Digestive system consists of an alimentary canal and digestive glands. Alimentary Canal: The alimentary canal is a straight tube from mouth to anus. It is lined by. The lancelets, also known as amphioxi (singular, amphioxus), consist of about 32 species of fish-like marine. The common lancelet, Branchiostoma lanceolatum, has been recorded from the . The digestive system of Branchiostoma consists of an alimentary canal and.

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The full-length cDNA transcriptome library of Akphioxus diverticulum: Data are available at: The digestive methods employed by amphioxus Branchiostoma —both intracellular phagocytic digestion and extracellular digestion—have been discussed since Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes.

Here, in Branchiostoma belcheriusing a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy sgstem all stages of its life.

Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostomathese amphioxys accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles.

We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion diegstive Branchiostomaexplain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver. Every organism requires energy to survive, and most heterotrophic unicellular organisms phagocytize food particles directly and break them down via intracellular digestion [ 1 ].

Phagocytic ampnioxus digestion is the evolutionary cornerstone of both the digestive and immune mechanisms of multicellular animals [ 2 — 6 ]. Currently, however, phagocytosis in deuterostomes is seen primarily as an immune process, and research into it focuses on the biological mechanisms used amohioxus eliminate pathogens, clear up cell debris and present antigens amphioxks 7 ]. It is commonly accepted that animals, especially deuterostomes, digest food using only extracellular digestion, which is the process of breaking down large food particles into small, water-soluble absorbable molecules by digestive enzymes in the lumen of a digestive organ [ 8 ].

Inhowever, van Weel observed that, in addition to general extracellular digestion, the digestive tract of Branchiostomaespecially its protruding part, the diverticulum, can phagocytize food particles directly. He, therefore, suggested that both extracellular and intracellular digestion can occur in Branchiostoma [ 9 ].

Nevertheless, because autolysis by intracellular digestive enzymes tends to occur during sample fixation, phagocytic intracellular digestion in the Branchiostoma diverticulum is difficult to observe digeetive using the typical fixatives used for light dgiestive election microscopy [ 10 — 14 ]. Recent studies of the Branchiostoma digestive tract focus on tracing the origin of the vertebrate immune mechanism [ 16 — 27 ], but neglect its original digestive function.

For instance, though variable region-containing chitin-binding proteins VCBPs have been shown to play important roles in the ov immune response [ 16 — 19 ] in Branchiostomathere has been little investigation into why the VCBP genes are expressed in endoderm-derived epithelial cells of the digestive tract but not in mesoderm-derived professional immune cells.

Consequently, determining the biological characteristics of the epithelial cells in the digestive tract, especially those in the diverticulum, is key to understanding the role of phagocytic intracellular digestion in Branchiostomaand could shed light on the origins of the vertebrate immune mechanism and liver.

Here, by using an improved method of tissue fixation for transmission electron microscopy TEMwe show that B. The gene expression profile of these cells reveals that they can support ssytem digestive and immune-like functions by expressing many kinds of immune genes and varying types and amounts of digestive enzymes.

Interestingly, qRT-PCR results demonstrate that the gene expression profile of the cells can present two different states: Our results shed light on the context in which phagocytic intracellular digestion occurs in Branchiostomaand suggest why the epithelial cells of the Branchiostoma digestive tract can express immune genes.

Phagocytic epithelia of the Branchiostoma digestive tract. The epithelial cells of the diverticulum and hindgut can phagocytize food particles directly, and the phagocytic capability of diverticulum cells is greater than that of the hindgut epithelial cells. Wild samples are assumed to be sated because of the plentiful algal resources in Zhanjiang Bay [ 28 ]. We sought a way to improve sample fixation so as to be able to visualize phagocytic intracellular digestion in the Brachiostoma digestive system.

We found that initial fixation including glutaraldehyde is insufficient to fix the phagocytic epithelial cells of the B. The annotated tissue-specific genes were verified by conserved homologous sequence using C lustal W and neighbour-joining trees using M ega 6 with bootstraps. The qRT-PCR assays for determining the gene expression profile of the diverticulum epithelial cells include all the tissue-specific unigenes in Full electronic supplementary material, table S2.


From 1-year-old adults, we dissected the empty epithelial cells of the diverticulum EDnatural sated epithelial cells of the diverticulum SDnatural sated gut SGempty gut EGas well as pharyngeal bar PB and notochord NT cells as contrast samples.

The empty samples were from animals that were starved for 3 days to ensure there were no residual algal cells in the epithelial cells of the digestive tract and that the gut had time to excrete all faeces completely. The total RNA isolation was processed in the same way as the construction of Full. All the primers were screened by electrophoresis and PCR product sequencing. The PCR products were purified with an E.

The most highly expressed genes of diverticulum phagocytic epithelial cells after 3 days of starvation. Two-tailed Student’s t -tests digestlve used to assess statistical significance. The most highly expressed genes of the diverticulum phagocytic epithelial cells in the natural sated diggestive. The methods of sample dissection, RNA isolation, sequencing and bioinformatics analysis electronic supplementary material, tables S7—S10 are same as those used for Full.

The in situ hybridizations were performed as described in [ 33 ] and in [ 34 ] with some modifications. The digoxigenin DIG -labelled sense and antisense riboprobes were synthesized with T7 or Sp6 RNA polymerase Roche, according to the manufacturer’s instructions. The hybridization experiment was performed in Netwell inserts Corning, To reduce the background noise, the probe concentration was decreased to 0. Images were combined using Adobe P hotoshop CS6.

Haematoxylin—eosin stain was used as previously described [ 16 ]. Images were acquired and processed using cell S ens xystem Olympus, Tokyo, Japan. Branchiostoma filters food particles from the water with its difestive cirri and pharyngeal bars.

Branchiostoma: Distribution, Structure and Systematic Position

Branchiostoma diverticulum epithelial cells can phagocytize algal cells directly. The ileo-colon ring sorts small xystem digestible cells into the diverticulum lumen. Almost all diverticulum epithelial cells exhibit a phagocytic function d.

The small arrowheads mark the algal cells.

Digestive Systems of Branchiostoma and Ascidia | Zoology

Di, diverticulum; Ir, ileo-colon ring; Lu, lumen; As, apical side; P, phagosome. The phagocytic epithelial cells of the diverticulum produce large numbers of lysosomes to support their intracellular digestive function. The small arrowheads in b mark the degrading algal cells. Ly, lysosomes; Lu, lumen; As, apical side. We also discovered that, just after the larval mouth opens at about 36 h, the epithelial cells of the diverticulum primordium can immediately begin to phagocytize algal cells electronic supplementary material, figure S3suggesting that phagocytic intracellular digestion occurs throughout all Branchiostoma life stages.

Additionally, the ileo-colon ring is crucial because it can control the size of the food particles that enter the diverticulum: In order to explore how the diverticulum epithelial cells deal with the phagocytized food particles, we constructed a full-length cDNA transcriptome library abbreviation: Full; GenBank Accession Number: The genome of B.

Thus, we discuss the analysis of individual intracellular digestive genes determined using the traditional Sanger method on a plasmid-based full-length cDNA library below.

The gene expression profile of the diverticulum epithelial cells overlaps with the patterns seen in both the digestive cells and phagocytes of vertebrates. The overwhelming majority of expressed gene markers belong to three families: The cathepsins are typical lysosomal proteases and, except for type K, most of them play major roles digestie intracellular protein hydrolysis divestive 20 ]. Ferritin is an indispensable intracellular protein that acts as a buffer to maintain the balance of iron [ 41 ].

The intracellular iron store can prevent the spread of infective agents by impeding their proliferation [ 42 ]. Among these genes, lysozymeVCBPs [ 16 — 19 ], tetraspanin, Gram-negative bacteria-binding proteinalpha2-macroglobulin [ 24 ], chitotriosidase 1-like protein [ 43 ], big defensin [ 16 ] and Toll-interacting protein are typical immune genes, and the other genes, except for methionine adenosyltransferaseare the main digestive or hydrolytic genes of both digestive cells and phagocytes.

The in situ hybridization experiments show that all the genes mentioned are expressed in diverticulum epithelial cells electronic supplementary material, figure S8. Next, to reveal the spatiotemporal pattern of gene expression of diverticulum epithelial cells, we compare digestve differences in gene expression between cells in empty after 3 days of starvation and sated states using qRT-PCR assays. In order to determine the results off the qRT-PCR assay more precisely, we selected both 18S and cytoplasmic actin as the reference genes [ 44 ].

This gene expression profile indicates that the cells are prepared to degrade phagocytized food particles.

Digestive System of Branchiostoma (With Diagram) | Chordata | Zoology

Moreover, sytem findings show that, though pancreatic lipase-like proteinplasminogen and peroxiredoxin V are highly expressed in diverticulum phagocytic epithelial cells, it is difficult to estimate whether they are highly expressed in either the empty or sated states because of the inconsistency caused by the two dugestive reference genes, as one gives a high expression ratio and the other gives a low one electronic supplementary material, figure S Plasminogen is the precursor of plasmin, which is a serine protease and can dissolve fibrin [ 45 ], whereas peroxiredoxin V has antioxidative and cytoprotective functions during oxidative stress triggered by immunogens [ 46 ].


The results of the qRT-PCR assays reveal that Branchiostoma phagocytic epithelial cells present different gene expression states in unfed versus sated states.

In fact, among all the annotated functional genes, only elastase I is obviously expressed amphioxks in the gut than in the diverticulum [ 47 ] electronic supplementary material, figure S Second, the suppression subtractive hybridization SSH results reveal that the gene expression profiles of the diverticulum and gut epithelial cells are similar, but that the diverticulum can express greater numbers of functional genes than the gut.

SSH technology allows for the PCR-based amplification of the cDNA fragments that differ between control driver and experimental tester transcriptomes [ 32 ]. Gut epithelial cells outside the diverticulum can phagocytize. The epithelial cells in the hindgut can also phagocytize algal cells chowever, the phagocytic capability of these cells seems more limited than that of diverticulum epithelial cells, which are longer and more numerous figure 2.

The small arrowheads in c mark the algal cells.

Phagocytic intracellular digestion in amphioxus (Branchiostoma)

Ap, atriopore; Ir, ileo-colon ring; Lu, lumen; Dgiestive, apical side. The phagocytic epithelial amphixous deal with this immune-like stimulation by expressing immune genes. Among aamphioxus immune genes, lysozyme can present in the cytoplasmic granules to destroy Gram-positive bacteria [ 41 ], VCBPs can present an adaptive-like immune character by exhibiting high levels of polymorphism [ 16 — 19 ], Gram-negative bacteria-binding protein can bind to lipopolysaccharides to elicit immune responses and alpha2-macroglobulin can prevent cell lysis by inactivating various proteinases [ 24 ].

In addition, big defensin is a small cysteine-rich peptide that can bind to the cell membranes of bacteria and form pore-like defects that can lead to the lethal efflux of cytoplasmic ions and nutrients [ 16 ]. The functions of these typical immune genes and large quantities of other endogenous degrading enzymes ensure that the phagocytic epithelial cells can not only degrade phagocytized food particles sufficiently, but also maintain their cellular integrity.

However, our findings demonstrate that the cell amphipxus, tissue structure and gene expression profile of the Branchiostoma diverticulum sysstem quite different from those of the vertebrate liver. Furthermore, hepatic products homologous to those in vertebrates, such as albuminalpha-fetoproteinaspartate aminotransferasealanine aminotransferase and miRwere not found in the Branchiostoma diverticulum through the tissue-specific expressions of hallmark genes—in fact, albuminalpha-fetoprotein and miRare ampuioxus from the Branchiostoma genome [ 2237 — 4048 ].

In summary, all of the aforementioned results suggest that the main physiological functions of the diverticulum are different from those of the liver. It digestiv be reckless to infer directly that the invertebrate chordate lacked a liver just because Branchiostoma performs phagocytic intracellular digestvie in its diverticulum.

On the one hand, the determination of the homology between the diverticulum and liver needs digestivve be supported by more evidence from evolutionary developmental biology investigations into the developmental gene regulatory networks of at least the liver, and preferably the whole endoderm [ 49 — 57 ].

On the other hand, because Branchiostoma cannot embody every feature of the ancestral chordate, and its diverticulum is possibly a specialized structure reflecting only Brachiostoma’s own evolutionary history or lifestyle, studies of echinoderms, hemichordates, urochordates and ammocoetes larval lampreys are indispensable to investigating this question [ 58 ].

Furthermore, determining the evolutionary histories of animal characteristics ddigestive be done well without direct palaeontological evidence [ 59 — 61 ], so the question of whether the last common chordate had a liver or not will remain open until new fossil evidence that provides a clear answer can be found [ 62 ].

The results of this study confirm the existence of phagocytic intracellular digestion in Branchiostoma and explain why epithelial cells in the Branchiostoma digestive tract express a number of typical immune genes.

The epithelial cells of the Branchiostoma digestive tract can phagocytize food particles, such as algal cells, directly. The phagocytic capability of diverticulum epithelial cells is greater than that of hind gut epithelial cells.

The major physiological function of the Branchiostoma diverticulum is also different from that of the vertebrate liver. Holland for technical and scientific guidance, E. Cui for scientific guidance, M. Gai for scientific guidance and drafting the manuscript, and K.

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