Journal: Frontiers in Zoology

Article Title: Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)

doi: 10.1186/s12983-018-0259-8

Figure Lengend Snippet: Primary and secondary antibodies used in the study

Article Snippet: All neuronal antibodies used here were combined with antibodies against monoclonal acetylated tubulin (Abcam, Cambridge, MA, USA).

Techniques:

Journal: Frontiers in Zoology

Article Title: Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)

doi: 10.1186/s12983-018-0259-8

Figure Lengend Snippet: FMRFamide-immunoreactivity (FMRFa-ir) in the trochophore and veliger larvae of C. gigas. Green—FMRFa-ir; magenta—cilia, acetylated tubulin immunoreactivity; blue—nuclei, DAPI. The apical pole is always upward; the ventral side is on the right. a Trochophore at 20 hpf. Dorsal (arrowheads) and ventral (asterisks) groups of early neurons located posttrochally. a1 Higher magnification of right dorsal neuron (dn); arrowheads indicate a ciliated dendritic knob (ck) and basal neurites (n). a2 and a3 Right and left ventral neurons (vn, asterisks), respectively. b Trochophore at 24 hpf. Faint dotted immunoreactivity appears at the apical region ( arrows ). A dorsal cell sends an axon towards the ventral side ( arrowheads ). Three ventral cells indicated by one arrow (flask-shaped cells) and asterisks (round cells). b1 and b2 Higher magnification of the right and left dorsal neurons (dn), arrowheads indicate the ciliated dendritic knob (ck) of each cell. b3 and b4 Higher magnification of right and left ventral groups (vn); each ventral group contains two flask-shaped cells ( arrows ) and two round cells (asterisks). c Trochophore at 28 hpf. Two flask-shaped cells appear in the apical organ (AO). Growth cone ( arrows , gc) of the right dorsal cell (dn), where a growing axon reaches the ipsilateral ventral cell (vn); only the right sides of the larvae are shown. c1-c4 Right and left dorsal and ventral groups of neurons with DAPI. c5 and c6 Two flask-shaped neurons of the AO at two different magnifications (asterisks). c7 Higher magnification of a growing axon of the right dorsal cell; arrowhead indicates a ciliated dendritic knob, and arrows indicate a growth cone of the dorsal neuron. d D-hinge veliger stage at 36 hpf. The cells of the AO are located at the top (asterisks); no connections exist between the AO and other early cells. d1 Two flask-shaped apical neurons (asterisks) and their basal neurites. d2 and d3 Long axons of both the right and left dorsal cells reach the ipsilateral groups of the ventral cells. e Ventral view of the veliger at 36 hpf. Arrowheads indicate projections from dorsal and apical cells towards the ventral neurons. insets Higher magnification of the left and right ventral groups. e1 Higher magnification of the apical neurons demonstrating long cilia at the end of their dendrite ( arrows ). f The veliger stage at 52 hpf and 60 hpf (insert). Dorsal (dn) and ventral (vn) cells stain in a punctate pattern and their axons form an anlagen of the ventral nerve cord. Small immunopositive neurons appear posteriorly (posterior neurons, arrow , pn). Insert: Four flask-shaped apical neurons (asterisks) of AO. f1 Higher magnification of the AO (asterisk). f2 Long axon of a dorsal neuron (dn) to the ventral neuron (vn). f3 Neurites extending from the AO (arrow, an) follow the path established by pioneer axons of the dorsal neurons. g The veliger stage at 96 hpf. AO/CG consist of six flask-shaped cells (asterisks) and basal processes of AO/CG neurons organize into a compact central neuropil ( arrow , np). A single neurite (n, arrowhead ) extends from the neuropil of the AO/CG toward the velum. Paired ventral cords (vnc) with the interconnecting commissures ( short arrows ) are clearly visible. The anlagen of the pedal ganglia (PG) appear along each ventral cord in the region of the developing foot. g1 Flask-shaped neurons (asterisks) with neuropil forming (Z12). g2 Flask-shaped neurons (asterisks) with neuropil forming (Z14). g3 and g4 Neurons within the PG under high magnification (asterisks). g5 Posterior neurons under high magnification (asterisk). h The late veliger stage at 9 dpf. Neurites extend from the AO/CG to the dorsal side and dorsal edge of the velum ( arrowheads ). The anlagen of the PG are located along the ventral cord (vnc). Paired ventral cords are connected by two commissures ( short arrows ). h1 Compact neuropil in the center of the AO/CG. h2 and h3 Right and left PG anlagen; arrows point to the neuronal nuclei. h4 and h5 Higher magnification of the right and left posterior neurons; the asterisk marks the cell nucleus. i 3D reconstruction of the AO/CG complex, paired ventral cords and PG anlagen of 9-dpf late-veliger larvae. j Summary diagram of the ontogeny of the FMRFa-ir-containing structures in C. gigas. Only one lateral side is shown. Arrowhead indicates an axon of a dorsal cell in the late trochophore. Additional abbreviations: a – anus, f – foot, m – mouth, pmo – presumptive mouth opening, pt – prototroch, st – stomach. Scale bars =20 μm

Article Snippet: All neuronal antibodies used here were combined with antibodies against monoclonal acetylated tubulin (Abcam, Cambridge, MA, USA).

Techniques: Staining

Journal: Frontiers in Zoology

Article Title: Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)

doi: 10.1186/s12983-018-0259-8

Figure Lengend Snippet: Serotonin immunoreactivity (5-HT-ir) in the trochophore and veliger larvae of Crassostrea gigas . Green—5-HT-ir; magenta—cilia, acetylated tubulin immunoreactivity. The apical pole is always upward; the ventral side is on the right. a The early trochophore at 20 hpf. The first two flask-shaped neurons ( arrows , an) are located in the anterior extreme of the larval body. Each cell has a ciliated short dendrite ( arrowheads ). b D-hinge veliger at 36 hpf. 5-HT-ir cells comprise a compact apical organ (AO) and comprise flask-shaped and round cells each with thin neurites ( arrowheads , n1) running to the velum. b1 two flask-shaped cells with thin neurites and neuropil (np). b2 single round cells (black asterisk). c The veliger stage at 52 hpf. Cells of the AO extend three long neurites to the velum ( arrowheads , n1), anterior−dorsal ( arrowheads , n2), and posterior−ventral ( arrowheads , n3). Acetylated tubulin-ir reveals the digestive system and green autofluorescent particles are visible in the stomach (st) in b, c, d, e. insets: High magnification of the AO cell composition. c1 two flask-shaped and one round cells (black asterisk) (Z5), c2 third flask-shaped cells and second round cell (black asterisk), and neuropil ( arrows , np) c3 merge picture. There are five cells: three flask-shaped cells and two round cells (black asterisk); d The middle veliger stage at 96 hpf. Two posterior−ventral neurites from cells of the AO run in parallel along the ventral part of the larval body organizing the ventral nerve cords (vnc). e The veliger at 9 dpf. Cerebral ganglia (CG) are located at the top, and pedal ganglia (PG) are detected in the middle of the ventral nerve cord. Small immunoreactive posterior neurons (pn) are located near the caudal end of each ventral cord. Neurites extending from the CG have multiple branches in the velum region. Note the solitary branch extending from the apical part of the ganglion ( arrowhead ) towards the dorsal edge of the velum. f The veliger at 15 dpf. Note the paired ventral nerve cords with two commissures ( short arrows ). Cerebral ganglia located on top of the ventral cords. The thickening of the upper portion of each ventral cord represents the anlagen of the pleural ganglia (PLG) and together with the CG they form a fused CG/PLG complex indicated as CPG. Right and left PG are located along the respective ventral cords at the middle of the foot. Posterior neurons are located caudally, and each sends an axon to the ipsilateral ventral cord. The velum is richly innervated by fibers extending from the CPG. Thin neurites extend to the foot, and digestive organs (mark as peripheral nerve system, PNS) extend from the PG region ( arrowheads ). insets: f1 High magnification of the CG/PLG region; f1 focus on CG and PLG parts of CPG, PG, ventral cord, and peripheral innervation. g Summary diagram of the ontogeny of the 5-HT-ir-containing structures in C. gigas . Only one lateral side is shown. Additional abbreviations: f – foot, m – mouth, pmo – presumptive mouth opening, pt. – prototroch, st – stomach. Scale bars =20 μm

Article Snippet: All neuronal antibodies used here were combined with antibodies against monoclonal acetylated tubulin (Abcam, Cambridge, MA, USA).

Techniques:

Journal: Frontiers in Zoology

Article Title: Nervous system development in the Pacific oyster, Crassostrea gigas (Mollusca: Bivalvia)

doi: 10.1186/s12983-018-0259-8

Figure Lengend Snippet: Characterization of VAChT antibodies and VAChT-ir (VAChT, magenta) in oyster, Crassostrea gigas , tissues. a Western blot of total protein lysates from adult oyster tissue probes stained with goat polyclonal antibodies against rat VAChT. The specific band is detected in all tested oyster tissues as well as in cell lysate from the mouse spinal cord. b Double-staining for VAChT/tubulin (VAChT/TUB) of frozen sections of adult oyster tissues. A strong positive signal is detected in the anterior adductor muscle (muscle), mantle, and gills at the structures corresponding to the nerve bundles. c-e Confocal images of the larvae stained with VAChT/tubulin, right side view; the anterior is always up. c D-hinge veliger. The apical organ (AO) contains two to three cells and their basal fibers. Paired solitary neurons located posteriorly on the right and left sides of the larval body (pn). Each posterior cell sends an anteriorly directed fiber along the ventral edge of the larval body ( arrows ) (an asterisk marks the neuron nuclei). c1 Bodies of two AO neurons; asterisks mark the nuclei. c2 Posterior neuron, an asterisk marks the nucleus. d In the veliger stage (92 hpf), a strong VAChT-ir signal is detected within the AO/CG complex, and single fibers appear to innervate the velum ( arrowheads ). Immunopositive fibers run in two parallel, ventral cords (vnc), and a solitary posterior cell is visible at the caudal end of each ventral cord. Immunopositive cells appear in the PG. e At the 7-dpf veliger stage, VAChT-ir fibers from AO/CG innervate the velum, and fibers from PG innervate the foot (f) and regions around the mouth. g Summary diagram of the ontogeny of the VAChT-ir-containing structures in C. gigas . Only one lateral side is shown. Additional abbreviations: a – anus, m – mouth. Scale bars =100 μm in b and 20 μm in c

Article Snippet: All neuronal antibodies used here were combined with antibodies against monoclonal acetylated tubulin (Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Staining, Double Staining

Journal:

Article Title: Dual Acylation Accounts for the Localization of α19-Giardin in the Ventral Flagellum Pair of Giardia lamblia

doi: 10.1128/EC.00136-09

Figure Lengend Snippet: Expression of α19-giardin in trophozoites of G. lamblia. (A) RT-PCR on cDNA, total RNA, and RNA treated with DNase I as negative controls. (B) Western blots of trophozoite extract with pellet (P) and supernatant (S) fractions, each immunodecorated with α19-giardin antiserum. (C) Western blots of isolated flagella immunodecorated with antiserum directed against α1-giardin, α14-giardin, α19-giardin, and tubulin (Tub) as a charge control.

Article Snippet: For immune decoration of the blots, rabbit antiserum against recombinant α1-giardin and α14-giardin in dilutions of 1:10,000, α19-giardin in a dilution of 1:10,000 to 1:100,000, and a monoclonal antibody against anti-acetylated tubulin (mouse immunoglobulin 2b [IgG2b] isotype; Sigma) in a dilution of 1:2,000 were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation