Grass Puffer Research Articles

Aggregating Behavior of the Grass Puffer,Takifugu niphobles, Observed in Aquarium During the Spawning Period

The grass puffer (Takifugu niphobles) exhibits unique spawning behavior. Large numbers of fish aggregate to coastal spawning beds several hours before high tide during the spring tide. In order to examine the environmental and physiological regulation of this semilunar-synchronized spawning rhythm, the aggregating and spawning behaviors of the grass puffer were observed in the field, and in an aquarium without tidal changes. The fish aggregated to a spawning bed in the rising tidal phases both in the morning and evening during the spring tide, and several days after the spring tide. Spawning occurred on several days when large numbers of fish (200-1000) aggregated to the spawning bed. The timing of aggregation and spawning was tightly connected to the tidal changes; aggregation occurred 2-3 h before high tide, and spawning occurred 2 h before high tide. In the aquarium, in which a slope was constructed with pebbles, small groups of mature fish aggregated on the slope only in the rising tidal phases during and after the spring tide, when the fish aggregated in the field. However, there was no spawning in the aquarium. The aggregating behavior observed in the aquarium without tidal changes suggests that the semilunar reproductive rhythm is endogenously maintained with surprising precision during the spawning period in grass puffer.

Brief migration of the grass puffer, Takifugu niphobles, to fresh water from salt water

The grass puffer, Takifugu niphobles, is a peripheral freshwater fish often seen in brackish water. In the Sai River, Miyazu, Japan, we found that the grass puffer migrate to fresh water from salt water in June and July. Most of the grass puffer stayed in fresh water for 3.6 h on average and returned to the sea during the day. A low salinity tolerance experiment indicated that the grass puffer can live in fresh water for 2 days but not for longer than 4 days. Based on these findings, we discuss the physiological effects and ecological implications of the migration.

Differential expression of three types of gonadotropin-releasing hormone genes during the spawning season in grass puffer, Takifugu niphobles

Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around new moon and full moon from spring to early summer. To investigate the role of gonadotropin-releasing hormone (GnRH) in the reproductive function, genes encoding three types of GnRHs, namely seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II) and salmon GnRH (sGnRH), were cloned and changes in their mRNA amounts were examined over the spawning season. In addition, changes in the pituitary gonadotropin subunit mRNAs and the plasma steroid hormones were examined over the spawning season. Fishes were assessed at four reproductive stages, i.e., in December (early maturation), in April (maturing), in May (spawning), and in July (post-spawning). Moreover, spawning fish just after releasing eggs and sperm were taken at a spawning bed. The amounts of sbGnRH mRNA were substantially elevated in May and the spawning fish in both sexes, concomitant with considerable elevations of follicle-stimulating hormone and luteinizing hormone β subunit mRNAs and plasma estradiol-17β (E 2) and testosterone (T) levels. There were strong positive correlations between the sbGnRH mRNA and the plasma E 2 and T levels over the spawning season in both sexes. The amounts of cGnRH-II mRNA showed no noticeable changes except for an increase in the post-spawning females. The amounts of sGnRH mRNA in the males were significantly increased in May, but they were low in the spawning males. In the females, sGnRH mRNA increased from the maturing stage and reached a maximum in the post-spawning stage, in which a positive correlation with the plasma cortisol levels was observed. These specific changes suggest that the expression of three types of GnRH genes is differentially regulated during the spawning season, and sex steroids may be important for the differential expression of GnRH genes.

Synchronized diurnal and circadian expressions of four subtypes of melatonin receptor genes in the diencephalon of a puffer fish with lunar-related spawning cycles

Multiple subtypes of melatonin receptors are expressed in neural and peripheral tissues to mediate melatonin actions on the regulation of circadian rhythms in vertebrates. To elucidate molecular basis of “circa” rhythms in the grass puffer Takifugu niphobles, which spawns synchronously with semilunar cycles, tissue distribution of four melatonin receptor subtype mRNAs (Mel 1a 1.4, Mel 1a 1.7, Mel 1b, and Mel 1c) were examined, and diurnal and circadian changes in their absolute amounts were examined in the retina, diencephalon, and optic tectum. Mel 1a 1.4, Mel 1a 1.7, and Mel 1b mRNAs were widely distributed in various brain regions, retina, pituitary, and peripheral tissues, whereas Mel 1c mRNA was mainly detected in the nervous tissues and pituitary. All subtype genes showed diurnal expressions with one or two peaks during nighttime. When the fish were reared under constant darkness, the retinal expressions of Mel 1a 1.7, Mel 1b, and Mel 1c genes were markedly diminished but still showed circadian variations. In contrast, increased and synchronized expressions of the four subtype genes were noticeable with one peak at circadian time 18 in the diencephalon. The circadian expression profiles in the optic tectum were different among the subtypes. The present results suggest that melatonin receptor gene expression is regulated by circadian clock and light, but the effects of light are different among the tissues. The synchronized expressions of the four subtype genes in the diencephalon may be related to the exertion of reproductive rhythmicity in this puffer species.

Structure of neurohypophysial hormone genes and changes in the levels of expression during spawning season in grass puffer ( Takifugu niphobles)

Vasotocin (VT) has been shown to influence various aspects of social and sexual behaviors in a broad range of vertebrate species, but less is known about the mechanisms through which this peptide modulates behavior. Additionally, much less is known about roles of isotocin (IT) in regulation of behavior. Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around the spring tide and is conducted by a group of 10–60 individuals, of which one is female. As a first step toward investigating the roles of VT and IT in this species’ spawning behavior, we determined the structures of the VT and IT genes from grass puffer using the genome resources of the closely related tiger puffer and green puffer. We then used these sequences to develop real-time PCR assays and examined changes in expression of the VT and IT genes over the spawning season. The structures of VT and IT genes are well conserved among three puffer species. Particularly, the sequence similarities between grass and tiger puffers were very high not only in the coding region (85–99%), but also in the non-coding regions (92–98%) that include the 5′-upstream regions. The levels of expression of VT gene increased in the brain of pre-spawning females. The levels of VT mRNA in the spawning females tended to be higher than that in the spawning males. In contrast, the levels of IT mRNA did not show such variation. The present results suggest that VT gene expression augments in the brain of females during the spawning period. The unique spawning behavior of grass puffer provides a useful model for studying the molecular mechanism of sexual behavior utilizing the genome resources of tiger puffer.

口白症トラフグ脳磨砕濾液を用いた人為感染試験によるブリでの発症

The causative agent of kuchijirosho (snout ulcer disease), an infectious disease of tiger puffer Takifugu rubripes, is filterable, detailed characteristics of the agent have not been clear. Here, susceptibility of eight fish species against kuchijirosho was studied by injection with the kuchijirosho agent. A hundred percent mortality with typical signs of kuchijirosho was observed in tiger puffer, grass puffer T. niphobles, panther puffer T. pardalis and blue-spotted boxfish Ostracion immaculatus, while no mortality occurred in Japanese parrotfish Oplegnathus fasciatus, largescale blackfish Girella punctata and red seabream Pagrus major. Moreover, yellowtail Seriola quinqueradiata was demonstrated to be susceptible to this disease, because cumulative mortalities of 100% and 60% were recorded in young (average 12 g in body weight) and adult (average 530 g in body weight) yellowtail, respectively. Affected young yellowtail exhibited convulsion with the curvature of the spine (scoliosis), although no typical disease sign was observed in the dead adult yellowtail. In experimental infection with the brain homogenate of the dead adult yellowtail, scoliosis was produced in young yellowtail.

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from ‘alien’ hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.

Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?

In May 2002, two parasitic copepods, Pseudocaligus fugu and Taeniacanthus sp., were collected from the body surface and gill of the grass puffer Takifugu niphobles, respectively, in Takehara city, Hiroshima Prefecture, faced with Seto Inland Sea located in the western part of Japan. To them was added 5 ml of 0.1% acetic acid, then the suspension was subjected to ultrasonic disruption with an ultrasonicator for 10 min. The resulting mixture was heated in a boiling water bath for 10 min, and then centrifuged. The supernatant was concentrated under reduced pressure, and loaded on to a Sep-Pak plus C18 Environmental Cartridge (Waters). The unbound fraction was analyzed by HPLC and gas chromatography–mass spectrometry (GC–MS) for tetrodotoxin (TTX). It was rather unexpectedly revealed from these results that this fraction was comprised of TTX and its analogues. As far as we know, this is the first record to show the existence of TTX in the copepods. In addition, relationships between the more and less than the average number of the two parasites and the toxicity of its skin mucus of the host were examined by student's t-test. In P. fugu, the average number per host was 13.9, and those are 520.7 ( n=9) and 269.0 MU/g ( n=22), respectively. A highly significant difference between them was detected at p-value 0.0011. In contrast, as for Taeniacanthus sp., the average number was 2.7, and those were 338.0 ( n=14) and 345.5 MU/g ( n=17), respectively. No significant difference was detected in Taeniacanthus sp. The high host-specificity of P. fugu on the toxic puffer and the present bioassay of its skin mucus suggest a possibility that TTXs may attract the parasite.

Experimental infection of several fish species with the causative agent of Kuchijirosho (snout ulcer disease) derived from the tiger puffer Takifugu rubripes.

Kuchijirosho (snout ulcer disease) is a fatal epidemic disease which affects the tiger puffer, Takifugu rubripes, a commercial fish species in Japan and Korea. To assess the possibility that non-tiger puffer fish can serve as reservoirs of infection, 5 fish species were challenged by infection with the extracts of Kuchijirosho-affected brains from cultured tiger puffer: grass puffer T. niphobles, fine-patterned puffer T. poecilonotus, panther puffer T. pardalis, red sea bream Pagrus major, and black rockfish Sebastes schlegeli. When slightly irritated, all these species, especially the puffer fish, exhibited typical signs of Kuchijirosho, i.e., erratic swimming, biting together and bellying out (swelling of belly), as generally observed in tiger puffers affected by Kuchijirosho. Although the mortalities of the 2 non-puffer species were lower, injection of the extracts prepared from the brains of both inoculated fish into tiger puffer resulted in death, indicating that the inoculated fish used in this experiment have the potential to be infected with the Kuchijirosho agent. Condensations of nuclei or chromatin in the large nerve cells, which is a major characteristic of Kuchijirosho, were histopathologically observed to some extent in the brains of all kinds of puffer fish species infected. These findings suggest that the virus can spread horizontally among wild and cultured puffers and even among fishes belonging to different orders.

魚篭の魚種選択性

To demonstrate the species selectivity of fish baskets, fishing experiment was conducted using baskets of iron frame covered with netting in a rocky ground. The baskets were baited with frozen sardine (baited basket) or partly covered with brushwood on their top (covered basket) and their catches were compared with that of the control (without bait and brushwood). The total catch from 156 hauls was much better in the covered baskets and baited ones than in the control. The influence of the bait and brushwood on the catch was fish species selective; the filefish was significantly dominant in the catch of the covered baskets, the grass puffer was caught only by the baited baskets, and no influence was seen in the wrasse and striped catfish. A tank experiment implied that the species selectivity was different between day and night; the rockfish and filefish ingressed preferably into the covered basket in daytime but into the control at night. An exploratory behavior was hypothesized for the ingress of the fishes into the control, and the brushwood was thought to enhance an exploratory drive of fish.

Changes in microflora of a puffer fish Fugu niphobles, with different water temperatures

Grass puffer Fugu niphobles, captured in November 1986 at Shimoda, Japan, and then reared at different temperatures ranging from 10 to 29°C, was examined for microflora changes in the skin, gill and intestines. At 10°C, the skin and gill were colonized mainly by Pseudomonas, Flavobacterium and Moraxella, while at 29°C Vibrio and Flavobacterium appeared abundantly. The intestinal microflora, consisting of Vibrio, Pseudomonas and Flavobacterium, revealed little temperature dependence, although the intrageneric composition of Vibrio changed conspicuously depending on the change of water temperature. Vibrio group 1, tentatively identified as V. alginolyticus, was detected in all tissues examined at 20 and 29°C. This, along with the fact that V. alginolyticus produces tetrodotoxin, suggests that the tetrodotoxin contained in puffer is, at least partly, accounted for by this bacterial species inhabiting intestines.