Longitudinal Observation of Japanese Lancelet , Branchiostoma japonicum , Metamorphosis

1 Department of Biological Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8526, Japan Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Center for Marine Environment Studies, Kumamoto University, 39-1 Kurokami 2-chome, Kumamoto, Kumamoto 860-8555, Japan


Introduction
Profound change of body plan under the same genomic background during life cycle is common in metazoans.It is called metamorphosis and found in at least some species of most extant phyla [1].Metamorphosis of lancelets (amphioxus) has been noticed from the �rst embryological study of Kowalevsky [2] and interested the following scientists such as Lankester and Willey [3], Willey [4], and van Wijhe [5].Like other marine invertebrates, lancelets change their life mode from pelagic to benthic passing through metamorphosis.However, their diet and feeding method are not changed principally.Lancelet ontogeny uniquely displays a long-term coexistence of cilia-and muscle-driving locomotion before metamorphosis [6,7].e metamorphosis takes place aer pelagic larval life, the duration of which depends on the environmental condition and species [8][9][10], and animals aer metamorphosis settle into sand substratum.
Metamorphic changes mainly occur in the pharyngeal region in lancelets.Metapleural folds appear �rst on the ventral trunk on both sides and fused ventrally covering the original body surface in the pharyngeal region and abdominal region anterior to the iliocolonic ring [3].Larval mouth that occupies from the second to the ninth myomere wide on the le body surface just before metamorphosis is shrunken forward during metamorphosis and �nally transformed into the velum as a median organ [4,11].Not only the mouth but also the digestive system as a whole undergoes remarkable modi�cation.A single row of gill pores, which expand from the midventral to the right side, get their counterparts above them on the right side, and both rows of gill pores are destined to the le-right pairwise positioning.During the pairing, each gill pore is divided into two by a tongue bar.Endostyle originally developed on the right side of the pharynx also moves to the ventral �oor and expands longitudinally.At the junction between the esophagus and stomach, the budding of hepatic diverticulum appears ventrally.
Although the aforementioned morphological changes are comprehensive and occur during relatively short period, lancelet metamorphosis is distinct from that of animals producing ciliate larvae such as lophotrochozoans.e latter animals completely change body plan, locomotion mode, and feeding behavior with metamorphosis as a trigger.In marine environment, as intermediate state during the transition of life mode causes higher risk to predation, the duration of metamorphosis is presumed to be optimally shortened [12].In chordates, all three members, cephalochordates, urochordates, and vertebrates, especially anuran amphibians, display metamorphosis, but each metamorphosis is distinct from others among chordate taxa and, except urochordates, also from that of other invertebrates.As a gene coding thyroid hormone receptor has been found in lancelets [13], a mechanism similar to that of anurans that takes weeks to months to complete metamorphosis [14] was expected, and it has been revealed that a thyroid hormone derivative (triiodothyroacetic acid) can induce lancelet metamorphosis [15].To advance studies on the lancelet metamorphosis, we have to understand basic time course of the metamorphosis based on longitudinal observations.We report here individual metamorphosis observed longitudinally under the culture condition.

Methodology
First generation of laboratory progeny of the Japanese lancelet (now assigned to Branchiostoma japonicum [16]) fertilized on 23 and 24 July 2011 at the Marine Station of Kumamoto University, Japan, was maintained as described previously [17].When metamorphic signs were observed in some precocious individuals at the end of August, about 200 animals were maintained in a small plastic container with 500 mL �ltered seawater.From these, �ve individuals that were developing metapleural folds were reared one by one in 92 mm Petri dish with �ltered seawater.e animals were fed daily with 20 mL and a few milliliters of well-grown culture medium of a diatome Chaetoceros gracilis for the plastic container and Petri dish, respectively.Seawater and plastic wares were changed every three days.e atmospheric temperature close to the culture dishes during the observation is shown in Figure 2, which can be regarded as being equivalent to water temperature because of small volume of culture dishes.Light-dark cycle was under the natural regime.Separately maintained �ve animals were photographed under the microscope daily from 17 September to 17 October 2011, and then body length measurement of the living animals with objective micrometer was started from 22 September and continued until 12 November 2011.e digital images were processed to produce montage for each animal with Adobe Photoshop CS4.Totally 31 images for each were used for F 1: Time course of individual metamorphosis in Branchiostoma japonicum at temperature �uctuated between 21.5 and 24.0 ∘ C. Note the individual variation of the duration and rapid expansion of ciliate surface by both opening of secondary gill series and division of each gill pore with tongue bar.e number of the animals corresponds to that in Figure 2.
making QuickTime movies by using Apple iMovie (Dataset Item 3 (Video)).e measurement of body length was also performed on the montage images.
Timing of metamorphosis was as follows.e observations were carried out directly on the living animals under the microscope and on the montage images.On 17 September, all individuals subjected to longitudinal observation were developing atrial chamber that had covered the posterior nine to ten primary gill pores (future le-side gill pores).e duration of metamorphosis showed individual variations under the culture condition.From the �rst observation of the thickened epithelium of the secondary gills (right-side gills), which is the most easily identi�able landmark in the early phase of metamorphosis, to that of elliptical windowlike perforation of the secondary gills, it took four to seven days, and from the budding of tongue bar in the secondary gill pore to the pairing of le and right gill slits, it took one to two days.en it took further four to eleven days to the formation of primary synapticles between branchial and tongue bars, at which metamorphosis is completed and thereaer animals are called juveniles [11].In total, the fastest individual took 13 days and the slowest 20 days from the thickening of the secondary gills to the primary synapticle formation between gill and tongue bars (Figure 1).e metamorphosis of lancelets is comparable only in habitat change from plankton to benthos with other metamorphic marine invertebrates, although there is no consistent account on the larval behavior.e larval behavior has been variously described as entirely planktonic [4], predominately planktonic under culture condition [18], vertical migrant between the sea bottom and surface with a day cycle [19,20], and dweller on the sea bottom until the 6-gill stage and then becomes planktonic [8].Under the culture condition, however, we observed that larvae stayed on the bottom in the glass tank frequently throughout their life.is suggests that the Japanese lancelet in larval life inhabits in the water  ).Arrows with specimen numbers denote when the specimen grows longer than the size at the beginning of observation.Growth lines are approximated by fourth-ordered regression curves.All specimens show growth arrest during metamorphosis.(b) Growth curve obtained by a cross-sectional sampling grouped according to developmental stages drawn from Dataset Item 2 (Table ).All specimens again display growth arrest during metamorphosis.In the sampling at the postmetamorphic stage, well-grown animals were selected to avoid mixture with younger stages, which causes size difference at the postmetamorphic stages between the isolated culture (a) and pooled culture (b).Each measurement shows the mean ± SD.Premeta: premetamorphic stage; M1: thickening of secondary gill series; M2: bisection of secondary gill pores; M3: pairing of gill slits.
column and on the bottom surface for a long period before metamorphosis, and it is interesting to know how long lancelets need for metamorphosis.Since all previous studies on lancelet metamorphosis are based on cross-sectional observations, there is little reliable data on this issue, in which the Florida lancelet took eight days at 22.5 ∘ C and about �ve days at 30 ∘ C under a culture condition [10].In the present study, as we started the observation when the animals developed atrium partially, it is not known how many days the studied animals took for metamorphosis exactly.However, animals with slow metamorphosis took 27 days and the fastest animal 15 days from the start of the observation to the formation of the primary synapticles under the conditioned temperature between 21.5 and 24.0 ∘ C. Lancelet larvae develop a large mouth occupying about a ��h of the body length and large gill openings during pelagic life.To change from pelagic to benthic, lancelet larvae required the strengthening and protection of the oral and pharyngeal regions because they settle into sand substratum when they are about 5 mm long.To meet this requirement, the mouth and gill pores are covered with preoral hoods and atrium during metamorphosis, respectively.At that time, however, the mouth changes to a very small opening and the number of gill pores reduces to about half [4,11].ese changes seem to hurt effective intake of seawater.To minimize this transitional discordance, lancelets develop the secondary gill series rapidly and divide all gill pores by tongue bar producing about twice large area for branchial cilia that create water �ow.During a long duration of metamorphosis compared with that of most marine invertebrates, which is usually a rapid process as hours or a few days [14], the modi�cation of the mouth and gill system took only two to four days.
Lancelet larvae are small and ciliate like the pelagic larva of most marine invertebrates.A difference from the others is found in that they develop functional muscles at the early larval stage [7,11].Against inherent danger for lancelet larvae as pointed out for other small pelagic larvae, there seems to be nothing to protect them in considering the anatomy and behavior.Lancelet metamorphosis thus seems to follow the need for speed hypothesis to avoid risk [14].However, the duration of the metamorphosis is not so short unlike other marine invertebrates.Rapid changes found in the mouth and branchial system seem to correspond with intrinsic requirements.Locomotion and feeding behavior that are subjected to external selection pressures do not change substantially between pre-and postmetamorphosis in lancelets.e uniqueness of lancelet metamorphosis will provide a research model to test what factors can determine the duration of metamorphosis [21].Growth during metamorphosis was as follows.It has been pointed out that the European lancelet Branchiostoma lanceolatum stopped growing or reduced the body length during metamorphosis [5,22,23].In contrast, the Florida lancelet B� �ori�ae was reported to continue growing [10].Although some of these studies were based on animals under the laboratory condition, none performed longitudinal observation.To con�rm whether or not the Japanese lancelet reduces the body length during metamorphosis, for �ve individuals we have traced longitudinally the change of the body length in living state under the dissection microscope.e result was that the Japanese lancelet did not grow during metamorphosis and reduced the size slightly (Dataset Items 1 and 2 (Table ); Figures 2 and 3).In the measurement on the individual images, compared with the size at the beginning of the observation (forming atrium), the size reduction was from 2.2% to 3.2% of the body length being observed when gill slits became paired (Figure 3(b)).As the body length of the premetamorphic stage is less than 6.0 mm, it seems to be rather difficult to depict exact size reduction by cross-sectional observation (Table 1).An interesting observation is that regardless of the �uctuated culture temperature, animals started metamorphosis when their body size was smaller than others tended to perform metamorphosis quickly and grew faster aer metamorphosis (Figure 2), which was also recognized in animals maintained in the 500 m� container, though not quanti�ed.
As gill pores were disappearing, the length of the pharynx was shortened and the esophagus was slightly elongated.is compensation was not balanced with the actual reduction of the pharynx, and thus the gut was pulled anteriorly (Figure 3).In this modi�cation of the alimentary canal, the iliocolonic ring was moved anteriorly, which was located posterior to the atriopore at the onset of metamorphosis and anterior to it by the �nal stage of metamorphosis.e reduction of the pharyngeal length and the anterior shi of the anterior gut thus were compensated by the elongation of the intestine (Figure 3).When the budding of the hepatic diverticulum occurred, the anterior gut region was reduced further even when the body length started growing again.e elongation of the intestine alone compensated this gut modi�cation.A chronological change during metamorphosis of a single animal for a month is shown in Dataset Item 3 (Video) that has been produced from the montage images taken daily.
Contrary to the European lancelet, the Florida lancelet did not show a stop of growth or shrinkage of the body size during metamorphosis [10].It is not known whether growth arrest found in the present study and pointed out in the European species depends on environmental factors such as temperature and food availability or growth arrest is a �xed character in some lancelet species.In the present culture method, the growth arrest continued for a month.Individual variations in the onset of metamorphosis exceed this period [17], and cross-sectional random sampling may hide the growth arrest.

Dataset Description
e dataset associated with this Dataset Paper consists of 3 items which are described as follows.

Concluding Remarks
Although the growth pattern during larval and postlarval periods in lancelets is heavily affected with environmental conditions [10,17], our �rst longitudinal observation of lancelet metamorphosis has found features that seem likely to be relatively irrelevant to external condition.e duration of metamorphosis varies among individuals even under a constant culture condition.Growth was arrested until when paired gill slits started to increase in number (called the third gill formation), and the maximum reduction of the body length was observed when gill slits became paired.It is an open question that whether the growth arrest can depend on external factors or not.Lancelet metamorphosis is a long process compared with that of other marine invertebrates [14].During this period, however, the oral transformation and the bisection of each gill pore took place within a few days.e reduction of pharyngeal and midgut length was solely compensated by elongation of the intestine, which may be related to digestive efficiency [24].ese characters suggest that internal requirements rather than predation pressures are important for the duration and mode of lancelet metamorphosis.e lancelet body plan displays some contradicting features.Its streamlined shape with massive myomeres makes us imagine a very active life like that of predatory vertebrates.�owever, lancelets depend completely on �lter feeding like sessile animals with some exceptional cases in larval life [20] and settled lancelets stay in sand substratum inactively throughout their life ( [25] and our observations in the culture system).Although we do not know how their body form and life mode have been acquired historically, the lancelet lineage seems to have diverged from the common stock with vertebrates by the Early Cambrian [26].e genomic study on the Florida lancelet gives a possibility that the lancelet body form has rather been stable since its divergence [27].e most remarkable feature of the lancelet metamorphosis is the transformation from a le-right asymmetry to a symmetric appearance.As mitochondrial DNA sequence comparison suggests that the asymmetric feature is a primitive character in lancelets [28], it is interesting to know how and, if possible, when the lancelet ancestor acquired metamorphosis.Despite the fact that lancelets seem to have no effective strategies, predation pressures did not affect strongly the feature of the lancelet metamorphosis, which is an interesting contrast with other marine invertebrates.Lancelets thus may retain the original metamorphic processes and provide unique clues to study the evolution of metamorphosis.

F 2 :
Growth curves and culture temperature.(a) Growth curves of �ve individuals and culture temperature drawn from Dataset Item 1 (Table

F 3 :
Size reduction and change of digestive system.(a) Chronological change of the specimen number 1 in Figure 2(a).Note the remarkable reduction of the pharyngeal length and moderate reduction of the esophagus and midgut region as well as remarkable elongation of the intestine.(b) Reduction of body length in �ve individuals compared with the length at the beginning of the observation.�ine colors are the same as those in Figure 2(a).Reduction of the body length is maximized when le and right gill rows become paired.Animal images numbered 1-5 are as follows: 1, on 17 September; 2, on 25 September; 3, on 30 September; 4, on 9 October; and 5, on 17 October.an: anus; ap: atriopore; at: atrium; ep: esophagus; hd: hepatic diverticulum; in: intestine; ir: iliocolonic ring; lm: larval mouth; pg: primary gills; sg: secondary gills; sgo: secondary gill openings; sy: synapticle; tb: tongue bars; ve: velum.
T 1: Number, Mean, and SD of body lengths (mm) of staged living individuals measured with objective micrometer on the date indicated, which were selected from pooled culture in a container.
). Body lengths (mm) of studied living individuals measured with objective micrometer and culture temperature ( ∘ C).
Column 8: Juvenile Stage on 12/11/2011 (mm) Dataset Item 3 (Video).Morphological change of Individual number 3 during metamorphosis from 17 September to 17 October.is video has been produced from 31 montage images taken one image every day by using Apple iMovie.