Amphioxus spawning behavior in an artificial seawater facility

Amphioxus Spawning Behaviorin an Artificial Seawater FacilityMARIA THEODOSIOU1, AUDREY COLIN2, JASMIN SCHULZ1,VINCENT LAUDET1, NADINE PEYRIERAS2,JEAN-FRANC-OIS NICOLAS3, MICHAEL SCHUBERT1�,AND ESTELLE HIRSINGER3�1Institut de Genomique Fonctionnelle de Lyon, Universite de Lyon (Universite Lyon 1, CNRSUMR5242, INRA 1288, Ecole Normale Superieure de Lyon), Lyon, France

2CNRS-NED, Institut de Neurobiologie Alfred Fessard, Gif-sur-Yvette, France3Department of Developmental Biology (CNRS URA2578), Institut Pasteur, Paris, France

Owing to its phylogenetic position at the base of the chordates, the cephalochordate amphioxus isan emerging model system carrying immense significance for understanding the evolution ofvertebrate development. One important shortcoming of amphioxus as a model organism has beenthe unavailability of animal husbandry protocols to maintain amphioxus adults away from thefield. Here, we present the first report of successful maintenance and spawning of Branchiostomalanceolatum adults in a facility run on artificial seawater. B. lanceolatum has been chosen for thisstudy because it is the only amphioxus species that can be induced to spawn. We provide a step-by-step guide for the assembly of such a facility and discuss the day-to-day operations required forsuccessful animal husbandry of B. lanceolatum adults. This work also includes a detaileddescription of the B. lanceolatum spawning behavior in captivity. Our analysis shows that theinduced spawning efficiency is not sex biased, but increases as the natural spawning seasonprogresses. We find that a minor fraction of the animals undergo phases of spontaneous spawningin the tanks and that this behavior is not affected by the treatment used to induce spawning.Moreover, the induced spawning efficiency is not discernibly correlated with spontaneousspawning in the facility. Last, we describe a protocol for long-term cryopreservation ofB. lanceolatum sperm. Taken together, this work represents an important step toward furtherestablishing amphioxus as a laboratory animal making it more amenable to experimental research,and hence assists the coming of age of this emerging model. J. Exp. Zool. (Mol. Dev. Evol.)316:263–275, 2011. & 2011 Wiley-Liss, Inc.

How to cite this article: Theodosiou M, Colin A, Schulz J, Laudet V, Peyrieras N, Nicolas J-F,Schubert M, Hirsinger E. 2011. Amphioxus spawning behavior in an artificial seawater facility. J.Exp. Zool. (Mol. Dev. Evol.) 316:263–275.

The invertebrate chordate amphioxus (phylum Chordata, sub-

phylum Cephalochordata, genus Branchiostoma, also referred to

as lancelet) is a marine filter feeder usually found in shallow,

sandy habitats of temperate and tropical seas (Yu and Holland,

2009). It is now widely accepted that, within the chordates,

amphioxus takes up the basal position and that the other

invertebrate chordate group, the tunicates, are the sister taxon

Published online 26 January 2011 in Wiley Online Library (wileyonline

library.com). DOI: 10.1002/jez.b.21397

Received 30 September 2010; Accepted 30 November 2010

Grant Sponsors: CNRS; Institut Pasteur; ANR (ANR-07-BLAN-0038; ANR-09-

BLAN-0262-02); EU FP6 NEST-Strep Embryomics; CRESCENDO, a European

integrated project of FP6.

Additional Supporting Information may be found in the online version of this

article.

Current address for Jasmin Schulz: Ludwig Institute for Cancer Research,

University of Oxford, Headington, Oxford OX3 7DQ, United Kingdom.

�Correspondence to: Michael Schubert, Institut de Genomique Fonction-

nelle de Lyon, Ecole Normale Superieure de Lyon, 46 allee d’Italie, 69364

Lyon Cedex 07, France. E-mail: [email protected] or�Correspondence to: Estelle Hirsinger, Unit of Molecular Biology of

Development, Department of Developmental Biology, Institut Pasteur, 25 rue

du Dr. Roux, 75015 Paris, France. E-mail: [email protected]

ABSTRACT

J. Exp. Zool.(Mol. Dev. Evol.)316:263–275, 2011

& 2011 WILEY-LISS, INC.

RESEARCH ARTICLE

of the vertebrates (Holland et al., 2008; Putnam et al., 2008).

Amphioxus and vertebrates share the so-called chordate features,

which are a dorsal, hollow nerve cord, a notochord, segmented

musculature, and pharyngeal gill slits. However, amphioxus lacks

some vertebrate-specific characters, such as definitive neural

crest and placodes (Holland and Yu, 2004; Schubert et al., 2006).

Moreover, the amphioxus and vertebrate genomes are also

organized similarly, although that of amphioxus has the

advantage of relatively little duplication (Schubert et al., 2006;

Holland et al., 2008; Putnam et al., 2008). This structural and

genomic simplicity in a vertebrate-like organism means that

amphioxus is of primordial importance for understanding the

evolution of the vertebrate body plan and genome from an

invertebrate chordate ancestor.

Owing to its phylogenetic position at the base of the

chordates, the cephalochordate amphioxus is thus a key

emerging model system to understand the evolution of vertebrate

development. For this reason, it is crucial to make this animal

more amenable to experimental biology and readily accessible to

the scientific community. The aim of this study was the

development of an amphioxus husbandry protocol leading to

the set-up of an artificial seawater (ASW)-based facility that does

not rely on proximity to the sea and allows for daily experiments.

Experimental studies on amphioxus have chiefly been based on

four different species: Branchiostoma lanceolatum (the European

amphioxus), Branchiostoma belcheri (the Chinese amphioxus),

Branchiostoma japonicum (the Japanese amphioxus), and Bran-

chiostoma floridae (the Florida amphioxus) (Yu and Holland, 2009).

The sexually mature adults of these four species all reproduce during

the summer months of each year, and the embryology of these four

species seems almost identical except for the optimal rearing

temperatures of the embryos and larvae (Yu and Holland, 2009).

The very first description of amphioxus embryology was

carried out by Kowalevsky in the second half of the 19th century

using B. lanceolatum (Kowalevsky, 1867), which was followed by

various other studies on B. lanceolatum development that

highlighted the importance of amphioxus for understanding

chordate evolution (Willey, 1891; Hatschek, 1893; Wilson, 1893;

Cerfontaine, ’06; Conklin, ’32). However, in the absence of an

amphioxus laboratory culture, the disappearance of the acces-

sible B. lanceolatum population in Naples, Italy, led to a stark

decline in developmental studies on amphioxus. In East Asia, the

amphioxus populations of B. belcheri and B. japonicum were first

used for developmental studies in the late 1950s (Tung et al., ’58;

Hirakow and Kajita, ’90). Research activity on developing

B. floridae really commenced only in the late 1980s, when the

first methods to induce spawning of ripe B. floridae adults in a

laboratory environment were developed (Holland and Holland,

’89). Although this electroshock-based protocol only triggers the

release of gonads on days the ripe amphioxus also spawn in the

field, its publication led to a veritable renaissance of amphioxus

as a model organism, with B. floridae as the species being most

commonly used by the research community. It is, hence, not

surprising that the first amphioxus genome sequenced was that

of B. floridae (Holland et al., 2008; Putnam et al., 2008).

The existing amphioxus facilities are located near the sea and/

or use natural seawater. Under these conditions, B. belcheri can be

maintained with natural seawater even in the absence of sand as

burying substratum (Yasui et al., 2007) and, in a natural seawater

system with sand, both B. belcheri and B. japonicum have been

successfully raised through two generations in a laboratory

environment (Wu et al., ’94; Zhang et al., 2007). Spontaneous

spawning of both B. belcheri and B. japonicum in the laboratory

has been reported, but it is currently impossible to induce

spawning in these two amphioxus species (Wu et al., ’94; Zhang

et al., ’99, 2001, 2007; Mizuta and Kubokawa, 2004; Yasui et al.,

2007). The only amphioxus species that can currently be induced

to spawn on any given day during the breeding season is

B. lanceolatum. A thermal stimulation (i.e. a temperature increase

from 181C to 231C) induces spawning after 36hr (Fuentes et al.,

2004, 2007). Similar to the Chinese and Japanese species,

B. lanceolatum can be maintained in a natural seawater-based

facility (Fuentes et al., 2004, 2007). Moreover, new aquaculture

techniques using natural seawater have been developed for

B. lanceolatum (Somorjai et al., 2008; Garcia-Fernandez et al., 2009).

This relative ease of use of B. lanceolatum makes this

amphioxus species a model of choice to develop a system that

allows the long-term culture of amphioxus in laboratories

without access to natural seawater. Here, we report the

maintenance and spawning behavior of B. lanceolatum in a

facility based on ASW and provide a protocol for long-term

cryopreservation of B. lanceolatum sperm.

MATERIALS AND METHODS

Aquarium Systems

We have set up comparable aquarium systems (Muller & Pfleger

GmbH & Co. KG, Rockenhausen, Germany) in Gif-sur-Yvette,

France, (system A) and in Lyon, France, (system B) utilizing ASW.

Deionized water is kept in a 300 L reservoir tank, which is

connected to a second 300 L tank, in which the ASW is prepared

(Fig. 1A). The ASW tank is connected to a pump used both for

providing the aquarium systems with ASW and for mixing

deionized water with salt crystals to prepare the ASW.

To make ASW, deionized water is mixed with salt crystals

(Sea Reef Crystals, Europrix Aquaria, Lens, France) to a final

salt concentration of 38g/L. To buffer the pH, the ASW is

supplemented with NaHCO3 (to a final concentration 250mM).

Deionized water and salt crystals are mixed for at least 1 hr and are

subsequently allowed to cool to facility temperature (181C) for 1hr

before entering the aquarium systems. The chemical composition

of the ASW is similar to that of the natural seawater in Argeles-sur-

Mer, France, where amphioxus adults are collected (Table 1).

THEODOSIOU ET AL.264

J. Exp. Zool. (Mol. Dev. Evol.)

Figure 1. Aquarium systems. (A) General description of the aquarium system setup. Pump 1 assists in the mixing of salt and deionized water

and also pumps the artificial seawater (ASW) into the lower reservoir (LR). Pump 2 brings water to the upper reservoir (UR), from where it is

distributed to the individual tanks. In system A, pump 3 serves to pump water in a time-dependent fashion into a separate compartment of the

upper reservoir, from where the water is then distributed to the individual tanks allowing for intermittent water distribution (a). In system B, this

pump is not utilized, allowing for constant water dripping (b). Water having passed through the individual tanks is eliminated into the drain,

although alternatively both systems can be converted to a closed configuration, in which water eliminated from the individual tanks is collected

in the lower reservoir and recycled through the system (�). (B) Individual tank within the aquarium system. Water is flowing out of the tank

through the slotted black disk, which prevents amphioxus adults from escaping the tank. (C) General layout and arrangement of individual tanks

and the light system within the amphioxus facility. (D) Light system-equipped incubator for thermal shock of amphioxus adults.

Table 1. Chemical analysis of artificial seawater.

Ca21 (mg/L) Mg21 (mg/L) NO�3 (mg/L) NO�2 (mg/L) Hardness (dGH) Alkalinity (dKH) pH

Cl2(mg/L)

Argeles-sur-Mer seawater 400 1,100 10 0 8 6 8 0

Artificial seawater (ASW) 405 1,008 10 0 8 6 8 0

AMPHIOXUS LABORATORY CULTURE 265


The aquarium system consists of an upper and lower water

reservoir. The ASW enters the system at the bottom reservoir and

subsequently passes through four filter sponges before being

pumped up to the top reservoir, which contains a trickle filter

system and a UV light for sterilization. From the top reservoir, the

ASW is distributed to the individual 2.7 L tanks. Our systems are

flexible in that they can be operated under two configurations.

In an open configuration, water runs through the individual

tanks and is subsequently evacuated (Fig. 1A). Water distribution

to the individual tanks is intermittent in system A or continuous

in system B. In system A, water is pumped in a time-controlled

fashion into a separate compartment before being distributed

to the tanks: a timer activates pump 3 for 1 min three times a day

to exchange 1 L of water per aquarium tank (hence, renewing 3 L

of ASW per day in each tank). In system B, pump 3 is not used

and water constantly drips through the individual tanks with a

rate of about 0.1 L/hr/tank (hence, exchanging about 2.4 L of

ASW per day in each tank). Both systems can also be converted to

a closed configuration, in which the water coming out from the

individual tanks flows into the bottom reservoir before it is

pumped back up into the top reservoir, where it is sterilized

by the UV light and then redistributed to the individual tanks

(Fig. 1A).

In system A, light is provided by neon lamps as well as by

blue and pink neon lights (Coralstar and Aquastar bulbs,

Sylvania, Europrix Aquaria, Lens, France) that mimic the light

spectrum at 9 m depth, where the amphioxus are found.

In system B, light is provided by neon lights and the light intensity

is controlled by the Profilux II computer system (GHL GmbH &

CoKG, Kaiserslautern, Germany). To mimic dusk, light is turned off

gradually over a period of 30min, and similarly, to mimic dawn,

the neon light intensity increases to 10% of its maximum capacity

over a 15min period. The light intensity at different points of each

level in the aquarium system was measured with a light meter

(Kato Electronics, Los Angeles) (Table 2).

Sand

Sand was collected from Argeles-sur-Mer, France. On reception

in the facilities, the sand was extensively washed with fresh

water. The sand was left to dry and was either autoclaved

(system A) or dry heated for 4 hr (system B).

Aquarium Tanks

Our aquarium system utilizes tanks with dimensions of

240 mm� 160 mm� 145mm that accommodate a total volume

of 2.7 L and about 15 amphioxus adults (Fig. 1B). A layer of

approximately 1 cm of washed and sterilized sand from

Argeles-sur-Mer, France, covers the bottom of each tank. Air is

constantly blown through a stone diffuser into the water in the

aquaria to ensure adequate oxygenation (Fig. 1B and C).

Algal Cultures and Amphioxus Feeding

The amphioxus were either fed with algae (system A) or with the

nutrient solution Preis-Microplan (system B) (Europrix Aquaria,

Lens, France). Starter algal cultures of Dunaliella tertiolecta,

Isochrysis galbana, and Tetraselmis suecica were a generous gift

from Michael Fuentes and Hector Escriva (Laboratoire Arago,

Banyuls-sur-Mer, France). Algae were grown in F/2 marine water

enrichment solution (Guillard and Ryther, ’62). For feeding, equal

volumes of each alga were mixed, centrifuged (4,000 rpm, 181C,

12min), and subsequently resuspended in ASW before being

distributed to the tanks. In the case of animals with full gonads, the

equivalent of 3mL of each algal culture (at a concentration of

1,500–5,000 cells/mL) was distributed to each tank twice a day. In

the case of animals with empty gonads, in an attempt to refill the

gonads, the equivalent of 6mL of each algal culture (at a

concentration of 1,500–5,000 cells/mL) was distributed in each

tank three times a day. For feeding amphioxus adults in system B,

Preis-Microplan food was diluted in ASW, following the instruc-

tions of the manufacturer (two drops in 25mL), and 1.5mL of the

diluted food was distributed to each tank twice a week.

Transport of Adult Amphioxus

Sexually mature amphioxus adults are collected by dredging at

Argeles-sur-Mer, France, and retrieved from the sand with a sieve

(Fuentes et al., 2004, 2007). In order to limit stress to the animals,

the amphioxus are then transported in 3 L plastic sleeves.

Each sleeve contains 1 L of rinsed sand from Argeles-sur-Mer,

France, 1 L of filtered natural seawater, 1 L of compressed air, and

15 animals. The sleeves are transported in a battery-run

Table 2. Light intensity measurements.

Points Light intensity (lx)

1 330

2 360

3 360

4 330

5 390

6 330

7 400

8* 2

9* 2

10* 2

* = lamp turned off



temperature-controlled cool box (CoolFreeze T32, Waeco, Hoeco

Handels GmbH, Austria) to ensure a transportation temperature

of about 181C. Upon their arrival in the facility, the animals are

transferred into their respective tanks (one sleeve per tank)

containing a 1:1 mix of filtered natural seawater (brought along

from Banyuls-sur-Mer, France) and ASW, as well as a 1 cm layer

of autoclaved sand. The tanks are then integrated into the

aquarium system to allow for gradual water changes, feeding

(system A and B), and antibiotic treatment (kanamycin at a final

concentration of 10mg/mL) (system A). Each tank is, hence,

initially populated with 15 mature amphioxus adults (males and

females are kept together) with gonads at gonadal stages 3 and 4

as defined in Fuentes et al. (2004). The animals are subjected to a

spring-like day/night light period (14 hr of light/10hr of absolute

darkness) in an inversed illumination cycle, where the lights turn

on at 23:00hr and off at 13:00 hr.

Thermal Shock

A thermal shock (from 181C to 231C) was applied, as previously

described (Fuentes et al., 2004, 2007). Males and females were

identified and kept in separate tanks for at least 3 days before the

thermal shock. To induce spawning, the animals were transferred

into tanks containing a reduced amount of sand (barely covering

the bottom) and approximately 1 L of ASW, fed with Preis-

Microplan food (system B), and placed in an incubator at 231C

(Fig. 1D). The incubator is equipped with a light system that

reproduces the light cycle of the amphioxus facility. The

procedure was initiated before 13:00 hr of day n and spawning

occurred after 13:00 hr of day n11. The animals, hence, spent

one full night/day cycle at the elevated temperature. Just before

13:00 hr of day n11, the animals were put into individual clear

plastic cups with approximately 10mL of filtered ASW at 181C to

prepare for spawning. Animals that did not spawn after thermal

shock were subjected to an additional cycle of thermal shock only

after a recovery period of at least 1 week.

Sperm Freeze

We have devised a method for the conservation of B. lanceolatum

sperm, which is significantly different from the protocol for

preserving B. belcheri sperm (Xu et al., 2009). Between 100mL

and 200mL of concentrated sperm is gradually and gently mixed

with an equal volume of cryopreservation solution made up in

ASW (final concentration: 100 mM Tris-HCl pH 8.0, 10% egg

yolk, and 10% DMSO). The cryotubes are then placed into a

cryocontainer (Nalgene, Thermo Fischer Scientific, New York),

which allows the gradual freezing of the sperm at a cooling rate

of �11C/min, when placed at �801C. Frozen sperm is stored at

�801C until used. For fertilization, sperm is thawed quickly at

ambient laboratory temperature (231C) and used sparingly (one to

two drops of sperm per 30mL egg culture, which amounts to a

sperm to ASW volume ratio of about 1:600) without prior

dilution. Once thawed, a given batch of sperm must be used

rapidly and cannot be refrozen. In general, it is best to use highly

concentrated sperm directly collected from a spawning male for

cryopreservation. However, it is possible to concentrate diluted

sperm before cryopreservation by centrifuging at 700 rpm for

90min at 41C.

Data Analysis and Presentation

Spawning and fertilization data were collected over the course of

the 2008 and 2009 amphioxus spawning seasons, beginning with

the arrival of the animals in the aquarium facility. In addition,

animal survival was assessed throughout and beyond the end of

the reproductive period. The raw data is provided in the

supplementary material (Table S1). Statistical analyses of the

data were carried out using Microsoft Excel 2004 for Mac

(Microsoft Corporation, Seattle) and the software platform R

version 2.10.1 (R Development Core Team, 2009).

Time-Lapse of Sperm Motility and Egg Fertilization

Sperm motility and egg fertilization were observed and captured

using a Zeiss Axiovert 100M microscope allowing time-lapse

analyses. To examine sperm motility, fresh and frozen sperm

(conserved for a year at �801C) were diluted in ASW (ten-fold

and five-fold, respectively). A drop of each of these diluted sperm

samples was mounted on a glass slide and time-lapsed for 30 sec

under the microscope with an image capture rate of 10 images

per second. Eggs were carefully pipetted into 2mL of ASW in a

six-well tissue culture plate (Falcon, BD biosciences, Le Pont de

Claix, France) coated with 1% low melting point agarose

(prepared with ASW). The eggs were then fertilized with either

freshly spawned or cryopreserved sperm. Fertilization and sub-

sequent development was time-lapsed using an image capture

rate of 2 images per minute. Movies were subsequently encoded

using both the Metamorph (MDS Analytical Technologies,

Toronto, Canada) and ImageJ (Abramoff et al., 2004) software

packages.

RESULTS AND DISCUSSION

Spawning is Obtained From Amphioxus Maintainedin an ASW-Based Facility

Work on living amphioxus has historically been carried out in

natural seawater in laboratories at or near the sea (Fuentes et al.,

2004, 2007; Garcia-Fernandez et al., 2009; Yu and Holland,

2009). In this report, we show that sexually mature amphioxus

adults can be cultured and induced to spawn in ASW. These

studies were carried out in parallel with two comparable

aquarium systems at the CNRS-NED in Gif-sur-Yvette, France,

(system A) and at the Institut de Genomique Fonctionnelle de

Lyon (IGFL) in Lyon, France, (system B).

The aquarium systems can be run in two different configura-

tions: an open system, where water is eliminated after one

passage through the individual tanks, or a closed system, where



the water that passes through the tanks is recycled (Fig. 1A).

We found that the closed system is associated with higher

mortality (data not shown). Therefore, although the closed

configuration is characterized by a significantly lower ASW

consumption, we decided to use the open configuration. Possible

explanations for this increased mortality include an increase in

salinity of the ASW, owing to evaporation, or an inefficient

sterilization of the recirculating water, which subsequently led to

cross-contamination of the tanks with disease-causing microbes.

Open systems can be run in one of two configurations:

intermittent water changes or constant drip, both leading to the

renewal of the entire volume of each tank per day. System A is

run with intermittent water changes, where 1 L of ASW is renewed

three times a day (Fig. 1A). In addition, antibiotics (kanamycin at

a final concentration of 10mg/mL) are added to the tanks after

each water change. System B is run with constant drip with a drip

rate of about 0.1L/hr/tank and no antibiotic is added (Fig. 1A).

Following the established protocols (Stach, ’99; Fuentes et al.,

2004, 2007), induced spawning of sexually mature amphioxus

adults was successfully obtained in both systems and traced over

the 2008 and 2009 reproductive periods (for system A) and for

the 2009 reproductive period (for system B). The average

spawning percentages in system A were 49.7% in 2008 and

30.3% in 2009, whereas in system B the average spawning

success in 2009 was 52.6% (Table 3). In system A, a subset of

shocked animals that did not spawn was successfully induced to

spawn, if reshocked. In system B, the health of shocked animals

deteriorated markedly and these animals were, therefore, not

subjected to additional thermal shocks. In general, reshocked

animals do not spawn as well as animals shocked for the first

time, the reshocked spawning percentage being 17.0% in 2008

and 15.7% in 2009 (Table 3).

Previous reports have suggested that long periods of

starvation result in the resorption of gonads in sexually mature

amphioxus (Fuentes et al., 2004). Moreover, it has been shown

that, when kept in tanks with natural seawater and fed with a diet

consisting of algae and homogenized artificial Tetra Microfood

food, spawned out B. lanceolatum adults can redevelop gametes,

and hence refill their gonads during the spawning season

(Fuentes et al., 2004). The animals in our facilities were on a

feeding regime of either algae (system A) or artificial Preis-

Microplan food (system B). Although we did not observe any

resorption of gonads in our facilities, we also did not find

evidence for gonadal refill in animals that had spawned out either

spontaneously or upon a thermal shock (data not shown).

These data suggest that the feeding regimes employed in our

systems, albeit sufficient for maintaining a given gonadal status,

are insufficient for gonadal refill. It remains to be established

whether a combination of algae and artificial food would lead to

redevelopment of gonads in empty adults in our facilities or

whether other factors, such as environmental cues, regulate

gonadal refilling of B. lanceolatum. The latter hypothesis has also

been put forth in an earlier report (Fuentes et al., 2004), which

points out that factors other than food are likely to play a role in

the accumulation of gonads in this amphioxus species.

Animal Health is Stable in an ASW-Based Facility

The health of the animals was monitored from their arrival in the

facilities over the 2009 amphioxus spawning season and beyond

(Fig. 2). At regular intervals, any dying animal identified in the

tanks was removed to prevent disease spreading to the rest of the

animals in the same tank. The percentages of dying animals were

plotted as a function of time (Fig. 2).

In system A, each animal collection as well as the population

of animals already subjected to a thermal shock or with the

nutrient solution was plotted separately (Fig. 2A). For each

collection (except the March 28 collection, red curve), the

percentage of dying animals increased for about two weeks and

then stabilized. We, hence, find that after an initial adjustment

period of approximately 2 weeks, animal health is stable under

the artificial conditions created in our facility (ASW, inverted

light cycle, a steady water temperature of 181C, and feeding

regimes based on algae). A similar adjustment period of 2 weeks

was observed for the animal collections of the 2008 season (data

not shown).

The final percentages of dead animals in the different

populations ranged from 5 to 22%, suggesting that health varies

significantly between animal collections. We also observe that for

animals collected closer to the natural spawning season (May

through early July) the final percentage of dying animals is higher.

For the reshocked population (purple curve; Fig. 2A), animals

exhibit mortality during the entire induced spawning season with

the percentage of dying animals reaching a plateau of about 10%

in August (after the end of the spawning season), which is within

the range of mortality for animals shocked only once.

For system B, the collections were not tracked separately, but

rather as one population and plotted as such (Fig. 2B). As can be

observed in the graph, the percentage of dying animals steadily

increases in May, June, and July, which correlates with the

amphioxus spawning season, and hence with the period of

regular thermal shocks, and reaches a plateau in August. The

Table 3. Efficiency of induced spawning during the amphioxus

reproductive season. The spawning percentage is the total number

of spawning animals over the total number of shocked animals.

2008 2009

System A System B System A System B

First shock 49.7% NA 30.3% 52.6%

Reshock 17.0% NA 15.7% ND

NA, Not applicable; ND, Not determined.



overall death rate in system B was significantly higher than that

in system A. However, the death rates in the two systems are

difficult to compare: the animal collections were pooled in

system B, the survey of dying animals in system B was not as

frequent as in system A, and antibiotics were used in system A.

Semi-Synchronized Pulses of Spontaneous Spawning Occurin the ASW Facility

It is well established that thermal shock can induce spawning in

B. lanceolatum adults (Fuentes et al., 2004, 2007). Nonetheless,

by assaying the presence of animals with empty gonads in the

four different animal collections plus the reshocked population

of system A, we found that, even when kept under steady

temperature and environmental conditions, a subset of the

animals spawn spontaneously and in semi-synchronized waves

from mid-April to mid-July (Fig. 3A). During the 2009

reproductive period, we observed an initial spontaneous

spawning period (April 18–April 22), which was limited to a

single population, and four additional periods of spontaneous

spawning characterized by spawning of at least two out of the

five animal populations for three or more days (May 11–May 13,

May 19–May 23, May 28–June 4, June 18–July 20). Based on

these observations, the 2009 reproductive season was divided

into ten periods, each of them corresponding to either a period of

substantial (periods 2, 4, 6, 8, and 10) or negligible (periods 1, 3,

5, 7, and 9) spontaneous spawning activity (Fig. 3B and C).

It is important to note that the reshocked animals follow the

spontaneous spawning pattern of the four animal collections,

suggesting that these spontaneous reproductive events are not

affected by thermal shocks. The synchronicity of these sponta-

neous spawning events in captivity is very intriguing and it

would certainly be interesting to correlate this behavior with the

natural spawning patterns in the field, the dynamics of which

vary significantly between spawning seasons (Fuentes et al.,

2004, 2007). Moreover, it remains to be established whether this

spontaneous spawning behavior in captivity can be reduced or

even prevented by alteration of the physical parameters of the

ASW facility, for example, by lowering the water temperature or

by altering the ambient light or feeding regime.

Induced Spawning Efficiency is Not Sex Biased, But Increases as theNatural Spawning Season Progresses

Based on our monitoring of animal health, after their arrival in

the animal facility the amphioxus adults were allowed to adapt

for at least 2 weeks before being shocked. Thermal shocks were

initiated in mid-April to assess when spawning can be induced

with this method. The spawning efficiencies of males and females

were recorded for both shocked and reshocked animals during

the 2009 season (Fig. 4A and B). The percentage of spawning

males (in blue) or females (in red) is plotted for each induced

spawning date. Although shocked males began to spawn on

April 18, shocked females did not begin to spawn until the

beginning of May (May 5). The date, on which both males and

females spawned (May 5), was defined as the beginning of the

induced spawning season (Fig. 4B). Using thermal shock,

spawning could be induced in both males and females until late

July (July 22). This induced spawning season thus exceeds the

natural spawning season in the sea that typically starts mid-May

and ends in early July (Fuentes et al., 2004, 2007).

We next analyzed, whether there is a sex bias in the efficiency

of induced spawning. For each of the four collections, the

average percentage of spawning males and females was

calculated for the totality of the spawning season (Fig. 5). We

find that there is no statistical difference in the spawning

efficiency of shocked males vs. shocked females for the March 20,

Figure 2. Animal health in captivity. (A) System A. The percentage

of dying animals is the cumulative number of dying animals on day

n over the maximal number of animals in a given population.

The four separate collections (March 20 in blue, March 28 in red,

April 24 in orange, and June 12 in green) and the reshocked

animals (in purple) are indicated. (B) System B. The three separate

collections are treated as one population. The percentage of dying

animals is the cumulative number of dying animals on day n over

the total number of collected animals.



April 24, and June 12 collections, as assessed by two-tailed

chi-squared and two-tailed Fisher’s exact test (P 5 0.8594

w2 5 0.031 and P 5 1.0000, P 5 0.4869 w2 5 0.483 and

P 5 0.3754, P 5 0.8426 w2 5 0.039 and P 5 1.0000, respectively).

In contrast, the population collected on March 28 exhibits

weak statistical support for males spawning more efficiently

than females (P 5 0.0627 w2 5 3.461 and P 5 0.0401). Unlike

animals shocked for the first time (inset in Fig. 4A), reshocked

males spawn at a higher frequency than reshocked females

(inset in Fig. 4B). This increased spawning performance is

statistically significant only for the reshocked population,

as assessed by two-tailed chi-squared and two-tailed Fisher’s

Figure 3. Spontaneous vs. induced spawning in captivity in system A. (A) Spontaneous spawning. The four separate collections (March 20 in

blue, March 28 in red, April 24 in orange, and June 12 in green) and the reshocked animals (in purple) are shown. The percentage of

spontaneous spawning is the cumulative number of empty animals on day n over the maximal number of animals in a given population.

(B) Characteristics of the ten periods. For each period, dates, length, and occurrence of substantial (1) vs. negligible (�) spontaneous spawning

are indicated. (C) Induced spawning. For each period, the induced spawning percentage is the total number of spawning animals over the total

number of shocked animals in a given period. The asterisk indicates statistically significant differences between the successive periods.



exact tests (P 5 0.4487 w2 5 0.574 and P 5 0.3965 for the

first shocks vs. P 5 0.0248 w2 5 5.036 and P 5 0.0262 for the

reshocks).

We next tested, whether there is heterogeneity in the

efficiency of induced spawning between the different animal

collections (males plus females) (Fig. 5). We found that the

Figure 4. Induced spawning throughout the reproductive period in system A. (A) Induced spawning percentages of males (blue) and females

(red) subjected to a single thermal shock over the course of the reproductive period. For each date, the spawning percentage is the number of

spawning animals (males or females, respectively) over the total number of shocked animals (males or females, respectively). The inset gives

the cumulative spawning percentages of males (blue) and females (red) for the entire induced spawning season. (B) Induced spawning

percentages of reshocked males (blue) and females (red) over the course of the reproductive period. For each date, the spawning percentage

is the number of spawning animals (males or females, respectively) over the total number of shocked animals (males or females, respectively).

The inset gives the cumulative spawning percentages of reshocked males (blue) and females (red) for the entire induced spawning season.

The asterisk indicates a statistically significant difference in spawning efficiency between reshocked males and females.



spawning percentages of the March 20 (animals shocked between

April 15 and July 22) and the March 28 collections (animals

shocked between April 23 and July 22) were not statistically

different (P 5 0.2398 w2 5 1.382 and P 5 0.2273). This was also

the case for the April 24 (animals shocked between May 29

and June 23) and June 12 (animals shocked between June 21 and

July 9) collections (P 5 0.7043 w2 5 0.144 and P 5 0.6498).

However, the induced spawning efficiency of the combined first

two collections (March 20 and March 28) was significantly lower

than that of the combined last two collections (April 24 and

June 12) (P 5 0.0058 w2 5 7.607 and P 5 0.0054). There is, thus, a

marked increase of the induced reproductive performance

between the first two and the last two animal collections.

Two hypotheses may account for this observation:

(i) Although spawning can be induced by thermal shock before

the beginning and after the end of the natural spawning season,

the efficiency of the induction may be dependent on the date

of the animal collection relative to the timing of the natural

spawning season. Thus, the difference in spawning performance

between the early and the late cohort may be due to an

environmental sexual maturation cue received in the field.

(ii) Alternatively, because animals from successive collections are

shocked at progressively later times in the spawning season, this

difference may be due to an intrinsic maturation mechanism that

makes amphioxus adults more amenable for spawning as

the reproductive period progresses. A visible readout of this

maturation might be the continuous production of gametes

during the natural spawning season, a process that has previously

been described both in the field and in captivity (Fuentes et al.,

2004, 2007).

Efficiency of Induced Spawning Does Not Correlate With Periodsof Spontaneous Spawning

After describing both spontaneous and induced spawning

dynamics of adult amphioxus in laboratory culture, we set out

to test whether the spontaneous spawning periods correlate with

phases of increased induced spawning efficiency. We grouped the

induced spawning data from the four animal collections plus the

reshocked population, and subdivided this pooled data set into

the ten periods defined on the basis of the spontaneous spawning

activity in the facility (Fig. 3B and C).

Statistical cross-comparisons (using two-tailed chi-squared

and two-tailed Fisher’s exact tests) of the induced spawning

efficiency between successive periods revealed that only a single

transition is statistically relevant (P 5 0.0224 w2 5 5.216 and

P 5 0.0176): the decrease of spawning efficiency between periods

nine and ten (June 5–June 17 and June 18–July 20). Interestingly

enough, whereas period nine, a time interval devoid of any

significant spontaneous spawning activity, is characterized by one

of the highest induced spawning percentages (37.1%), period ten,

which is marked by substantial spontaneous spawning, exhibits

an induced spawning efficiency of only 23.6%. Induced spawning

efficiency during periods of spontaneous spawning (37.5, 34.3,

and 30.9% in periods 4, 6, and 8, respectively) seems higher than

that during periods without spontaneous spawning (19.2, 20.9,

and 15.6% in periods 3, 5, and 7, respectively). However, these

fluctuations are not statistically significant (data not shown). This

failure of statistical support suggests that the efficiency of induced

spawning is not correlated with spontaneous spawning in

captivity. The significant decrease in induced spawning efficiency

between periods nine and ten corresponds to the end of the

induced spawning season, as previously defined (Fig. 4).

Males Tend to Spawn Before Females After Thermal Shock

We have also monitored the dynamics of male and female

spawning after thermal shock. Because animals from the four

different collections as well as the reshocked population exhibit

similar behavior on days of spawning (data not shown), we were

able to pool the individual data sets to assess the overall

spawning dynamics of the animals after thermal shock (Fig. 6).

By plotting (in 30min intervals) the spawning percentages of

males and females in the course of a spawning session, we found

that males generally start spawning earlier than females. Males

show a peak of spawning activity around 2 hr after the lights

Figure 5. Comparison of spawning efficiencies between animal

collections in system A. For each collection, males (blue) and

females (red) are shown separately. The spawning percentage

indicated is the number of spawning animals (males or females,

respectively) over the total number of shocked animals (males or

females, respectively). The asterisk indicates a statistically

significant difference in spawning efficiency between the March

20/March 28 and the April 24/June 12 cohorts.



have gone off, whereas females commence spawning shortly

thereafter and over a longer period of time (1–5hr after dark).

Our analysis also indicates that a small percentage of males and

females continue to spawn more than 5 hr after dark (Fig. 6).

A similar behavior was observed during the 2008 spawning

season (data not shown). These spawning dynamics are in partial

disagreement with a previous report suggesting that amphioxus

adults subjected to thermal shock spawn within 2 hr after dark

(Fuentes et al., 2007). However, this discrepancy might be due to

differences in the experimental setup used to determine the

spawning behavior after thermal shock. Whereas our animals

were subjected to artificial light conditions and spawning was

monitored in 10mL of filtered ASW in plastic cups, Fuentes et al.

(2007) used natural light and natural seawater and filmed the

spawning adults with an infrared camera in 12.5 cm2 plastic

bottles associating spawning behavior with locomotory behavior

of the amphioxus adults.

85% of Females and 70% of Males Provide Good Quality Gametesby Induced Spawning

After evaluation of the overall induced spawning efficiency of

amphioxus adults, the quality of the obtained eggs and sperm

was assessed. Egg quality was evaluated after fertilization by

normal development of the resulting embryos through neurulation.

Eggs were qualified as of good quality when more than 50%

of the embryos in a clutch developed normally. Sperm quality

was examined by its ability to successfully fertilize a large

fraction of a clutch leading to subsequent first cleavage of the

fertilized eggs. Based on data from the 2008 and 2009 spawning

seasons, we consistently obtain good quality eggs and sperm.

We found that 85% of spawning females provide eggs of good

quality. After spawning, eggs can be used for only about 1 hr

before their quality starts to deteriorate. Moreover, 70% of

spawning males provide good quality sperm. Concentrated

sperm can be kept at 41C for up to 6 days, whereas once diluted

in ASW, sperm can only be kept for up to 3 days at 41C.

Successful Fertilization of Amphioxus Eggs With Frozen Sperm

To extend the period of sperm availability during the amphioxus

spawning season, we established a protocol for conserving

B. lanceolatum sperm by cryopreservation. Although different

solutions for conserving frozen sperm had previously been tested

in B. belcheri (Xu et al., 2009), we developed a sperm-freezing

protocol for B. lanceolatum that differs significantly from these

previously tested methods and that is based on a solution

containing tested, Tris-HCl buffer, egg yolk (an extracellular

cryoprotectant), and DMSO (an intracellular cryoprotectant).

Although DMSO is a widely used cryoprotectant owing to its

Figure 6. Dynamics of male and female spawning behavior after thermal shock in system A. Based on data from four different animal

collections plus the reshocked population over the course of the reproductive season, the global spawning dynamics after dark is shown for

males (blue) and females (red). The spawning session is subdivided in 30 min intervals, except for the first (spawning during thermal shock)

and the last (after 19:30 hr) time slots. The spawning percentage is the number of animals (males or females, respectively) that spawned in a

given time slot over the total number of spawning animals (males or females, respectively).



fast penetration into spermatozoa and its stabilizing interactions

with phospholipids of the sperm plasma membrane (Suquet et al.,

2000), egg yolk is an important component of cryopreservation

solutions for fish sperm, as its absence or surplus negatively

affects the fertilization capability of spermatozoa after freeze

thaw (Cherepanov and Kopeika, ’99). This effect of egg yolk is

likely the result of alterations in the formation and size of ice

microparticles during the freezing process, which positively

impacts spermatozoid survival during cryopreservation by

preventing damage to the plasma membrane (Suquet et al.,

2000; Andreev et al., 2009). Importantly, it has been shown that

intracellular and extracellular cryoprotectants interact to improve

the overall cryoprotection of sperm from marine animals (Suquet

et al., 2000).

The study on the cryopreservation of B. belcheri sperm

identified a solution containing NaCl, KHCO3, CaCl2, MgSO4,

glucose, and DMSO as best suited to preserve B. belcheri sperm

upon freezing (Xu et al., 2009). Moreover, although our sperm

freeze protocol is based on a cooling rate of�11C/min from room

temperature to �801C and storage of the frozen sperm in a

cryocontainer in the �801C freezer, the published method for

freezing B. belcheri sperm suggests a cooling rate of about

�101C/min from room temperature to �1201C with subsequent

storage in liquid nitrogen (Xu et al., 2009). Thus, the only obvious

agreement between the cryopreservation solution we developed

for B. lanceolatum and the one previously described for

B. belcheri is the suggested final concentration of the cryo-

protectant DMSO, which is 10% for the B. lanceolatum and 12%

for the B. belcheri protocol (Xu et al., 2009).

During the 2009 season, sperm frozen with our cryopreserva-

tion protocol was used on six separate occasions to fertilize eggs

and, with one exception of low sperm activity, the obtained

fertilization rate was good with, as defined above, a large fraction

of the fertilized eggs undergoing first cleavage (Table S1).

Subsequent development of all clutches fertilized with frozen

sperm was normal from early cleavage stages through neurula-

tion (data not shown). This contrasts with the cryopreservation

method published for B. belcheri sperm, which, although leading

to good fertilization rates of more than 50%, yielded a maximal

hatching rate of only 15.4% (Xu et al., 2009). This divergence

between fertilization and hatching rate might be due to sub-

optimal cryopreservation conditions inherent in the B. belcheri

sperm freeze protocol (Xu et al., 2009).

We then tested the activity of the frozen sperm after 1 year of

storage at �801C: enough spermatozoids were still alive and

motile (Movies S1, S2) to efficiently fertilize eggs, which

subsequently underwent normal cleavage (Movies S3, S4).

In summary, our experiments show that concentrated

B. lanceolatum sperm can be stored in cryopreservation solution

at �801C until needed. With this new method, amphioxus sperm

can thus be stored from one spawning season to the next without

losing its capacity to fertilize eggs and trigger normal development.

Conclusions

Amphioxus is a very important emerging model system for

studies focusing on the evolution of developmental mechanisms.

In this study, we report for the first time the successful

maintenance and spawning of B. lanceolatum in a facility

running on ASW and the successful long-term cryopreservation

of amphioxus sperm. With this aquarium system in hand, the

next steps should be oriented toward extension of the length of

the spawning season with the ultimate goal being the complete

deseasonalization of amphioxus spawning behavior.

ACKNOWLEDGMENTSThe authors thank Hector Escriva, Michael Fuentes, and the

Laboratoire Arago in Banyuls-sur-Mer, France, for providing

adult amphioxus, algal cultures and helpful advice on amphioxus

husbandry. We are grateful to Jr-Kai Yu, Nicholas D. Holland,

Linda Z. Holland, Hector Escriva, and Ricard Albalat for critical

reading of this manuscript. We are indebted to Jurgen Kispert

(Muller & Pfleger GmbH & Co. KG, Rockenhausen, Germany) for

help with aquarium system design and helpful advice for

operating the animal facility. We thank Morgane Roulot for her

precious help with animal care and the Animalerie Centrale

(Gif-sur-Yvette, France) for technical support. We are also

grateful to Florent Campo-Paysaa and Jie Chen for technical

assistance and to Gabriel V. Markov and Marie Semon for

support with statistical analyses using R. We wish to thank the

imaging platform PLATIM at the Ecole Normale Superieure de

Lyon, France. Jean-Franc-ois Nicolas is from the Institut National

de la Recherche Medicale (INSERM).

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Amphioxus spawning behavior in an artificial seawater facility