Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Xenacoelomorpha is an early-branching bilaterian lineage of aquatic worms. Animal icons are from phylopic.org , and are in the public domain; the dashed line reflects uncertainty in the consensus phylogeny ( ; ; ; ). ( B ) Dorsal view of an adult Hofstenia miamia . ( C ) Ventral view of an adult H. miamia ; most reproductive structures are visible in this view. ( D ) Schematized view of the ventral surface of a worm with known reproductive structures illustrated. ( E ) Time course of a representative worm through development, from hatchling to reproductively mature adult. ( F ) Schematic of time course shown in ( E ) with key reproductive developments illustrated. The first appearance of each organ is highlighted in red. ( G ) The length of worms increases over time ( R 2 = 0.91, p < 0.0001), and ( H ) worms grow proportionally: their length scales with their width ( R 2 = 0.85, p < 0.0001). Error band shows 95% confidence interval. ( I–K ) The length of each reproductive organ scales with increases in body size (penis: R 2 = 0.70, p < 0.0001; seminal vesicle: R 2 = 0.63, p < 0.0001; ovaries: R 2 = 0.84, p < 0.0001). Error band shows 95% confidence interval, with zero values excluded from these regressions. ( L ) Worms with delayed feeding increases had significant delays in the appearance of their seminal vesicle and ovaries, but not the penis (Welch’s t -test for date of appearance for penis: p = 0.08, seminal vesicle: p = 0.04, ovary: p < 0.0001; n ≥ 15). ( M ) Worms with delayed feeding increases had a smaller body length when a penis and seminal vesicle appeared, but not when ovaries appeared (Welch’s t -test for length on date of appearance for penis: p = 0.005, seminal vesicle: p = 0.03, ovary: p = 0.74; n ≥ 15). Asterisks indicate statistical significance. ( N ) Ranking the order in which reproductive organs appear ( y -axis) in developing worms reveals a stepwise pattern of reproductive differentiation. The x -axis shows individual worms. dpl = days post laying. Scale bars: 1 mm.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques:

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) The male reproductive system includes the male gonopore, penis, prostatic vesicle, and seminal vesicle. ( B ) H. miamia ’s ovaries are lateral and organized along the anterior–posterior axis. There is no visible organization of oocytes by size or stage within the ovaries. ( C ) A cluster of zygotes is visible medially, and immediately posterior to the male copulatory apparatus. ( D ) Representative time course of embryonic development of embryos dissected from the central cavity. Scale bars: 1 mm.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques:

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Schematic of regeneration of the penis, seminal vesicle, and ovaries following three different amputations. Shading indicates the tissue that regenerates. ( B–J ) Growth dynamics of reproductive organs (within column) for each of three amputations (within row). Error bands show SEM. ( K ) Time course of a starving worm undergoing de-growth and stepwise loss of reproductive organs. ( L ) Schematic of reproductive organ degradation as seen in ( K ) over the course of starvation-induced de-growth. ( M ) Worm length decreases over the course of de-growth ( R 2 = 0.85, p < 0.0001). Error band shows 95% confidence interval. ( N ) Worms shrink as they grow; their lengths and widths decrease proportionally ( R 2 = 0.73, p < 0.0001). Error band shows 95% confidence interval. ( O ) Across different growth contexts, reproductive organs appear or disappear at roughly consistent body lengths. Ranking the order in which reproductive structures are lost in worms undergoing de-growth ( P ) and gained in regenerating worms ( Q ) shows that organs are gained and lost in roughly the same order in all growth contexts. The x -axis shows individual worms in these plots. dps = days post onset of starvation. dpa = days post amputation. Scale bars: 1 mm.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques:

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A, C, E ) Representative time course of regenerating tail tip, sagittal cut, and head, and ( B, D, F ) schematized version with reproductive structures shown. ( G ) Regenerating heads and tail tips increase in length following amputation while sagittally cut fragments shrink and then begin to grow. Error bar shows SEM. ( H ) Ovaries scale with body length in sagittally cut fragments. ( I ) Ovaries regenerate once a tail fragment reaches a certain length. Scale bars: 1 mm.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques:

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Worm length decreases over time when worms are starved (starved: R 2 = 0.85, p < 0.0001; fed control: R 2 = 0.004, p = 0.48). Error band shows 95% confidence interval. ( B–D ) Penis length (starved: R 2 = 0.54, p < 0.0001; fed control: R 2 = 0.04, p = 0.03), seminal vesicle length (starved: R 2 = 0.60, p < 0.0001; fed control: R 2 = 0.03, p = 0.06), and mean ovary length (starved: R 2 = 0.67, p < 0.0001; fed control: R 2 = 0.02, p = 0.15) decrease over time for worms experiencing de-growth but do not change in worms that are continuously fed. Error band shows 95% confidence interval. ( E ) The number of zygotes in the central cavity decreases over time in both starved and fed worms, likely because these worms are isolated (starved: R 2 = 0.48, p < 0.0001; fed control: R 2 = 0.33, p < 0.0001). Error band shows 95% confidence interval. ( F ) When controlling for the effect of body size, the residuals of ovary length (regressed against body length) are strongly correlated between ovaries within worms over the course of de-growth (Pearson correlation coefficient = 0.75, p < 0.0001). Error band shows 95% confidence interval. ( G–I ) The size of reproductive structures scales similarly (see ) across development, de-growth, and regeneration (penis: R 2 = 0.70, p < 0.0001; seminal vesicle: R 2 = 0.50, p < 0.0001; ovary: R 2 = 0.76, p < 0.0001). Error band shows a 95% confidence interval. All data here are from regenerating fragments at least 2 weeks post-amputation, when worms begin regenerating their reproductive organs. ( J–L ) The residuals of organ length (regressed against body length) generally converge toward zero over the course of regeneration despite sometimes displaying large initial deviations. Data from regenerating fragments prior to 2 weeks omitted. ( M ) Sagittal fragments degrade their existing ovary during initial regeneration at a faster rate normalized to body size (slope = −0.015) than adult worms experiencing de-growth (slope = −0.003) (Wald’s test on slope of linear regression of ovary length and day: p = 0.0006). Error band shows 95% confidence interval.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Control, Isolation

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) A schematized view of the ventral surface of the worm with male reproductive structures highlighted in red. ( B ) Schematic of male reproductive structures with the copulatory apparatus (excluding the seminal vesicle) highlighted. ( C ) Labeling with an actin dye (white) labels the male gonopore, sheath, penis stylet, and prostatic vesicle. ( D ) A histological section also reveals these organs. ( E ) Schematic of the male reproductive system, with the penis stylet highlighted. ( F ) The stylets are a bundle of needles labeled by actin. ( G ) The posterior of the penis sheath terminates in a ring of hair-like projections, also labeled by actin. ( H ) Schematic of the male copulatory apparatus, with the prostatic vesicle highlighted. ( I ) Actin staining with a nuclear label (Hoechst) shows that the prostatic vesicle is enveloped by a thin epithelium-like layer, and contains densely packed sperm. ( J ) Schematic of the male copulatory apparatus, with the seminal vesicle highlighted. ( K ) The morphology of the copulatory apparatus in mature, adult worms is similar to that of early adults (previous panels). ( L ) The seminal vesicle of this adult worm contains densely packed sperm. ( M ) Dissecting out an adult seminal vesicle allows labeling of individual sperm cells, showing their distinctive morphology. ( N ) Schematic of a transverse view of an adult worm’s anterior, showing the relative organization of the seminal vesicle and testes. ( O ) Transverse sections show that testes appear as a continuous structure that spans the dorsal surface of the worm. ( P ) The testes extend through the dorso-ventral axis of the worm and wrap around the head. The pharynx (labeled and circled with a dotted line) contains residual food. ( Q ) Schematic of the male copulatory apparatus, with the testes highlighted. ( R–T ) Nuclear staining on an adult worm, cut sagittally, reveals the testes, which contain dense bundles of sperm organized around clusters of cells in the parenchyma. ( U ) Histological sections confirm this organization of the testes. Scale bars: 20 μm ( C, U ), 10 μm ( F–G, M ), 50 μm ( D, I, S, T ), 100 μm ( K, L, R ), 200 μm ( O, P ).

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Labeling, Staining

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) The gross morphology and location of the reproductive system can be seen in sagittally cut worms with nuclear staining. ( B, C ) Penis stylets are arranged in a bundle, seen with differential interference contrast (DIC). ( D ) Schematic of the male reproductive structures with the copulatory apparatus highlighted. ( E ) Antibody staining for FMRFamide shows that the penis sheath is surrounded by presumptive neurons. ( F ) Immunofluorescence for tropomyosin, and actin and nuclear labeling enables visualization of the musculature surrounding the male structures. ( G ) A packet of sperm ejected during mating made up of ( H ) sperm cells. ( I ) Schematic of the male reproductive structures with the testes highlighted. ( J ) Sperm in the testes develop in clusters interspersed with the muscle of the body, visualized by immunofluorescence for Tropomyosin, a muscle marker. Scale bars: 500 μm ( A ), 100 μm ( B, G ), 50 μm ( C, E, J ), 25 μm ( F ), 10 μm ( H ).

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Staining, Immunofluorescence, Labeling, Marker

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) The male reproductive system in a sagittal section of a juvenile worm has a male gonopore but lacks a clear male copulatory apparatus, prostatic vesicle, and seminal vesicle. ( B ) A longitudinal section showing the male gonopore. ( C–E ) Sagittal sections through the male reproductive system along the medial–lateral axis showing a channel of sperm (yellow arrows) connecting the prostatic vesicle and seminal vesicle. ( F–H ) Transverse sections moving along the anterior–posterior axis show the positions of the prostatic vesicle and seminal vesicle and the musculature and glands surrounding the seminal vesicle. Scale bars: 100 µm.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques:

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Fluorescence in situ hybridization (FISH) for the male germline marker pa1b3-2 results in two regions of ventrolateral expression that extend toward the dorsal surfaces. ( B ) Hoechst and pa1b3-2 mRNA expression label sperm cells. ( C ) Nuclear staining of cross-sections at different points along the anterior–posterior axis within the head shows that the testes emerge as two lateral lobes that gradually form a continuous cylindrical structure. Cartoons depict the plane of sectioning (red line) and reproductive structures (white). Scale bars: 250 μm ( A ), 50 μm ( B ), 200 μm ( C ).

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Fluorescence, In Situ Hybridization, Marker, Expressing, Staining

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Actin-dye labeling shows how the male reproductive system changes over the course of post-embryonic development (shown here from left to right). The sheath and stylet emerge first, followed by the appearance of the prostatic vesicle. ( B ) Fluorescence in situ hybridization (FISH) for the male germline marker pa1b3-2 results in two regions of ventrolateral expression that extend along the dorsal–ventral axis to different depths. Images are organized by pseudo-time: from least-developed (and smallest) on the left, to most-developed (and largest) on the right. Panels in ( C ) show depth-coloration, showing that the testes extend through the dorso-ventral axis. ( D ) Cross-sections of worms at different points in reproductive development stained with nuclear dye show that testes grow toward the dorsal surface and eventually meet to form one continuous structure. ( E ) Cartoon schematic of cross-sections shown in ( D ). Scale bars: 20 μm ( A ), 100 μm ( B, C ), 200 μm ( D ). Estimated worm lengths (wl) are noted under each panel.

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Labeling, Fluorescence, In Situ Hybridization, Marker, Expressing, Staining

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) A schematized view of the ventral surface of the worm with female reproductive structures highlighted. ( B ) Eggs near the pharynx of the worm (within the red circle) are fertilized and mature while oocytes in ovaries (red arrow) are immature or unfertilized, with a visible germinal vesicle. ( C ) A sagittal histological section shows that the ovaries contain oocytes of varied size and maturity embedded in the parenchyma. ( D ) Fluorescence in situ hybridization (FISH) shows that cgnl1-2 labels immature oocytes in the ovaries. ( E ) Oocytes in ovaries are also labeled by a Piwi-1 antibody. ( F ) A histological transverse section of an immature oocyte encircled by follicular cells. Inset: sperm cells appear to be trapped in the follicle. ( G ) Piwi-1 immunofluorescence confirms the organization of follicular cells, and nuclear staining sometimes identifies sperm apparently trapped in its surface (inset). Histology also shows that immature oocytes may have irregular shapes ( H ), contain a germinal vesicle ( H, I ), and possess an abundance of (likely yolk) granules ( I, J ). Blue arrows label germinal vesicles in all relevant panels; yellow arrows label sperm; white arrows label follicular cells. Scale bars: 100 μm ( B , C (inset), D–F ), 500 μm ( C ), 50 μm ( G–J ).

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Fluorescence, In Situ Hybridization, Labeling, Immunofluorescence, Staining

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) Sequence of images from a video of egg laying through the mouth. Eggs in the pharynx and emerging through the mouth are shaded blue. ( B ) Schematic showing presumed process of embryo traveling from the cavity beneath the pharynx to the pharynx and then out through the mouth. ( C ) Histogram showing the timing of eggs laid by adult worms living in communal tanks and then isolated. ( D ) Histogram showing the timing of eggs laid by worms that undergo reproductive development in isolation and then self-fertilize. ( E ) Histogram showing the timing of eggs laid by worms that are allowed to mate once. ( F ) Scatterplot of the percentage of eggs found on the floor of communal tanks ( n = 30). This is significantly different from the expected percentage of eggs based on tank surface area ( t -test, p < 0.0001). ( G ) Kernel density estimate of egg locations on a subset of tank surfaces with similar dimensions ( n = 2144 eggs). ( H ) Distance matrix of egg coordinates, with density-based spatial clustering, from a representative tank surface shows that eggs are often laid in clutches. Number of eggs in a clutch is shown in white; cluster identity is shown on the x and y axes. ( I ) In some culturing conditions, worms lay clutches of up to 145 eggs. ( J ) New worms add eggs to pre-existing clutches laid by other worms. ( K ) Worms that are unfed for 4 days lay fewer eggs than fed worms ( n = 9 tanks, t -test, p < 0.0001). ( L ) Unfed worms that are subsequently fed lay more eggs than worms that are continuously fed ( n ≥ 8 tanks, t -test, p < 0.0001).

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: Sequencing, Isolation

Journal: eLife

Article Title: Developmental, regenerative, and behavioral dynamics in acoel reproduction

doi: 10.7554/eLife.105712

Figure Lengend Snippet: ( A ) The life cycle of Hofstenia miamia , with major reproductive events displayed. ( B ) Family-level phylogeny of Acoelomorpha (Nemertodermatida, Acoela) showing anatomical and reproductive life history traits : position of the mouth, whether gonads are mixed or separated by sex, penis type, paired or unpaired testes, paired or unpaired ovaries, presence or absence of a female gonopore, presence or absence of a seminal bursa, the number of associated bursal nozzles, egg-laying mode, mode of sexual reproduction, alternative reproductive strategies, and regenerative capacity (see for definitions of terms and categories). Schematic diagram of the reproductive anatomy of representative species from each family within Acoelomorpha with specific structures colored: male copulatory organ (purple), sperm in testes and/or seminal vesicle (blue), oocytes (red), female or shared gonopore and/or bursa (green) . White boxes represent unknown phenotypic states, and in the case of asexual reproduction, its possible absence. Phenotypic classifications are from ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; and . Phylogeny of Acoelomorpha from and .

Article Snippet: In order to study the regeneration of reproductive systems, we isolated adult worms from communal tanks, anesthetized them in 15% tricaine (ethyl 3-aminobenzoate methanesulfonic acid), and then amputated them with micro knives (Fine Science Tools #10316-14).

Techniques: