Influence of social stability on the sex determination process in false clown anemonefish (Amphiprion ocellaris)

ABSTRACT

To evaluate the involvement of plasma cortisol in sex determination in hermaphrodite protandrous sex-changing fish, sexually immature false clown anemonefish (Amphiprion ocellaris) were kept in groups of 3 individuals for 360 days. Observations of time-course changes of diurnal behaviour and plasma concentrations of cortisol, oestradiol, and 11-ketotestosterone were evaluated. The social hierarchy of the experimental groups was assumed to become stable during the middle period of the experiment, but unstable at the beginning and at the end according to the increase or decrease in the frequencies of agonistic behaviours. The plasma concentration of cortisol increased as the social hierarchy became unstable and oestradiol showed the opposite trend to cortisol. These results suggest that in protandrous anemonefish sex determination induced by social interaction is mediated by cortisol. The stability of the social hierarchy is considered to be very important for the sex determination process.     

Eleven microsatellite loci for the saddleback clownfish Amphiprion polymnus

We report nine (CA)_12?49, one (GATA)₃₉, and one (TCTA)₂₆ microsatellite loci for the saddleback clownfish Amphiprion polymnus, isolated using an enrichment cloning procedure. A large number of alleles (range 6–30), and high levels of observed heterozygosity (mean = 0.6810) were resolved in 100 individuals of a single population, indicating that these markers should be useful to assess genetic structure and population dynamics over fine spatial scales.     

Social and mating systems of the protandrous anemonefish Amphiprion perideraion under the influence of a larger congener

The effect of a larger anemonefish Amphiprion clarkii (Bennett) on the social and mating system of a smaller congener Amphiprion perideraion (Bleeker) was investigated on a coral reef at Okinawa, Japan, where both species use the same host sea anemone Heteractis crispa (Ehrenberg). The population of A. perideraion consisted of adult, subadult and juvenile groups. Adult groups, which consisted of one or two adults and a varying number of subadults and juveniles, usually did not share the host with A. clarkii. In contrast, subadult and juvenile groups, which included no adults, always cohabited with A. clarkii. In the heterospecific groups, subadult A. perideraion were able to mature histologically, and changed to female when they were the largest among conspecific members, although their reproduction was suppressed by A. clarkii. After all members of A. clarkii emigrated or disappeared from a heterospecific group, adult A. perideraion could begin breeding. I suggest that A. perideraion in both heterospecific and conspecific groups adopt a mating strategy that involves waiting for vacated breeding posts because of their low mobility and a low host density.     

Response to underwater laser pointer in the Orange-finned Anemonefish Amphiprion chrysopterus and three-spot damselfish Dascyllus trimaculatus

Response of orange-finned anemonefish Amphiprion chrysopterus and three-spot damselfish Dascyllus trimaculatus to red laser-pointer light was studied in Mo'orea, French Polynesia. Four magnificent anemones Heteractis magnifica and their resident fish were observed for typical behaviours (biting, chasing, hiding, posing, lunging and retreating) with and without exposure to laser-pointer light. Lunging behaviour increased significantly for both fish species upon exposure to laser-pointer light; none of the other behaviours changed significantly. We advance the hypothesis that orange-finned anemonefish and three-spot damselfish interpret laser pointer stimulation as a territorial threat.     

Eight microsatellite loci in Clark's anemonefish,Amphiprion clarkii

To determine the genetic relationship between different colour morphs (orange and black morphs) of Clark's anemonefish (Amphiprion clarkii) in Taiwan, we isolated eight polymorphic microsatellite loci. A large number of alleles (range, 6–30), and high levels of observed heterozygosity (range, 0.1231–0.8358) were resolved in 71 individuals from two populations, indicating that these markers should be useful in assessing the relationship between the two colour morphs of A. clarkii.     

No evidence of shelter providing a metabolic advantage to the false clown anemonefish Amphiprion ocellaris

There was no evidence that shelter conveyed a metabolic advantage to the false clown anemonefish Amphiprion ocellaris in terms of standard and routine rates of oxygen uptake. The metabolic and fitness benefit of shelter might not, therefore, be widespread among all fish species.     

Consistent behavioural traits and behavioural syndromes in pairs of the false clown anemonefish Amphiprion ocellaris

Using the social clown anemonefish Amphiprion ocellaris, whether individuals exhibited consistency in activity levels, boldness and sociability in a paired context, and whether these three behavioural traits were positively correlated within a single behavioural syndrome, was investigated. The results highlight that consistent individual differences in behaviour are expressed in a social fish and suggest that consistent behavioural traits and behavioural syndromes could influence the structure and functioning of their societies.     

Ontogeny of the Digestive and Feeding Systems in the Anemonefish Amphiprion Melanopus

Ontogenetic growth and development in the anemonefish Amphiprion melanopus (Pomacentridae) is very rapid when compared to other tropical and temperate fish species. A. melanopus hatched with a highly differentiated digestive tract and the ability to capture and ingest prey items. Their alimentary tract changes rapidly throughout the larval period. Concurrent with yolksac absorption at three days after hatching was the development of the stomach followed by calcification of the jaw structures. This period of acute structural change may be a critical period in their development. Metamorphosis coincided with settlement at 8 days after hatching and was marked by calcification of fins and acquirement of adult coloration. The rapid development found in this species may be a specialisation to enhance the return of larvae to a patchy and highly specific settlement habitat.     

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue‐specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better‐known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10–15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.     

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630nm), green (peak at 530nm), and blue (peak at 450nm) within a visible light. We investigated the oxidative stress induced by starvation for 12days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish.     

Territorial Behavior and Ecology of the Anemonefish Amphiprion melanopus on Guam1)

Field observations and experiments on the Indo-Pacific anemonefish Amphiprion melanopus at Guam show that it colonizes the aggregating sea anemone Physobrachia douglasi extensively and nearly exclusively. There are an average of four fish per colony. The total standard length of anemonefish at each aggregation is highly correlated with the total area covered by resident anemones, suggesting both a carrying capacity and some form of social control of growth of anemonefish²). Inter-colony migration of juveniles, as well as larval recruitment, may contribute to maintenance of the optimum colony size. Adults do not migrate. Both juveniles and adults defend territory. Juvenile territories are mutually exclusive areas within the confines of the anemone aggregation, while adult territories are considerably larger than the area covered by the anemone aggregation. Though territories of mated ♀♀ and ♂♂ overlap completely, female emphasis is peripheral relative to the ♂. Adult conspecific intruders are attacked more heavily and at greater distances than are juveniles. Intraspecific territoriality in juveniles probably reflects the limited availability of critical habitat. In adults it may function to protect the pair bond. Interspecific aggression is less intense and appears to protect both the spawn and host anemones from various predators.     

Mobility of Stichodactyla Gigantea Sea Anemones and Implications for Resident False Clown Anemonefish, Amphiprion Ocellaris

For reef fishes that do not move between habitat patches following settlement, habitat selection is expected during settlement. Although false clown anemonefish, Amphiprion ocellaris, are sedentary following settlement, they are not especially discriminating during settlement, and are commonly found occupying anemones at which no apparent nest site exists. In this study I report on mobility of Stichodactyla gigantea sea anemones, including anemones with resident false clowns. I argue that anemone mobility can help explain why settling false clowns are not more discriminating: although the per annum probability of an anemone moving is low, the probability of that anemone moving over the course of a resident's life is considerably higher. Therefore, an anemone's current microhabitat may not be a good predictor of its microhabitat and suitability as a host in the future.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Characterization of reproductive cycles and adrenal activity in the black‐footed ferret (Mustela nigripes) by fecal hormone analysis

The reproductive cycle of the black‐footed ferret (Mustela nigripes) was characterized by enzyme immunoassay (EIA) analysis of ovarian fecal steroids (estradiol, progestins) in 29 females over two consecutive breeding seasons. Estrous status was determined by measuring the vulva size and examining the percentage of superficial cells in vaginal lavages. Mean fecal estradiol concentrations were correlated with vulval area (r = 0.370, P < 0.0001) and the percentage of superficial cells (r = 0.380, P < 0.0001). Ovulation resulted in a rise in fecal progestin concentrations 5 days after breeding that differed (P < 0.05) between pregnant (n = 14) and pseudopregnant (n = 12) females during the late luteal phase (days 12–40), with concentrations remaining higher in pregnant animals. Gestation length was 41.3 ± 0.7 days with 3.6 ± 0.4 kits produced per female. Litter size correlated significantly (P < 0.05) with fecal estradiol, but not progestins during the 12 to 40 days after breeding. Females failing to breed (n = 3) remained in estrus for 31 ± 6.2 days before ovulation induction with human chorionic gonadotropin. Adrenal activity in male (n = 4) and female (n = 6) black‐footed ferrets was monitored by quantifying fecal corticoid metabolites after a series of manipulations (physical restraint, intramuscular saline, intramuscular gel adrenocorticotropic hormone (ACTH), intramuscular liquid ACTH). A significant (P < 0.0001) increase in fecal corticoids above the pre‐treatment baseline occurred 20 to 44 hours after restraint (five of 10 animals), saline (six of nine), gel ACTH (seven of 10), and liquid ACTH (nine of 10) treatments. Immunoreactivity of high‐performance liquid chromatography–separated fecal elutes was compared using antibodies against cortisol and corticosterone. The cortisol EIA demonstrated immunoreactivity that co‐eluted with ³H‐cortisol, whereas a corticosterone radioimmunoassay detected a metabolite peak that co‐eluted with ³H‐corticosterone in addition to a slightly less polar and one considerably more polar peak. Despite recognizing different metabolites, both assays produced similar temporal profiles of corticoid excretion after manipulation. This study provides new information on the black‐footed ferret regarding differences in fecal steroid excretion patterns between pregnancy and pseudopregnancy and the potential application of fecal corticoid metabolite monitoring for evaluating responses to stressors associated with practices used in breeding management. Zoo Biol 20:517–536, 2001. © 2002 Wiley‐Liss, Inc.     

First description of the neuro‐anatomy of a larval coral reef fish Amphiprion ocellaris

The present study described the neuro‐anatomy of a larval coral reef fish Amphiprion ocellaris and hypothesized that morphological changes during the transition from the oceanic environment to a reef environment (i.e. recruitment) have the potential to be driven by changes to environmental conditions and associated changes to cognitive requirements. Quantitative comparisons were made of the relative development of three specific brain areas (telencephalon, mesencephalon and cerebellum) between 6 days post‐hatch (dph) larvae (oceanic phase) and 11 dph (at reef recruitment). The results showed that 6 dph larvae had at least two larger structures (telencephalon and mesencephalon) than 11 dph larvae, while the size of cerebellum remained identical. These results suggest that the structure and organization of the brain may reflect the cognitive demands at every stage of development. This study initiates analysis of the relationship between behavioural ecology and neuroscience in coral reef fishes.     

Coexistence of two anemonefishes,Amphiprion clarkii and A. perideraion,which utilize the same host sea anemone

Synopsis Social structure and interactions between the anemonefishes, Amphiprion clarkii and A. perideraion, which utilize the same host sea anemone Radianthus kuekenthali, were investigated on a coral reef of Okinawa Islands, Japan. In an 87 × 373 m² study area, 98 sea anemones were inhabited by both species (32.5%), by only A. clarkii (48.9%), or by only A. perideraion (18.6%). A group of A. clarkii often occupied two or more individual hosts, and group members often interchanged. However, a group of A. perideraion usually used only one host and migration between groups was rare. The larger A. clarkii suppressed reproduction of A. perideraion in cohabiting groups, while A. perideraion suppressed settlement of Juvenile A. clarkii to its own hosts. Juvenile A. clarkii settled on small hosts as well as on large hosts, whereas juvenile A. perideraion settled only on large hosts. Coexistence appears to be possible in part by differences in settlement patterns between juveniles of the two anemonefishes.     

Spacing pattern and body size composition of the protandrous anemonefish Amphiprion frenatus inhabiting colonial host anemones

The protandrous anemonefish Amphiprion frenatus often forms a group consisting of a large female, a small male, and a smaller nonbreeder at an isolated single host anemone, where home ranges of subordinates were covered with the female's home range. Within the group, the dominant individuals suppress the growth of subordinates, resulting in large size differences. The spacing pattern and body size composition of A. frenatus on colonial hosts were investigated in Ishigaki Island, Okinawa, Japan. Six breeding pairs and 14 nonbreeders inhabited a colony of 157 anemones. Each pair maintained a territory in which pair members used different hosts. Nonbreeders had unstable home ranges on the outskirts of or in the pairs' territories. Body size differences between males and females in pairs and between males and nonbreeders were small. The small size differences in the colony of hosts are caused by reduced suppression of growth of subordinates by the dominant individuals. The total area of host anemones largely affects spacing pattern and social suppression of the anemonefish.     

A bovine-specific FSH enzyme immunoassay and its application to study the role of FSH in ovarian follicle development during the postnatal period

The primary aim of this study was to develop a FSH enzyme immunoassay (EIA) for the bovine species. The newly developed EIA was validated for FSH determination in bovine plasma by comparison with an existing bovine FSH radioimmunoassay. The EIA detected bovine FSH with a high sensitivity (0.1 ng/ml). Cross-reactivity of the EIA was 0.01% with bovine LH, 51% with ovine FSH, <0.1% with porcine FSH and <0.01% with equine FSH. Using this EIA on different time series of plasma in cows, we have confirmed the presence of a FSH pre-ovulatory peak at estrus, of periodic FSH fluctuations accompanying the waves of terminal follicular development, and of FSH pulses, mainly asynchronous with LH ones, in the peri-ovulatory phase of the cycle. In a second objective, the EIA was used to assess the role of FSH in regulating the development of ovarian follicles up to the small antral stage in young calves. To answer this question, six calves were submitted to weekly blood sampling during their first 3 months of life, and FSH changes were studied concomitantly to those of anti-Müllerian hormone (AMH), a well-established endocrine marker of the ovarian population of small antral follicles in cows. In the ovaries of 3-month calves, the population of 3 to 5 mm follicles contained the highest intra-follicular AMH amounts, and the number of 3 to 5 mm follicles on ovaries was closely correlated with AMH concentrations in the plasma of calves at this age (r_s = 0.94). Before 3 months of age, only two out of six calves showed a clear postnatal FSH peak in plasma, and no correlation was found between plasma FSH and AMH concentrations. These results indicate that female calves undergo different patterns of FSH secretion and that postnatal activation of follicular growth up to the small antral stage appears independent and not directly related to circulating FSH levels.     

Acquisition of Breeding Space by Nonbreeders in the Anemonefish Amphiprion clarkii in Temperate Waters of Southern Japan

Behavior of Amphiprion clarkii, especially of nonbreeders, was investigated at the shore of boulders and rocks in Uwa Sea, southern Japan. All individuals except small ones under one year were tagged. Movements of A. clarkii between host sea anemones were usual in this study area where host anemones were abundant, unlike in the general habitat (coral reefs) of Amphiprion, where movements between hosts are restricted. Monogamous pairs established almost contiguous territories containing all hosts except small ones. Nonbreeders had home ranges on the fringes of the pairs' territories and sheltered in relatively small hosts. Breeding spaces were saturated with breeders and were available for nonbreeders only after disappearance of one or both members of an established breeding pair. Home ranges of nonbreeders of similar body size were spaced out, due perhaps to competition for breeding spaces. It is suggested that large nonbreeders refrained from becoming females to keep their gonads ambosexual, so that they could replace either sex in a territory as breeding space became available, or, in pairing with other nonbreeders, the larger fish of the pair could take the female role.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.