Sea Anemones through X-rays: Visualization of Two Species of Diadumene (Cnidaria, Actiniaria) Using Micro-CT Author Gusmão, Luciana C. Author Grajales, Alejandro Author Rodríguez, Estefania text American Museum Novitates 2018 2018-09-17 2018 3907 1 47 http://www.bioone.org/doi/10.1206/3907.1 journal article 7883 10.1206/3907.1 545af53d-7f24-4445-be19-8da7966398d8 0003-0082 4566328 Diadumene manezinha , new species Figures 1 , 6 –9, table 2 MATERIAL: Holotype . BRAZIL , Santa Catarina , Florianópolis , dock located in the Barra da Lagoa Channel , 27°35′4.49″S ; 48° 26′10.45″W , collected November 19, 2011 , by L. Gusmão and A. Grajales (0 m). MZUSP 002504 ( 1 specimen ). Material examined for comparison: Diadumene neozelanica ZMUC ANT-000076 ( 4 specimens ; syntypes ); Locality: New Zealand, Kaipara, North Island , Slipper Island , -37.05, 175.94, collected January 8, 1915 , by Th. Mortensen “Hinemoa” ( Th. Mortensen’s Pacific Expedition ). Diadumene sp. MNRJ 7055 ( 6 specimens ); Locality: Flutuante da Vaca , RJ, Brazil, collected November 1, 1990 , by D.O. Pires. Diadumene sp. ( 8 specimens ); Locality: Praia do Boqueirão , Ilha do Governador , RJ, Brazil, collected August 10, 1987 , by INSB- E. Martins. Diadumene sp. MNRJ 7169 ( 20 specimens ); Locality: Praia da Guanabara , Ilha do Governador , RJ, Brazil, collected July 11, 1987 , by INSB-E. Martins. DIAGNOSIS: Individuals with inconspicuous cinclides arranged in longitudinal rows on scapus. Fighting tentacles with holotrichs of two types may be present. No anatomical irregularity due to asexual reproduction; 24 pairs of mesenteries in three cycles of mesenteries at midcolumn; third cycle never with distinct retractor or filaments. Actinopharynx with small p -mastigophores A (18–38.7 × 2.7–5.0 µm); acontia with three categories of p -mastigophores B2a (20.9–33.2 × 4.0– 5.4 µm ; 35.0–53.9 × 5.8–10.0 µm; 57–69.0 × 7.9–10.7 µm ). EXTERNAL ANATOMY (fig. 6): Live and preserved specimens up to 11.0 mm in length (fig. 6A–C). Most preserved specimens with oral disc relaxed exhibiting visible tentacles (fig. 6B–D). Pedal disc flat, circular, adherent, diameter 1.5–5.5 mm in diameter in preserved specimens (fig. 6A–C). Column cylindrical, smooth, divided into long scapus and short capitulum (fig. 6A–C). Capitulum usually not distinct in fully extended live specimens (fig. 6A) and well- relaxed preserved ones (fig. 6C) or retracted into scapus in contracted preserved ones (fig. 6B); margin of capitulum tentaculate. Scapus with inconspicuous cinclides not positioned on top of raised projections (fig. 6A–C); cinclides arranged in 12 longitudinal rows with 2–4 cinclides per row distributed on proximal to distal scapus but more conspicuous in proximal half of scapus in live (fig. 6A) and preserved specimens (fig. 6C). Column olive green with mesenterial insertions visible as beige lines on column from limbus to distal scapus (fig. 6A–C). Column diameter 2.0–7.0 mm and length 1.0–11.0 mm in preserved specimens. Oral disc circular, small, as wide or slightly wider than column, olive green becoming lighter close to base of inner tentacles with large orange, central mouth exhibiting 12 distinct lobes in live specimens (fig. 6D). Oral disc diameter 1.0– 4.5 mm in preserved specimens (fig. 6E). Tentacles 89–92, smooth, long, slender and pointed, arranged in five cycles (6+6+12+24+ n ) in outer half of oral disc in both live (fig. 6D) and preserved specimens (fig. 6E). Tentacles of first and second cycles olive green somewhat darker than the light green or yellow tentacles in outer cycles (fig. 6A, D); tentacles with no markings in live specimens (fig. 6D). All tentacles translucent beige in preserved specimens (fig. 6B, C, E). Inner tentacles longer than outer ones in both live (fig. 6A, D) and preserved specimens (fig. 6B, C, E); longest tentacle up to 3 mm in live and preserved specimens. Fighting tentacles large, with blunt tip and broad base, exhibiting different color from feeding tentacles, observed in three preserved specimens (fig. 6E). Six fighting tentacles of first cycle observed in two specimens; six tentacles of first cycle and one of second cycle observed in one specimen (fig. 6E). One fighting tentacle of the specimen with seven of them had its tip autotomized (fig. 6E, arrow). FIG. 6. External anatomy of Diadumene manezinha , sp. nov. A, lateral view of an extended living specimen showing row of cinclides (arrows); B, lateral view of preserved specimen showing capitulum retracted into scapus (arrow); C, lateral view of preserved specimen showing cinclide on scapus with protruding acontium (arrow) D, oral view of living specimen; E, oral view of preserved specimen showing fighting tentacles; notice fighting tentacle with autotomized tip (arrow). Scale bars: A, 3.0 mm; B, 2.5 mm; C, 3.5 mm; D, 1.5 mm; E, 2.0 mm. INTERNAL ANATOMY AND HISTOLOGY (figs. 7, 8): Body short and broad in preserved specimens (fig. 8A) with wall thickness varying along column: all three body layers thicker in scapus than capitulum; limit between scapus and capitulum gradual (fig. 8B, C) with transition zone visible in histological sections (figs. 7A). Cinclides mostly inconspicuous, not positioned on top of raised projections, but easily observed in histological sections (fig. 7B). Cinclides distributed in endocoels corresponding to first and second cycle pairs of mesenteries. Longitudinal endodermal musculature of column strong (fig. 7C). Actinopharynx up to 2 mm in length, approximately one third of column’s length (fig. 8B), longitudinally sulcated throughout; with thick and highly glandular epidermis (fig. 7E). Specimens with two differentiated siphonoglyphs (fig. 8E) exhibiting thin gastrodermis and mesoglea, but glandular epidermis as in actinopharynx (fig. 7E). Longitudinal musculature of tentacles ectodermal (figs. 7D, 8D). Mesenteries hexamerously arranged in three cycles (6+6+12 = 24 pairs) spanning most of body length: first cycle perfect, including two pairs of directives, each associated with one siphonoglyph (fig. 8E); second and third cycles imperfect (fig. 8E). A few specimens exhibited irregularities in the distribution of mesenteries but only proximally (fig. 8F, G). More mesenteries distally than proximally (fig. 8G). All mesenteries of first and second cycles, including directives, fertile and with filaments (figs. 7A, 8F); those of third cycle sterile and without filaments (figs. 7F, 8F). Species gonochoric: major axis of oocytes 16.4–31.3 µm in diameter; major axis of spermatic cysts 87.0–221.0 µm in diameter in specimens collected in November. Retractors of first and second cycles strong, most diffuse but some restricted (figs. 7F, 8F); those of third cycle very weak (figs. 7F, G, 8E, F). Parietobasilar musculature very weak in all mesenteries, with no free mesogleal flap (fig. 7G); not visible in micro-CT images (fig. 8I). Basilar musculature of mesenteries weak (figs. 7H, 8J). CNIDOM (fig. 9): Spirocysts, basitrichs, p -mastigophores A, p -mastigophores B1, p -mastigophores B2a, and holotrichs. Acontia contain two types of nematocysts: basitrichs and p -mastigophores B2a. See figure 9 and table 2 for size and distribution. DISTRIBUTION AND NATURAL HISTORY: Specimens were collected attached to a rope tied to a dock in the channel that connects Lagoa da Conceição to Praia da Barra da Lagoa (closer to the latter). Individuals formed large aggregations with specimens of variable sizes. ETYMOLOGY: The species epithet refers to the popular name of inhabitants of the type locality of the species, Florianópolis , Santa Catarina , Brazil (i.e., manezinhos da ilha ) . MOLECULAR PHYLOGENETIC ANALYSIS: Similar sequence lengths were obtained for all Diadumene specimens studied: approximately 600 bp were sequenced for 12S, 400 bp were sequenced for 16S mitochondrial rDNA, while approximately 1500 bp were obtained for 18S nuclear rDNA. The phylogenetic relationships recovered are depicted in figure 10: within Metridioidea , clade Metridina of Rodríguez et al. (2012) was recovered with high support (99%), and included a well-supported monophyletic Diadumenidae (100%) sister to a clade containing members of Metridiidae and Acricoactinidae Larson, 2016 . The genus Diadumene was recovered as monophyletic with high support (100%) and was composed of two major clades: one formed by the two specimens of D. leucolena (from the coasts of Brazil and the United States ) found as sister taxa to D. manezinha with high support (100%), and the second formed by the remaining species of Diadumene included in this study ( D. cincta , D. paranaensis , Diadumene sp., and D. lineata ). MICRO-CT SCANNING: Different tissue types were successfully stained with osmium tetroxide and micro-CT scanning resulted in high-contrast images consistent between the two species of Diadumene examined (figs. 4, 8). Fine details of external and internal anatomical characters traditionally used in the taxonomy of sea anemones were readily observed in the 2D micro-CT images and 3D volumetric renderings of each species. 2D MICRO-CT RECONSTRUCTIONS: Most external anatomical features were easily identified in the 2D micro-CT images: pedal disc morphology (figs. 4B, 8B), column morphology and its division into scapus and capitulum (figs. 4B, 8B, C), columnar specializations (i.e., cinclides, 3D MICRO-CT RECONSTRUCTIONS: All external features observed in the 2D micro-CT scans were also seen in the computer-aided 3D reconstructions for Diadumene leucolena and D. manezinha (figs. 4A, 8A). Column morphology and its division into scapus and capitulum was accurately visualized only for D. leucolena (fig. 4A), due to the degree of contraction seen in the specimen of D. manezinha scanned (fig. 8A). Likewise, while the morphology, number, and arrangement of feeding tentacles and fighting tentacles in D. manezinha (fig. 8A, D), and feeding tentacles of D. leucolena was easily observed in the 3D reconstruction (fig. 4A), the tentacles of D. leucolena displayed lower resolution in certain regions corresponding to areas where 2D micro-CT images exhibited low contrast (fig. 4F). Similarly, all internal characters observed in the micro-CT images were observed in the 3D reconstructions and certain characters were especially prominent as a result of the tridimensionality from the 3D reconstructions: mesentery growth pattern (fig. 8I), number of mesenteries distally and proximally (figs. 4G–I, 8E–G), distribution of filaments (figs. 4H, 8F), and gametogenic tissue (fig. 8F). In addition, establishment of characters such as position of cinclides (e.g., endocoels of first and second cycles in D. FIG. 7. Internal anatomy and microanatomy of Diadumene manezinha , sp. nov. A, longitudinal section through column showing differentiation between scapus (dotted line) and capitulum (solid line) with limit between the two indicated by horizontal bar; notice cinclide on scapus (arrow); B, longitudinal section through distal column showing a cinclide in detail (arrow); C, longitudinal section through mid-scapus showing endodermal musculature (arrow); D, cross section through a feeding tentacle showing ectodermal longitudinal musculature; E, cross section through distal scapus showing retractors of directive mesenteries and spermatic cysts; F, cross section through proximal scapus showing cycles of mesenteries (indicated by numbers) and a pair of directives; note mesenteries of first and second cycles with oocytes; G, detail of mesenteries of third cycle (indicated by number); H, cross section through pedal disc showing basilar musculature (arrows). Abbreviations: ca, capitulum; di, directive mesenteries; ep, epidermis; ga, gastrodermis; oo, oocytes; sc, scapus; si, siphonoglyph; sp, spermatic cysts. Scale bars: A, 0.25 mm; B, C, 0.05 mm; D, 0.025 mm; E, G, H, 0.2 mm; F, 0.4 mm. fig. 4B–E) and feeding (figs. 4F, 8D) and fighting tentacles (fig. 8D). The tentacles of Diadumene leucolena , however, exhibited low contrast (fig. 4F). The ectodermal musculature of tentacles could not be not visualized in the images (figs. 4F, 8D). The distribution of cinclides in endocoels of first and second cycles was particularly easy to establish in micro-CT images from D. leucolena (fig. 4C–E). Internal features were also readily visualized in micro-CT scans, including actinopharynx size and morphology (figs. 4B, G, 8B, E), number and distribution of siphonoglyphs (figs. 4G, 8E), presence and distribution of filaments (figs. 4B, H, 8B, F), and gametogenic tissue in D. manezinha (fig. 8B, F, H, I), the only species found fertile. The gametogenic tissue exhibited particularly high contrast to osmium tetroxide, which facilitated the recognition of fertility patterns in the specimen of D. manezinha scanned. Although general musculature shape and morphology was accurately established from micro-CT scans for both species examined (e.g., retractors, figs. 4B, G, H, J, 8E, F, H, I; basilar musculature, figs. 4L, 8J), the resolution of the images obtained was not sufficient to visualize details of musculature (e.g., number and branching pattern in retractors). FIG. 8. Micro-CT volumetric rendering and sections of Diadumene manezinha , sp. nov. A, lateral view of 3D rendering of whole specimen showing external anatomy showing; note the long fighting tentacle (arrow); B, longitudinal section through whole specimen showing external and internal anatomy; C, cross section through distal column showing detail of capitulum; D, cross section through fighting tentacle and two feeding tentacles; E, cross section through distal scapus showing pairs of mesenteries of three cycles (indicated by numbers) including directive mesenteries; F, cross section through midscapus showing cycles of mesenteries (indicated by numbers) and gonads on first and second cycles; notice unpaired mesentery of third cycle (arrow); G, cross section through column at different levels showing unpaired mesentery of third cycle only distally (arrow); H, cross section through proximal scapus showing a pair of mesenteries of first cycle with diffuse retractor and no visible parietobasilar musculature; notice oocytes on pairs of mesenteries of second cycle; I, cross section through proximal scapus showing detail of a pair of mesenteries of second and third cycles; parietobasilar musculature is not visible (arrow); J, cross section through pedal disc showing basilar musculature (arrows). Abbreviations: ac, actinopharynx; ca, capitulum; di, directive mesenteries; fe, feeding tentacle; fi, filament; fit, fighting tentacle; ga, gastrovascular cavity; oo, oocytes; re, retractor musculature; sc, scapus. Scale bars: A, 1.5 mm; B, 2.0 mm; C, D, 0.4 mm; E, G–J, 1.0 mm; F, 2.0 mm; I, 0.8 mm. Table 2. Size ranges of the cnidae of Diadumene manezinha , sp. nov. Ẍ, mean; SD, standard deviation; S, proportion of specimens in which each cnidae was found; N, Total number of capsules measured; F, frequency; +++, very common; ++, common; +, rather common; -, rare.
Categories Range of length and width of capsules (µm) Ẍ ± SD S N F Diadumene neozelanica (ZMUC-ANT000076)
COLUMN
Basitrichs I (A) 08.1–16.0 x 1.3–2.5 10.5±1.1 x 1.8±0.2 186 6/6 +++ 13.1–17.0 x 1.8–2.8
Basitrichs II (B) 11.6–21.5 x 2.3–4.5 17.3±2.0 x 3.4±0.4 289 6/6 +++ 13.8–24.1 x 3.0–3.9
p -mastigophores B2a (C–D) 09.7–27.3 x 2.6–5.5 16.6±4.2 x 4.3±0.6 399 6/6 +++ 07.1–23.0 x 2.8–5.3
Holotrichs (E) 12.0–17.6 x 3.2–4.9 14.8±1.2 x 3.9±0.4 61 5/6 ++ 14.1–17.1 x 4.6–6.0
TENTACLES
Spirocysts (F) 12.3–26.0 x 2.1–6.0 19.9±2.9 x 3.5±0.7 269 5/5 +++ 10.0–17.0 x 1.9–5.2
Basitrichs G) 08.5–22.0 x 1.4–4.0 17.4±3.2 x 2.2±0.5 153 5/5 +++ 11.7–19.7 x 1.8–2.4
p -mastigophores B2a (H) 15.3–35.6 x 2.7–5.4 29.0±4.1 x 4.5±0.5 185 5/5 +++ 07.7–15.4 x 2.3–4.2
FIGHTING TENTACLES
Holotrichs I (I) 13.7–14.6 x 3.2–3.8 14.1±0.5 x 3.5±0.3 52 3/5 ++ 22.6–38.4 x 4.1–5.6
Holotrichs II (J) 17.0–29.0 x 3.0–4.9 20.9±2.3 x 3.3±0.4 65 3/5 ++ 21.2–36.3 x 8.3–12.5
ACTINOPHARYNX
Basitrichs (K) 10.6–24.4 x 1.4–2.8 19.4±1.9 x 2.0±0.2 181 6/6 +++ 14.2–28.6 x 2.0–3.0
p -mastigophores A (L) 18.0–38.7 x 3.0–5.6 26.5±5.5 x 4.1±0.6 87 6/6 ++ 14.1–21.7 x 2.2–3.9
p -mastigophores B2a I (M) 08.9–20.0 x 2.7–5.0 14.5±2.3 x 3.7±0.5 75 5/5 ++ 10.4–18.0 x 2.7–4.4
p -mastigophores B2a II (N–O) 16.9–42.0 x 3.0–6.0 26.8±2.9 x 4.2±0.6 313 6/6 +++ 21.6–32.4 x 2.8–4.9
FILAMENT
Basitrichs (P) 08.4–20.4 x 1.3–3.5 11.1±3.6 x 1.8±0.6 55 4/5 + 11.3–15.7 x 1.3–3.0
p -mastigophores A - 17.2–19.4 x 3.7–3.9
p -mastigophores B1 I (Q) 5.8–10.4 x 3.2–5.4 8.4±0.9 x 4.2±0.3 246 5/5 +++ 09.1–13.5 x 3.7–5.4
p -mastigophores B1 II (R) 11.6–19.6 x 3.3–5.7 15.0±1.4 x 4.4±0.4 129 5/5 +++ 15.2–22.7 x 4.3–6.0
p -mastigophores B2a (S) 10.5–33.9 x 3.1–5.2 15.4±4.0 x 3.8±0.4 100 5/5 ++ 10.1–23.6 x 2.7–4.7
p -mastigophores B2a - 30.8–39.7 x 4.8–5.8
ACONTIA
Basitrichs (T) 09.7–16.8 x 1.2–3.0 14.3±0.4 x 2.1±0.4 124 6/6 +++ 09.2–18.7 x 2.1–3.4
p -mastigophores B1 - 17.0–23.0 x 2.5–3.2
p -mastigophores B2a I (U) 20.9–33.2 x 4.0–5.4 26.7±3.1 x 4.9±0.5 110 6/6 ++ -
p -mastigophores B2a II (V) 35.0–53.9 x 5.8–10.0 44.4±3.5 x 7.5±0.9 170 6/6 +++ -
p -mastigophores B2a III (X) 57.0–69.2 x 7.9–10.7 61.7±3.1 x 9.1±0.7 54 6/6 + 51.2–77.0 x 5.1–8.2
FIG. 9. Cnidom of Diadumene manezinha , sp. nov. A, B, G, K, P, T, basitrich; C, D, H, M, N, O, S, U, V, W, p -mastigophore B2a; E, I, J, holotrich; F, spirocyst; L, p -mastigophore A; Q, R, p -mastigophore B1. Scale bar: 15 µm . leucolena ) was considerably facilitated by the use of virtual sections from 3D volumetric renderings (fig. 4A–E). ARTIFACTS: Artifacts did not affect the accuracy of micro-CT images, posterior 3D representation, or establishment of characters for the two species examined. Among the artifacts, we observed low contrast of micro-CT images of the gastrovascular cavity core in the specimen of Diadumene manezinha compared with more peripheral areas (fig. 8B, E, F). Similarly, micro- CT images of the distal part of the column corresponding to the capitulum of D. manezinha suggested imperfect penetration of the stain in this specimen (fig. 8E). Probably resulting from imperfect penetration of the stain, air pockets inside the gastrovascular cavity of D. leucolena were seen as black voids in the micro-CT images (fig. 8B). Air pockets, however, were small and did not affect the morphology of internal features or compromise the recognition or orientation of surrounding anatomical characters. A more pervasive artifact related to air pockets caused a greater technical problem for the method: movement of the specimen during scanning. Air pockets created inside the specimen before scanning may result in slight movements if the specimen is not securely positioned, leading to blurry images and posterior problems in the alignment of multiple scans (results not shown).