Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum)

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model.     

Preliminary observations on the spawning conditions of the European amphioxus (Branchiostoma lanceolatum) in captivity

Members of the subphylum Cephalochordata, which include the genus Branchiostoma (i.e. amphioxus), represent the closest living invertebrate relatives of the vertebrates. To date, developmental studies have been carried out on three amphioxus species (the European Branchiostoma lanceolatum, the East Asian B. belcheri, and Floridian-Caribbean B. floridae). In most instances, adult animals have been collected from the field during their ripe season and allowed (or stimulated) to spawn in the laboratory. In any given year, dates of laboratory pawning have been limited by two factors. First, natural populations of these three most studied species of amphioxus are ripe, at most, for only a couple of months each year and, second, even when apparently ripe, animals spawn only at unpredictable intervals of every several days. This limited supply of living material hinders the development of amphioxus as a model system because this limitation makes it more difficult to work out protocols for new laboratory techniques. Therefore we are developing laboratory methods for increasing the number of amphioxus spawning dates per year. The present study found that a Mediterranean population of B. lanceolatum living near the Franco-Spanish border spawned naturally at the end of May and again at the end of June in 2003. Re-feeding experiments in the laboratory demonstrated that the gonads emptied at the end of May refilled with gametes by the end of June. We also found that animals with large gonads (both, obtained from the field and kept and fed at the laboratory during several weeks) could be induced to spawn in the laboratory out of phase with the field population if they were temperature shocked (spawning occurred 36 hours after a sustained increase in water temperature from 19 degrees C to 25 degrees C).     

Methods for Generating Year-Round Access to Amphioxus in the Laboratory

Cephalochordates, commonly known as amphioxus, are key to understanding vertebrate origins. However, laboratory work suffers from limited access to adults and embryonic material. Here we report the design and experimental validation of an inland marine facility that allows establishing stable amphioxus colonies in the laboratory and obtaining embryos at any time of day and over almost the entire year, far exceeding natural conditions. This is achieved by mimicking the natural benthic environment, natural day- and moon- light, natural substrate and by providing a strictly controlled and seasonally fluctuating temperature regimen. Moreover, supplemented algae diets allow animals to refill their gonads in consecutive years. Spontaneous spawning, a major problem in previous setups, no longer occurs in our facility; instead, all breeding is induced and fertilization occurs fully in vitro. Our system makes amphioxus a standard laboratory animal model.     

Non-synchronous spawning behavior in laboratory reared amphioxus Branchiostoma belcheri Gray

Spontaneous spawning in amphioxus (Branchiostoma belcheri Gray) was observed in a tank and the characteristics of spawning were analyzed by video recording under infrared light conditions during summer of 2001. Everyday when spawning was observed, without exception a male first swam up from the bottom and released sperm near the water at the surface of the tank. The initiation time of the male spawning gradually became earlier as days passed. Spawning males and females individually swam up at various intervals and released gametes. However, at the population level, the spawning pattern of amphioxus was considered to be synchronous because both males and females intensively spawned around 90 min after the spawning of the first male. The act of releasing eggs or sperms of individuals was shorter than 10 min in most of the spawning animals.     

A snapshot of the population structure of Branchiostoma lanceolatum in the Racou beach, France, during its spawning season

A methodology for inducing spawning in captivity of the lancelet Branchiostoma lanceolatum has been developed recently with animals collected at the Racou beach, in the southern coast of France. An increasing amount of laboratories around the world are now working on the evolution of developmental mechanisms (Evo-Devo) using amphioxus collected in this site. Thus, today, the development of new aquaculture techniques for keeping amphioxus in captivity is needed and the study of the natural conditions at which amphioxus is exposed in the Racou beach during their spawning season becomes necessary. We have investigated the amphioxus distribution, size frequency, and population structure in the Racou beach during its natural spawning season using multivariate methods (redundancy analysis and multiple regression). We found a clear preference of amphioxus for sandy sites, something that seems to be a general behaviour of different amphioxus species around the world. We have also estimated the amphioxus growth rate and we show how the animals are preferentially localized in shallow waters during April and June.     

Duration of larval and spawning periods inPagrus auratus (Sparidae) determined from otolith daily increments

Synopsis Counts of pre-metamorphic and post-metamorphic daily increments in the sagittae of settled juvenilePagrus auratus were used to determine duration of the larval period and to back-calculate spawning dates. The duration of the larval period was 18–32 days, and was longer for snapper spawned early in the spawning season, when water temperatures were low, than for snapper spawned later in the season when temperatures were high. Sagitta size at metamorphosis was unrelated to duration of the larval period or temperature, and mean increment width during the larval period increased with temperature. These results suggest that metamorphosis is size- rather than age-dependent. Back-calculated spawning dates ranged from September to March, and peaked in November-January. Maximum spawning season duration was five months. Spawning onset was earlier when spring water temperature was higher than normal, and first spawning occurred at 14.8–15.6 °C over three seasons, indicating that spawning onset is temperature-dependent.     

Laboratory culture of the Oriental lancelet Branchiostoma belcheri

To overcome difficulties in getting research materials of cephalochordate lancelets, which has severely hampered experimental studies of this animal, we have attempted to establish a culture system in the laboratory. Adult animals collected from the wild were maintained in 2.5-L plastic containers filled with natural seawater without sand substratum. They were fed daily with unicellular algae. About 25% of the animals collected in 2003, 2004, and 2005 developed gonads in our culture system. Some of the sexually mature animals collected in the breeding seasons in 2005 and 2006 spawned spontaneously in the plastic containers of this system. Broods obtained in 2005 were maintained longer than a year in a glass tank without sand substratum. The progeny born in the laboratory showed great individual variation in growth but metamorphosed normally, and some of them started to develop gonads around 10 months after fertilization. Our mass culture methods for both adults and their progeny made daily observation possible and allowed the constant spawning of animals collected from the wild, at least in the summer season. Our culture method saves labor in maintenance and is easily set up without any specific demands except for running seawater, though still required to better survival rate and spawning control. Lancelet populations maintained in the laboratory can promote studies on these animals across disciplines and especially contribute to elucidation of the evolutionary history of chordates.     

Gonadal state of wild amphioxus populations and spawning success in captive conditions during the breeding period in Japan

Adult individuals of amphioxus (Branchiostoma belcheri) were collected by dredging from a research vessel at selected stations in two areas off the coast of Japan in July 2000: Deyama and Takamatsu so named by local fishermen in the Enshu Nada Sea. The number of males collected exceeded that of females at all the stations in Takamatsu and at four of five stations in Deyama. The over all sex ratio (males : females) of the collected animals was 1.2 : 1. The animals showed various maturational stages of the gonad, and approximately 70% had mature gonads. However, post-spawning animals were identified only at two stations in Takamatsu. Mature animals were placed in laboratory tanks. These animals remained in good conditions for about two months, and many animals spontaneously spawned in the tanks. This is the first report of spontaneous spawning of B. belcheri in Japan.     

Sexual Maturation, Annual Reproductive Cycle, and Spawning Periodicity of the Shore Scorpionfish, Scorpaenodes littoralis

The ovarian structure, sexual maturation, annual reproductive cycle, and spawning periodicity of the shore scorpionfish, Scorpaenodes littoralis, in Uchiura Bay, central Japan, were examined using specimens collected between May 1995 and March 1998 and fishes reared in laboratory. The ovarian stroma and blood vessels run longitudinally through the center of each ovarian lobe. The ovarian peduncles radiate from the central stroma. During the spawning season, gelatinous material is secreted from the epithelia of both the ovarian peduncle and ovarian wall, and the epithelia show morphological changes accompanying the ovarian maturation cycle. The minimum standard length at maturity was 55.2mm for males and 40.2mm for females. Males with mature testes were collected from March to November. Females in the mature or post spawning stages were collected between May and October, when the mean gonadosomatic indices were also high. This indicates that the spawning season of this species occurs between May and October. Four successive types of oocytes were grouped in the mature ovary, comprised of mature, late and early vitellogenic and previtellogenic oocytes respectively, suggesting that this species is a multiple spawner. Four captive females spawned repeatedly at intervals of 2–8 days over a prolonged period (4–8 months); a 2-day spawning interval was the most common for all females. This suggests that female S. littoralis have a 48-h spawning cycle in captivity.     

Assessment of reproductive potential in multiple-spawning fish with indeterminate fecundity: a case study of yellow sea bream Dentex hypselosomus in the East China Sea

This study examined the spawning season, spawning frequency and batch fecundity of yellow sea bream Dentex hypselosomus in the East China Sea to reassess the previously reported reproductive characteristics of the species. Time-course sampling showed that this species had a diurnal ovarian maturation rhythm. Late tertiary yolk-stage oocytes appeared 2 days before spawning, starting the process of germinal vesicle movement and breakdown. On the day of spawning, ovulation and subsequent spawning occurred in the early morning (0400–0800 hours). Postovulatory follicles disappeared from the ovaries within c. 24 h of ovulation. Seasonal changes in the ovarian conditions indicated that this species spawned more or less throughout the year, with the peak ranging from spring to autumn. The compositions of the developing oocytes and degenerating postovulatory follicles in the ovaries suggested that most females spawned repeatedly over 2 to 3 consecutive days during the peak of the spawning season. Somatic body condition did not have a significant effect on batch fecundity, but there was a significant relationship between batch fecundity and fork length according to spawning status. Females spawning on consecutive days were more fecund than those spawning every other day. The findings show that this species has much greater reproductive potential than previously estimated.     

Spawning and maternal‐care behaviours of a copulating sculpin, Radulinopsis taranetzi

The fertilization mode, and spawning and egg‐care behaviours of the sculpin Radulinopsis taranetzi were investigated in the laboratory. Embryonic development began only after the eggs came into contact with sea water. Females spawned c . 1000 eggs and covered them with sand using their pectoral and caudal fins. Unlike other cottids, the females guarded the egg masses after spawning. During the parental period, the supramaxillary lamina and mandibular lamina of females extended to form a disc‐like structure, which was used to 'suck' water from near the surface of the egg mass. The frequency and duration of this 'sucking' behaviour increased gradually until hatching, which occurred after 23–26 days at 8° C. The oxygen consumption of the embryos was positively related to the 'sucking' activity. All females in this study spawned only once during the spawning season, in contrast with the paternal‐care copulating cottids, which are multiple spawners.     

Temporal and Spatial Trends in Spawning Aggregations of Camouflage Grouper, Epinephelus Polyphekadion, in Pohnpei, Micronesia

The aims of this investigation were to document the temporal and spatial dynamics of aggregated camouflage grouper, Epinephelus polyphekadion in Pohnpei, Micronesia, and to assess seasonal and daily patterns of spawning and reproductive behavior. Camouflage grouper aggregated and spawned adjacent to the Kehpara Marine Sanctuary (KMS), Pohnpei, Micronesia, during two consecutive months in 1998 and 1999. A combination of gonadosomatic indices, oocyte diameter analysis, and histological evidence confirmed that camouflage grouper spawned 1–2 days prior to full moon over an 8–9h period after dusk in each of the four study months. Males entered the site approximately 7 days prior to females and color variations were frequently observed. Significant differences were detected in mean size between the sexes. A series of management initiatives was initiated by the government in the 1980s to reduce fishing during aggregation periods, such as a March–April grouper sales ban, but these were largely ineffective. Following the 1998–1999 survey, several recommendations were made to halt aggregation fishing, including a commercial and subsistence catch and sales ban during spawning months, and the extension of the KMS to incorporate two additional grouper aggregations directly adjacent to it.     

Natural spawning,egg and fry production of milkfish,Chanos chanos (Forsskal), broodstock reared in concrete tanks1

Gonadal maturation, rematuration, and spawning of nine- (1982 stock) and ten-year-old (1981 stock) milkfish reared in 150 and 200 m³ concrete tanks were observed in 1990 and 1991. From 23 September to 9 November 1990, the 1981 stock spawned 15 times with an average daily collection of 61,000 ± 21,000 eggs. From 4 larval rearing trials, the mean overall survival rate (from eggs to 21 -day-old fry) was 9.68 ± 4.3%. From 25 Aril to 31 October 1991, the stock spawned a totaf of 108 times. Mean daily egg collection increasef to 305,000 ± 32,000. Successful rearing trials conducted during the latter part of the spawning season had a mean overall survival rate of 17 ± 3% (n = 17) indicating that mass fry production techniques can be improved. Spawning of milkfish broodstock in concrete tanks provides an alternative to other existing methods of seed production.     

Spawning frequency, fecundity, egg weight and spawning type of silver pomfret, Pampus argenteus (Euphrasen) (Stromateidae), in Kuwait waters

Silver pomfret, Pampus argenteus, were collected by fishing with drift gillnets on one spawning ground in Kuwait waters during 1998–2000. Fish size frequency, sex ratio, maturation cycle, spawning frequency, fecundity and egg weight were assessed. The length–weight relationship differed between sexes whereby females were significantly bigger than males. Spawning started in mid‐May and continued until early October. During this time the water temperature ranged from 26.0 to 32.8°C, salinity was ? 39.0‰ and water depth ranged between 5 and 12 m. Large females spawned earlier than young spawners and the overall percentage of males during the spawning period was 70.3%. Spawning occurred after 13.00 h, with peak spawning between 15.00 and 18.00 hours during outgoing tide. Mean daily spawning frequency amounted to 63.2%. Spawning activity was found to be associated with the lunar cycle and spawnings were concentrated during the first and third quarters of the moon period, indicating a semilunar reproduction cycle. It was concluded that a female would spawn at least six times during the season. No change was observed in relative fecundity during the peak spawning season (June–August). Average relative batch fecundity was 176.3 eggs g^?1 somatic weight (SW), corresponding to a relative total fecundity of 1058 eggs g^?1 SW, which is 1.5 times higher than estimates obtained from counting the standing stock of oocytes. Bigger fish produced heavier eggs and the egg weight decreased as the spawning season progressed. Based on gonadal cycles, oocyte size frequency distribution and total fecundity, we concluded that silver pomfret is a multiple batch spawner with indeterminate fecundity.     

Maturation, fecundity, and intertidal spawning of Pacific sand lance in the northern Gulf of Alaska

Pacific sand lance Ammodytes hexapterus in Kachemak Bay, Alaska, showed no sexual dimorphism in length-to-weight (gonad-free) ratio or length-at-age relationship. Most matured in their second year, males earlier in the season than females, but females (31%) attained a higher gonadosomatic index than males (21%). Sand lance spawned intertidally once each year in late September and October on fine gravel or sandy beaches soon after the seasonal peak in water temperatures. Sand lance in Cook Inlet and Prince William Sound displayed similar maturation schedules. Schools were dominated 2: 1 by males as they approached the intertidal zone at a site where spawning has taken place for decades. Sand lance spawned vigorously in dense formations, leaving scoured pits in beach sediments. Fecundity of females (93–199 mm) was proportional to length, ranging from 1468 to 16 081 ova per female. About half of the overall spawning school fecundity was derived from age group 1 females (55% of the school by number). Spawned eggs were 1·02 mm in diameter, demersal, slightly adhesive, and deposited in the intertidal just below the waterline. Sand lance embryos developed over 67 days through periods of intertidal exposure and sub-freezing air temperatures.     

Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis. This sperm motility-initiating activity was also found in the supernatant of seawater in which immotile sperm from the testis were incubated overnight. These suggest that in the amphioxus, a sperm motility-initiating substance resides in the sperm, and upon spawning, the substance is transformed into a free and active form to activate the sperm. Partial purification of the substance revealed it as a small and heat-stable substance with maximum UV absorbance at 234 nm.     

Reproductive ecology and early life history of a lacustrine sculpin, Cottus extensus (Teleostei, Cottidae)

Using a variety of sampling techniques and observations we describe aspects of the reproductive ecology and early life-history of Bear Lake sculpin, Cottus extensus, a species endemic to Bear Lake, Utah-Idaho. Adult sculpin spawned in shallow water (0.5–6.0 m depths) in cavities beneath large cobbles and boulders. During 1993, egg mass densities were highest (> 4.0 m²) at 1.0–2.0 m depths. Electivity indices verified substrate selection and also indicated an avoidance of s and- and gravel-embedded materials. During years of low water elevation, suitable spawning substrates were restricted to one or two limited areas of the lake and comprised < 0.004% of the total benthic area. Disturbance from turbulence, as would be produced by storm-induced waves, appeared to initiate hatching. After hatching, embryos passed through a pelagic interval that lasted for approximately one week. Laboratory experiments confirmed the short duration of this surface-swimming behavior. The pelagic behavior of free embryos appears to aid in dispersal of fish via currents from limited spawning habitats to areas throughout the 282 km² lake.