塩基配列決定を行った。その結果、スケトウダラにはこれまで日本近海から報告されているA. simplex (s.s.), A. pegreffii以外に、A. paggiaeとA. brevispiculataが寄生していることが明らかになった。この2種が日本近海の魚類から見つかったのは初めてである。

模式産地である地中海のA. pegreffiiからは、極東で採集されたA. pegreffiiの5.8S rDNAの塩基配列と2塩基異なる配列が報告されていた。そこで、あらためて地中海産のA. pegreffiiを採集し、その塩基配列を決定したところ、地中海産のA. pegreffiiも極東の種と同じ配列をもち、形態学的にも識別できないことが明らかとなった。

本研究で採集し同定した結果ならびに既報のデータをもとに魚類に寄生するアニサキス属線虫の分布状況を調べたところ、日本近海ではA. simplex (s.s.)とA. pegreffiiが卓越しており、その地理的分布に違いがあり、前者は北海道北部海域と太平洋の魚類に、後者は日本海と東シナ海の魚類に高頻度に寄生していることが明らかになった。

(2)実験感染による研究

A. simplex (s.s.)とA. pegreffiiはどちらも海産魚類に寄生が認められるものの、魚体内での寄生部位の違いはわかっていない。そこで、この2種の寄生部位を明らかにするため感染実験を行った。具体的には、この2種をニジマスとヒラメの2種の魚類に別々に経口投与し、虫体の生残、魚体内分布を観察した。その結果、A. simplex (s.s.)はどちらの魚種でも筋肉内に移行したのに対し、A. pegreffiiはどちらの魚種でも体腔内にとどまり、ニジマスでは最終的に魚体内から消失した。これらの結果から、この2種の生残と寄生部位は魚種によって異なることが示唆された。

以上、本研究により、水産学的に重要でありながら研究が進んでいなかった海産魚類に寄生する2つの分類群の線虫類について、分類から生態にわたる様々な新しい知見が集積された。これらの成果は、学術上、応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。