Taxonomy Of Genus Branchiostoma In Xiamen Waters And Continuous Breeding Of Two Lancelets In The Laboratory

The Cephalochordata, commonly called amphioxus or lancelets, are considered to be basal chordates and have long occupied a key place in studying the origin and evolution of vertebrates. The lancelet is considered to be a promising laboratory model organism. Xiamen has long been famous for having abundant lancelets. Since it was taxonomically identified as Branchiostoma belcheri in 1932 by Boring & Li, the lancelets living there have not received much taxonomic attention for the past several decades. However, in 2005, a comparison on 12S rRNA gene sequences of lancelets from two localities (Oucuo and Huangcuo) in Xiamen waters indicated that there are two separate species, B. belcheri and B. tsingtauense, and then the scientific name of the latter was corrected to B. japonicum, according to the priority rules of nomenclature. In the present study, we observed thousands of specimens from Xiamen waters in detail and herein describe the morphological characters of the two species. Our observations showed that at least three morphological characters distinguish the two species: 1) the rostral fin is slightly round with the end obtuse in B. belcheri but elliptic with the end cuspate in B. japonicum; 2) the number of preanal fin-chambers is more than 80 in B. belcheri but less than 70 in B. japonicum, and the chambers are slender in the former but stout in the latter; 3) the caudal fin of B. belcheri is narrower than that of B. japonicum, and the angle between the dorsal and super-caudal fins, and between preanal and sub-caudal fins, is obtuse in B. belcheri but acute in B. japonicum. The two separate species were also confirmed further by cross breeding experiments.The gonad development of two lancelets B. belcheri and B. japonicum in Xiamen were observed and the two lancelets were cultured in the laboratory. At the population level, the two lancelets develop and mature their gonads in batches. The gonads of B. belcheri begin to develop in late April and mature firstly in late May, the matured peak appeare in June and August, and the spawning occurs from June through September. For B japonicum, the gonads begin to develop in second half of November, the matured gonads appear firstly in late December or early January of next year and arise up to a peak in April, and the spawning occur in the first period from late April to early May. The matured gonads appear in another period from late July through early September, and the second spawning period presents from August to late September or early October. The ovary of B. belcheri develops appreciably later than the testis, while the ovary of B. japonicum dose significantly earlier than testis and also regresses earlier after spawning. So the ratio of female to male is various in sex-differentiable individuals of B. japonicum collected in different period. Some individuals of two lancelets, at the age of two- or three-year-old, can spawn and then ripen and spawn again in the same breeding season.We used multiform containers in shape, volume and character for the laboratory culture of lancelets. The containers were laid with about 5～10 cm layer of sand at the bottom and filled with seawater at a depth of 5～10cm over the sand. The sand were obtained from the lancelet habitat and sifted through 0.5～1 mm wire sieve and the fresh seawater were filtered through sand layer. The stocking density, depending on the body size and settlement surfaces, varied from 2,000 to 3,000 inds/m~2. The containers were not covered and maintained at ambient photoperiod. The continuous air supply was provided. The seawater temperature was controlled by air-conditioners between 10℃in winter and 30℃in summer. To supply enough food, we cultured several species of unicellular algae, including Dicrateria zhangjiangensis, Isochrysis galbana, Chaetoceros muelleri, Chlorella sp. in f/2 medium under 24-hr artificial illumination in the laboratory. The animals were fed with algal mixture twice daily. The proportion of each alga was not strictly controlled and varied greatly depending on the growth status of algae. The total quantity of algae depended on the number and the body size of individuals and the criterion was that the water was still slightly turbid 3 h later after feeding. A portion of seawater was changed before feeding. To keep settlement substratum clean, the sand was cleaned monthly. Under the culture conditions above, the two lancelets grew well and matured gonad, then spawned in large quantity in 2006.The lancelets cultured in the laboratory persist to spawn for long period with different interval. The spawning of B. belcheri persisted for 30 nights from 16 June to 9 September at intervals of 0～17 days in the laboratory in 2006, and 53 nights for B. japonicum from 2 May to 23 October at the same intervals. The spawning overlapped 11 nights between two lancelets. The spawning dates are not significantly correlated with lunar phase or seawater temperature or fluctuation in salinity or change of weather or volume of container or size of population. Only if sufficient foods were supplied and the gonads developed well, the lancelets would spawn in summer in the laboratory.The first filial generations (F1) of two lancelets were produced in our laboratory in 2005. After culturing for 12 months for B. belcheri and 9.5 months for B. japonicum, a part of F1 generations developed gonads matured and spawned in 2006, and the secondary filial generations (F2) were produced successfully. Our study is the first report on rearing lancelets for a complete reproductive life cycle in the laboratory.The two lancelets experience the similar developmental events and also grow in the same pattern; however the differences in development and growth are mostly likely to involve minor difference in relative timing of events. The developmental and growth rates of both species connect with the culture temperature. At the same temperature, the rates of B. belcheri are slower than those of B. japonicum and the body length of B. belcheri is smaller slightly than that of B. japonicum at the same age. The relative rates of two lancelets vary depending on the temperature. The developmental and growth rates among individuals of either B. belcheri or B. japonicum, produced at the same time and cultured at the same conditions, are also different significantly. That maybe indicate the rate is influenced by genetic or environmental factors except temperature.The lancelets can spawn eggs in large quantity and they possessed very high fertilizing and hatching rates in the laboratory, but the newly hatched neurula are very delicate and about two-thirds of them die before mouth-opening. After mouth open, only ten percent of the larvae can keep alive through metamorphosis stage. And only two-thirds of newly metamorphosed juveniles can survive till the stage of onset of gonad development. The reason for the death of juveniles perhaps mainly owes to their bad developmental status of hepatic diverticulum.Finally, the embryonic and somatic metaphase chromosomes of two lancelets are prepared by using improved protocol. The diploid choromosome numbers of B. belcheri is 40 while that of B. japonicum is 36, verifying further their taxonomic status as two separated species.