2026 年 61 巻 1 号 p. 23-31
The development of first to third stage larvae of Philometroides seriolae, the type species of the genus, was monitored. Hatched larvae were obtained from gravid females collected from Japanese amberjack Seriola quinqueradiata. The larvae in the uterus consisted of the first stage (18.4%) and second stage (81.6%). Larvae obtained from the uteri were exposed to the harpacticoid copepod Tigriopus japonicus, employed as a surrogate intermediate host. Infected copepods were maintained at 20°C and at 25°C for 28 days, during which larvae in the hemocoel were periodically examined. All larvae had developed into second stage by 5 days post exposure (5 dpe). Third stage larvae first appeared at 13 dpe at 20°C and at 8 dpe at 25°C. First to third stage larvae possessed two unicellular oesophageal glands located in the posterior region of the oesophagus. The second stage larvae possess single papilla-like projections at the surface of cephalic region and the caudal end. In the third stage larvae, these projections were liberated from the surface with peeling of the second stage sheath. This study provides the first description of the second and third stage larvae of P. seriolae and documents their experimental development in a copepod host.
Japanese amberjack Seriola quinqueradiata is one of the most important fishery resources in Japan, and, at the same time, one of the most studied fish species in Japan. Many parasites have been recorded from its skeletal muscle (Ogawa, 2023). Among them, the large philometrid nematode Philometroides seriolae, which can reach a body length of up to 50 cm, has been frequently reported in wild populations (Ishii, 1931; Yamaguti, 1935; Moravec et al., 1998) and, less commonly, in cultured stocks (Nakajima and Egusa, 1969; Moravec and Ogawa, 2019). Although P. seriolae is not a zoonotic parasite (Moravec, 2004), its conspicuous presence in the muscle tissue severely diminishes the commercial value of infected fish, posing a serious problem for the Japanese fisheries industry.
Only limited knowledge is available about the life cycle of P. seriolae. Philometrid nematodes, including P. seriolae, are known to require intermediate hosts to complete their life cycles (Moravec, 2004; Moravec and de Buron, 2013). The natural intermediate host(s) of P. seriolae has not been specified. Following Sakaguchi and Fukuhara (1975), Ogawa et al. (2023) employed the harpacticoid copepod Tigriopus japonicus as a surrogate intermediate host. When larvae of P. seriolae were given to the copepod, they successfully developed to the infective stage within the hemocoel. Subsequently, when these infected copepods were fed to Japanese amberjack fingerlings, mature worms were recovered from their skeletal muscle after eleven months.
In this study, we conducted a series of experimental infections using freshly obtained hatched larvae to investigate the developmental progression of P. seriolae within the surrogate copepod host. This paper provides the first detailed description of larval development within the copepod and offers novel insights into the life cycle of P. seriolae.
Hatched larvae of P. seriolae were obtained from the uteri of gravid female worms collected from a Japanese amberjack (66.2 cm in fork length, 3.13 kg in body weight). The fish was captured off the coast of Shirahama, Wakayama Prefecture, in November 2023. The nematodes were carefully dissected, and the larvae released from their uteri were collected and transferred to sterilized sea water. These larvae exhibited vigorous motility and were immediately used for the subsequent infection experiments involving T. japonicus. Additionally, hatched larvae obtained from gravid nematodes collected from Japanese amberjack cultured in Kamae, Oita Prefecture, in May 2023 were used as supplementary materials for figure preparation.
The copepod T. japonicus were collected from tide pools in Shirahama, Wakayama Prefecture, and initially maintained at room temperature in a 5 L plastic tank filled with UV-treated, sand-filtered seawater. Microscopic examination of subsamples (>300 individuals) confirmed the absence of natural infections. The copepods were periodically fed the phytoplankton Tetraselmis tetrathele to maintain their condition. Copepods reproduced in the containers and newly emerged individuals with lighter body coloration were used as much as possible in the experiment.
For the infection trials, active copepods were transferred to clear 200 mL plastic containers (200–300 individuals per container) containing sterilized seawater. First stage larvae of P. seriolae were added to the container for experimental exposure.
Experimental designNumerous larvae of P. seriolae (several to tens of thousand individuals), freshly obtained from the gravid females, were gently pipetted into the containers containing the copepods. The exposed copepods were then maintained at 20°C or 25°C, with seawater changes every few days and occasional feeding with T. tetrathele.
At 2 days post exposure (2 dpe), infection status was assessed. Copepods were anesthetized with 500 ppm ethyl 3-aminobenzoate methanesulfonate (MS 222, Nacalai Tesque, Inc.) (Shin and Hur, 2006), placed on a glass plate, and individually examined under a stereomicroscope to detect nematode larvae within the hemocoel. Copepods harboring visibly motile larvae in the hemocoel were considered infected (Fig. 1). These individuals were transferred to new containers and maintained under the same conditions.
Larval development within the copepods was monitored periodically from 2 to 28 dpe. Infected copepods (n = 3 to 23 per observation) were gently compressed between a glass slide and an 18 × 18 mm cover slip to expel the larvae from the hemocoel. Expelled larvae were carefully transferred onto a glass slide with 5 μL of seawater and heat-fixed by briefly passing the slide over a flame. Subsequently, 5 μL of 10% formalin was added for fixation, followed by placement of a coverslip and application of a few drops of 30% glycerin beneath the coverslip for permeation.
Portions of larvae from the uteri of P. seriolae females obtained from Japanese amberjack at Shirahama and Kamae were separately processed. Larvae from Shirahama were incubated at 25°C for one day before being fixed using the same method as those recovered from copepods. Remaining larvae were used for infection experiments using T. japonicus. In contrast, larvae from Kamae, used as supplementary materials for determination of the developmental stages of the larvae in the uterus and drawing figures, were fixed immediately upon retrieval from uteri, without prior incubation (Fig. 2A, D).
Fixed larval specimens were observed and photographed under a compound microscope. Their morphological parameters, including total body length, body width, oesophagus length, and tail length, were measured from the digital images using the image analysis software ImageJ (image processing program available at: https://imagej.net/ij/). All measurements were given in micrometers, as mean values, followed by ranges in parentheses.
A total of 142 individuals of larvae (2–23 per sampling across 11 occasions) maintained at 25°C, and 102 individuals (11–16 per sampling across 7 occasions) maintained at 20°C were used for developmental stage identifications. Additionally, 141 individuals maintained at 25°C and 90 individuals at 20°C were used for morphological observations and morphometric analysis. All figures were drawn with the aid of a drawing tube attached to a light microscope.
Statistical analysesThe proportions of first, second, and third-stage larvae were compared across observation days and between the 20°C and 25°C temperature groups using the chi-square test. Morphometric measurements, including total body length/body width, oesophagus length/total body length, and tail length/total body length, were analyzed for each developmental stage and between two temperature conditions on the same observation day using t-test, or ANOVA followed by Tukey’s HSD post-hoc test. The significance level was set at 0.05. All statistical analysis was performed using JMP ver. 11 (SAS Institute).
Body short and slender, 515 (405–595) long, maximum width 17.4 (14.5–20.1). Body length/maximum width 29.8 (24.6–33.6). Oesophagus 165.6 (125.5–202) long, 32.2 (25.2–40.6) % of body length. Oesophageal anterior dilation absent. Muscle of oesophagus weakly developed. Two unicellular oesophageal glands present at posterior region of oesophagus. Nerve ring located at anterior region of oesophagus. Excretory pore unobservable. Intestine and rectum visible. Tail long and tapering, 107.7 (76.3–134.8) long, 20.4 (15–23.0) % of body length.
Body short and slender, 536 (368–650) long at 20°C, 509 (311–635) long at 25°C. Maximum width 18.1 (10.9–27.6) at 20°C, 17.2 (10.6–24.7) at 25°C. Body length/maximum width 31.2 (16.3–54.8) at 20°C, 30.5 (15.3–54.9) at 25°C. Most of specimens ensheathed, single sheath present. Some in the process of shedding sheath. Single papilla-like projection presents at cephalic region. Oesophagus 175 (121–271) long, 33.7 (22.4–49.1) % of body length at 20°C, 162 (108–260) long, 32.5 (22.9–45.4) % of body length at 25°C. Oesophagus possessing anterior dilation. Muscle of oesophagus developed. Two unicellular oesophageal glands present at posterior region of oesophagus. Nerve ring located at anterior region of oesophagus. Excretory pore unobservable. Intestine and rectum visible. Tail long and tapering, 80.8 (62.3–149.3) long, 15.2 (11.7–23.5) % of body length at 20°C, 77.4 (45.1–143.1) long, 15.4 (9.4–25.3) % of body length at 25°C. Single small papilla-like projection presents at caudal end.
Third stage larvae (based on 77 specimens: 43 larvae maintained at 20°C, and 34 larvae maintained at 25°C; Fig. 2C, F; Fig. 3E, F)Body short and slender, 583 (441–668) long at 20°C, 547 (440–693) long at 25°C. Maximum body width 12.8 (9.21–18.8) at 20°C, 13.4 (9.3–20.7) at 25°C. Body length/maximum width 47.3 (31.7–67.6) at 20°C, 41.5 (30.1–58.3) at 25°C. Most of specimens ensheathed, one or two sheaths present. Cephalic and caudal papilla-like projections lost with second-stage sheath. Oesophagus 157.9 (109.6–234.1) long, 27.2 (17.8–42.4) % of body length at 20°C, 158.2 (110.3–227.7) long, 30.2 (18.9–38.9) % of body length at 25°C. Oesophagus possessing anterior dilation. Muscle of oesophagus developed. Two unicellular oesophageal glands present at posterior region of oesophagus. Nerve ring located at anterior region of oesophagus. Excretory pore unobservable. Intestine and rectum visible. Tail long and tapering, 66.7 (48.5–93.4) long, 11.4 (8.3–14.3) % of body length at 20°C, 64.3 (49.1–95.8) long, 11.9 (9.8–16.0) % of body length at 25°C.
Discrimination of developmental stageLarvae without evidence of a newly formed cuticle layer (i.e. lacking a sheath) were classified as first stage. Larvae having the following morphological characteristics were identified as second stage: 1) presence of a new cuticle layer beneath the old one, 2) papilla-like projections at the cephalic and/or caudal ends on the surface of the new cuticle, and 3) a single peeled sheath. Larvae bearing double peeled sheaths, or those with a single peeled sheath that bore papilla-like projections at the cephalic and/or caudal ends were classified as third stage. Additionally, after 5 dpe, larvae without a sheath and without papilla-like projections at cephalic and/or caudal ends were also considered third stage.
Comparison of morphometrics among first, second and third stage larvae (Fig. 4)At 25°C, the tail and the ratio of tail length to body length of second stage larvae was significantly shorter and lower, respectively, than those of the first stage larvae. The other morphometrics of second stage larvae showed no significant differences compared to first stage larvae at 25°C.
At 20°C, the body length of third stage larvae were significantly longer than that of the second stage. The body width, oesophagus length and tail length of third stage larvae were significantly shorter than those of the second stage larvae. The ratios of oesophagus length to body length and tail length to body length were significantly lower in third stage larvae than in the second stage larvae. The ratio of body width to body length of third stage larvae was significantly higher than that in the second stage larvae.
At 25°C, the body width and tail length of the third stage larvae were significantly shorter than those of the second stage larvae. The body length to body width ratio of third stage larvae was significantly higher, while the tail length to body length ratio was significantly lower compared to second stage larvae. Other morphometrics showed no significant difference between second and third stage larvae.
A total of 167 larvae, directly obtained from the uteri of female nematodes and fixed without incubation, were examined to determine their developmental stage. Of these, 31 (18.6%) were identified as first stage larvae, and 136 (81.4%) as second stage larvae, characterized by the presence of both old and newly formed cuticle layers, along with a single papilla-like projection at cephalic and/or caudal regions. No third stage larvae were detected.
Development of larvae in the hemocoel of Tigriopus japonicusLarvae of P. seriolae exhibited limited growth within the hemocoel of T. japonicus. Following infection to the copepod, larval body length increased by only approximately 4% over 24 days at 25°C and 12% over 28 days at 20°C (Fig. 5). In contrast, body width decreased substantially, by 35% at 25°C and 23% at 20°C over the same periods. No apparent differences in the temporal changes of body length or width were observed between the two temperature conditions.
Despite minimal somatic growth, the larvae successfully progressed to the third development stage during the observation period. Notably, some larvae had already begun transitioning to the second stage after incubated for 1 day in sea water, as evidenced by the presence of a papilla-like projection at cephalic and/or caudal regions. After infecting T. japonicus, second stage larvae were observed as early as 3 dpe (Fig. 6), and the proportion of second stage larvae increased rapidly, with no first stage larvae were observed after 5 dpe (Fig. 6). Third stage larvae were detected as early as 8 dpe at 25°C and 13 dpe at 20°C (Fig. 6). By 15 dpe, over 60% of the larvae had developed into the third stage, with their proportion increasing thereafter. Although the observation period differed between the two temperature groups, temperature appeared to have no significant effect on the overall larval development.
The genus Philometroides Yamaguti, 1935 currently comprises 15 valid species (WoRMS, 2025), with P. seriolae designated as the type species. As the natural intermediate host of P. seriolae remains unknown, we employed the harpacticoid copepod T. japonicus as a surrogate intermediate host. This allowed us to obtain third stage larvae suitable for experimental infection to the definitive host S. quinqueradiata (Ogawa et al., 2023). In the present study, we characterized developmental progression of P. seriolae larvae through periodic sampling from the copepod host.
Ogawa et al. (2023) successfully recovered adult P. seriolae from S. quinqueradiata following the administration of larvae that had developed in T. japonicus at 25°C for 23–27 days. Our current observations confirmed that these larvae had reached the third stage at the time the infecting copepods were administered to the fish, as shown in Fig. 6.
Only a few previous studies have documented the development of Philometroides species in copepods (Uhazy, 1977; Moravec, 2023). By comparing the developmental changes of P. seriolae with those of its congeners, we identified morphological features unique to this species. Specifically, single papilla-like projections appeared at the surface of both the cephalic and caudal ends of the second stage larvae. These projections were liberated along with the shedding of second stage sheath during the development into the third stage, leaving the third stage larvae devoid of such structures. In contrast, other Philometroides species viz. P. nodulosus, P. sanguineus and P. huronensis, exhibit different patterns (Uhazy, 1977; Moravec, 2023). For example, P. nodulosus develops two minute caudal extensions upon reaching the third stage (Moravec, 2023). P. sanguineus possesses a single minute dorsal tooth at the cephalic end in the first stage (Moravec, 2023) and P. huronensis develops two pairs of projections at the caudal end in the second stage, which are retained in the third stage (Uhazy, 1977). Thus, the presence of a single papilla-like projection at both cephalic and caudal regions exclusively in second stage larvae represents a distinctive characteristic of P. seriolae and serves as a reliable marker for staging its larval development.
Presence of two unicellular oesophageal glands located at the posterior region of their oesophagus is a common feature among first to third stage larvae of P. seriolae. Interspecific variation in the number of these glands has been reported: P. huronensis possesses three, while P. sanguineus has only one (Uhazy, 1977; Moravec, 2023). These differences further support the utility of unicellular oesophageal gland counts as a possible diagnostic trait for species differentiation within the genus.
In the present study, the majority of larvae obtained from the uteri were already in the second stage. This indicates that first stage larvae of P. seriolae hatch within the uterus and rapidly develop into the second stage before being released. This developmental speed appears faster than that observed in other species: second stage larvae of Philometroides cyprini were first observed at 3–4 days post infection, and those of P. sanguineus at 5–6 days post infection, when kept at 18–19°C (Moravec, 2023). Uhazy (1977) further reported that molting to the second stage in P. huronensis occurred between 14–18 days at 10°C in Cyclops bicuspidatus, and 6–9 days at 20–23°C in Acanthocyclops vernalis. While the developmental rate within T. japonicus observed in our study may partly reflect the influence of higher rearing temperature (20°C and 25°C), the presence of second stage larvae within the uteri indicates that P. seriolae undergoes rapid development even before release into the environment.
The present results demonstrate that P. seriolae developed to the stage infective to the final host S. quinqueradiata in the copepod T. japonicus. Although development within a surrogate host may not fully replicate one in the natural intermediate host, the use of such surrogate systems may also be applicable to studies of other fish nematodes with unknown life cycles.
We express our gratitude to the members of Fish Disease Lab, Aquaculture Research Institute of Kindai University, for their assistance in data collection. Part of this study was presented at the annual meetings of Japanese Society of Fish Pathology held online on March 5, 2022 and in Tokyo, September 3, 2023. This study was partially supported by a Grant-in-Aid for Scientific Research (C) (no. 21K05735) from the Japan Society for the Promotion of Science.
