European Amphioxus Research Articles

Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individualsm- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.

Parallel evolution of amphioxus and vertebrate small-scale gene duplications

BackgroundAmphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates.ResultsWe present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements.ConclusionsIn contrast to their slower molecular and morphological evolution, amphioxus’ small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

Spawning Induction and Embryo Micromanipulation Protocols in the Amphioxus Branchiostoma lanceolatum.

In the last decades, the cephalochordate amphioxus has reached a peculiar place in research laboratories as an excellent animal model to answer Evo/Devo questions. Nevertheless, mainly due to its restricted spawning season and to the small size of its embryos, only a few basic techniques in developmental biology could be used until recently. Fortunately, these last years, and thanks to the development of high-throughput techniques, new technical approaches have been possible, such as comparative transcriptomics and/or genomics. However, classic micromanipulation techniques are still difficult to apply. Here we present simple protocols for the manipulation of amphioxus embryos. First, we present the spawning induction method used with the European amphioxus species Branchiostoma lanceolatum. Second, we explain simple methods to manipulate the developing amphioxus embryo during the first steps of its development (before the hatching stage). These methods open many technical possibilities for future functional studies. Thus, we present here a simple technique to efficiently dechorionate a large number of embryos, we detail a protocol for the dissociation of cells during the first steps of the embryonic development and, finally, we describe micromanipulation approaches for tissue isolation during the gastrula stage.

Keeping amphioxus in the laboratory: an update on available husbandry methods

Cephalochordates, commonly known as amphioxus or lancelets, are small, marine animals that can be found in coastal habitats of temperate, subtropical, and tropical waters. Together with vertebrates and tunicates, the cephalochordates belong to the chordate phylum, whose members are characterized by a number of conserved morphological features, such as a dorsal nerve cord, a notochord, a pharynx, a segmented musculature as well as a post-anal tail. Due to their basal position within the phylum, cephalochordates have become essential models for studying the evolutionary origin and diversification of vertebrates. Here, we present the currently available methods for maintaining and rearing cephalochordates in a laboratory environment, focusing on five species: the European amphioxus (Branchiostoma lanceolatum), the Florida amphioxus (Branchiostoma floridae), the Chinese amphioxus (Branchiostoma belcheri), the Japanese amphioxus (Branchiostoma japonicum), and the Bahamas lancelet (Asymmetron lucayanum). In addition to reviewing the protocols for capture, transport, aquaculture, and feeding of adults, we discuss methods for controlling gonad development and spawning, as well as for growing embryos and larvae. This information is complemented by observations from our animal facility on the European amphioxus (Branchiostoma lanceolatum). In sum, this work summarizes the latest advances in cephalochordate animal husbandry and highlights challenges for improving the use of these animals as laboratory model systems.

Amphioxus regeneration: evolutionary and biomedical implications.

Regeneration is a variable trait in chordates, with some species capable of impressive abilities, and others of only wound healing with scarring. Regenerative capacity has been reported in the literature for 5 species from two cephalochordate genera, Branchiostoma and Asymmetron. Its cellular and molecular bases have been studied in some detail in only two species: tail regeneration in the European amphioxus B. lanceolatum; and oral cirrus regeneration in the Asian species B. japonicum. Gene expression analyses of germline formation and posterior elongation in cephalochordate embryos provide some insight into regulation of progenitor and stem cell function. When combined with functional studies of gene function, including overexpression and knockdown, these will open the door to amphioxus as a good model not only for understanding the evolution of regeneration, but also for biomedical purposes.

The opsin repertoire of the European lancelet: a window into light detection in a basal chordate.

Light detection in animals is predominantly based on the photopigment composed of a protein moiety, the opsin, and the chromophore retinal. Animal opsins originated very early in metazoan evolution from within the G-Protein Coupled Receptor (GPCR) gene superfamily and diversified into several distinct branches prior to the cnidarian-bilaterian split. The origin of opsin diversity, opsin classification and interfamily relationships have been the matter of long-standing debate. Comparative studies of opsins from various Metazoa provide key insight into the evolutionary history of opsins and the visual perception in animals. Here, we have analyzed the genome assembly of the cephalochordate Branchiostoma lanceolatum, applying BLAST, gene prediction tools and manual curation in order to predict de novo its complete opsin repertoire. We investigated the structure of predicted opsin genes, encoded proteins, their phylogenetic placement, and expression. We identified a total of 22 opsin genes in B. lanceolatum, of which 21 are expressed and the remaining one appears to be a pseudogene. According to our phylogenetic analysis, representatives from the three major opsin groups, namely C-type, the R-type and the Group 4, can be identified in B. lanceolatum. Most of the B. lanceolatum opsins exhibit a stage-specific, but not a tissue-specific, expression pattern. The large number of opsins detected in B. lanceolatum, the observed similarities and differences in terms of sequence characteristics and expression patterns lead us to conclude that there may be a fine tuning in opsin utilization in order to facilitate visually-guided behavior of European amphioxus under various environmental settings.

Identification, evolution and expression of an insulin-like peptide in the cephalochordate Branchiostoma lanceolatum.

Insulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF- and insulin-like functions in amphioxus supporting the idea of a common ancestral protein.

An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri

Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200 mM KCl, 100 ng/μl actin-LacZ or 100 ng/μl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8 % expressed exogenous gene LacZ and 80.5 % exhibited foreign RFP expression, which were driven by a promoter from amphioxus β-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.

Broken colinearity of the amphioxus Hox cluster

BackgroundIn most eumetazoans studied so far, Hox genes determine the identity of structures along the main body axis. They are usually linked in genomic clusters and, in the case of the vertebrate embryo, are expressed with spatial and temporal colinearity. Outside vertebrates, temporal colinearity has been reported in the cephalochordate amphioxus (the least derived living relative of the chordate ancestor) but only for anterior and central genes, namely Hox1 to Hox4 and Hox6. However, most of the Hox gene expression patterns in amphioxus have not been reported. To gain global insights into the evolution of Hox clusters in chordates, we investigated a more extended expression profile of amphioxus Hox genes.ResultsHere we report an extended expression profile of the European amphioxus Branchiostoma lanceolatum Hox genes and describe that all Hox genes, except Hox13, are expressed during development. Interestingly, we report the breaking of both spatial and temporal colinearity for at least Hox6 and Hox14, which thus have escaped from the classical Hox code concept. We show a previously unidentified Hox6 expression pattern and a faint expression for posterior Hox genes in structures such as the posterior mesoderm, notochord, and hindgut. Unexpectedly, we found that amphioxus Hox14 had the most divergent expression pattern. This gene is expressed in the anterior cerebral vesicle and pharyngeal endoderm. Amphioxus Hox14 expression represents the first report of Hox gene expression in the most anterior part of the central nervous system. Nevertheless, despite these divergent expression patterns, amphioxus Hox6 and Hox14 seem to be still regulated by retinoic acid.ConclusionsEscape from colinearity by Hox genes is not unusual in either vertebrates or amphioxus and we suggest that those genes escaping from it are probably associated with the patterning of lineage-specific morphological traits, requiring the loss of those developmental constraints that kept them colinear.

Amphioxus makes the cut—Again

The cephalochordate amphioxus is now established as an important model system for understanding the evolution of vertebrate novelties from an invertebrate chordate ancestor. It is also emerging as a serious candidate for studies of organ regeneration. We extend here our previous observations on the European amphioxus´ extensive adult regenerative capacity. The expression of Wnt5 and the presence of β-catenin protein in the early bud-stage blastema support a role for Wnt signaling during tail regeneration in amphioxus. We also present data showing that Branchiostoma lanceolatum continues to regenerate well after repeated amputation of the post-anal tail. These results are discussed in relation to vertebrate regeneration and other stem cell systems, and in the context of regeneration decline with aging.

From the American to the European amphioxus: towards experimental Evo-Devo at the origin of chordates

Pallid anchovy fillet, friendly filtering, peacefully laying and little lancelet are some of the nicknames and adjectives the cephalochordate amphioxus has received throughout the last two centuries. Traditionally regarded as the living representative of the last ancestor of vertebrates, amphioxus has recently been promoted to the privileged position of being the most ancient chordate. The preliminary analysis of its prototypical genome is nearly completed, and its hidden secrets towards the understanding of the primitive chordate and deuterostome genomes will soon see the light. Amphioxus embryonic development and body plan have remained in evolutionary stasis since the cephalochordate lineage split from the chordate ancestor about 500 million years ago. In contrast, amphioxus research is far from being at a standstill; in Europe, thanks to the international cooperation and the Banyuls Oceanographic Station, amphioxus embryos are obtained on demand during the spawning season. We summarise here our progress towards the dream of the experimental manipulation of the amphioxus embryo, to enter the era of Experimental Evo-Devo.

Unexpectedly large number of conserved noncoding regions within the ancestral chordate Hox cluster

The single amphioxus Hox cluster contains 15 genes and may well resemble the ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of the European amphioxus Branchiostoma lanceolatum and compared it to the American species, Branchiostoma floridae, by phylogenetic footprinting to gain insights into the evolution of Hox gene regulation in chordates. We found that Hox intergenic regions are largely conserved between the two amphioxus species, especially in the case of genes located at the 3' of the cluster, a trend previously observed in vertebrates. We further compared the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus.

Development of a semi-closed aquaculture system for monitoring of individual amphioxus ( Branchiostoma lanceolatum), with high survivorship

The European amphioxus Branchiostoma lanceolatum is becoming an important model for developmental studies, and as such requires more study both in the field and in the laboratory. We present an experimental set-up with temperature and flow rate control that allows easy care and monitoring of individual amphioxus. Over the course of several months, 98/100 individuals in two size categories and experiencing different levels of handling stress survived. Flow cytometry and gut contents indicate that the system meets the nutritional needs of the amphioxus. This simple and effective system for separate aquaculture of individual amphioxus prevents infections due to crowding. It should be particularly useful for future breeding, genetic, behavioural and life history studies, and can be easily adapted to other marine organisms.

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.

D-Aspartic acid and L-amino acids in the neural system of the amphioxus Branchiostoma lanceolatum

The lancelet (amphioxus), a cephalochordate, is the closest invertebrate relative to vertebrates, with a simple vertebrate-like body plan and a prototypical genome. We have determined D-aspartic acid (D-Asp) and major free L-amino acids (L-AAs) content in the nervous system (neural tube) of the European amphioxus Branchiostoma lanceolatum, and have compared these values with those of molluscs and human brain. The B. lanceolatum neural tube contains relatively high amounts of L-Glu, L-Asp, L-Ala and L-Gly. Thus, the amphioxus neural tube has in common with the molluscan and human nervous systems the presence of appreciable amounts of L-Glu and L-Asp, which suggests that they are the most common neurotransmitters among these phylogenetically distant animal groups. The relatively high concentration of L-Ala in amphioxus is consistent with that found in molluscs and the low concentration of taurine is consistent with that described in the human brain. The D-Asp concentration, very high in the molluscan nervous system, was rather low in amphioxus, although a little higher than the extremely low amounts observed in the human brain. Our data on free amino acids composition is in agreement with the intermediate phylogenetic position of cephalochordates, in terms of the evolutionary transition from simple to complex neural systems.

An electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense: Cleavage.

Development of the Asian amphioxus, Branchiostoma belcheri tsingtauense, was investigated by scanning and transmission electron microscopy (SEM and TEM) from the fertilized egg through the blastula stage. The fertilized egg is spherical (mean diameter 115 μm after SEM preparation) and is covered with microvilli. Throughout cleavage, the second polar body remains attached to the animal pole. The cleavage type in this species is essentially radial, as revealed by SEM observations. At the third cleavage or 8-cell stage, and at later stages, a size difference between blastomeres in the animal and the vegetal halves is clearly discernible, but less marked than that reported for the European amphioxus, B. lanceolatum. During the period spanning the third to the fifth cleavage (8-32-cell) stages, blastomeres are arranged in tiers along the animal-vegetal axis. After the sixth cleavage, or 64-cell stage, the tiered arrangement of the blastomeres is no longer seen. At the 4-cell stage, the blastocoel or cleavage cavity is seen as an intercellular space, opening to the outside. The blastocoel remains open at the animal and the vegetal poles in later stages. Throughout early development, the cytoplasm of the blastomeres includes yolk granules, mitochondria, Golgi complexes, and rough and smooth endoplasmic reticulum. Chromatin in the interphase nucleus is not clearly demonstrated, and chromosomes in the mitotic phase are also extremely difficult to detect. As yet, regional differences have not been found in distribution and organization of cytoplasmic components with respect to prospective ectodermal, mesodermal, and endodermal areas in the fertilized egg and later cleaved embryos, although there are possibly fewer yolk granules in the region of the animal pole than in the vegetal polar zone.