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Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos and larvae


Abstract and Figures

Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in other fields. They are characteristic for their acoustic communication, and develop hearing and sound production while young juveniles. They lay large benthic eggs (>5mm) with a thick chorion and adhesive disk and slow development, which are particularly challenging for studying embryology. Here we took advantage of a classical tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series of embryos and larvae from 3 to 30mm in length, which allowed detailed 3D anatomical reconstructions non-destructively. We documented some of the developmental stages (early and late in development) and the anatomy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other techniques accessible to developmental biology labs, OPenT provided advantages in terms of image quality, cost of operation and data throughput, allowing identification and quantitative morphometrics of organs in larvae, earlier and with higher accuracy than is possible with other imaging techniques.
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Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus
didactylus embryos and larvae
Pedro M. Felix
, Ania Gonçalves
, Joana R. Vicente
, Paulo J. Fonseca
, M. Clara P. Amorim
José L. Costa
, Gabriel G. Martins
MARE Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal
Instituto Gulbenkian de Ciência, R. Quinta Grande, 6, 2780-156 Oeiras, Portugal
MARE Marine and Environmental Sciences Centre, ISPA - Instituto Universirio, 1149-041 Lisbon, Portugal
cE3c Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
abstractarticle info
Article history:
Received 4 July 2015
Received in revised form 21 February 2016
Accepted 4 March 2016
Available online xxxx
Batrachoidids, which include midshipman and toadsh are less known among embryologists, but are common in
other elds. They are characteristic for their acoustic communication, and develop hearing and sound production
while young juveniles. They lay large benthic eggs (N 5 mm) with a thick chorion and adhesive disk and slow
development, which are particularly challenging for studying embryology. Here we took advantage of a classical
tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series
of embryos and larvae from 3 to 30 mm in length, which allowed detailed 3D anatomical reconstructions non-
destructively. We documented some of the developmental stages (early and late in development) and the anat-
omy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other
techniques accessible to developmental bio logy labs, OPenT provided advantages in terms of image quality,
cost of operation and data throughput, allowing identication and quantit ative morphometrics of organs in
larvae, earlier and with higher accuracy than is possible with other imaging techniques.
© 2016 Elsevier Ireland Ltd. All rights reserved.
Inner ear
Swim bladder
Optical tomography
1. Introduction
Fishes are the largest extant group of vertebrates and exhibit a tre-
mendous diversity of features and adaptations (Nelson, 2006), includ-
ing many homologous to vertebrate tetrapods (e.g. Bass et al., 2008).
The study of embryonic development presents a unique opportunity
to investigate those homologies. Most of what we known of sh embry-
ology derives from work on the model organisms zebrash and medaka
(Kimmel et al. 1995; Iwamatsu, 2004), whose transparent and small
embryos are easily studied with conventional microscopy. Fish embryos
vary considerably both in size and shape (Richardson et al., 1997), with
zebrash and medaka falling at the small end of the spectrum. On the
other end, larval stages (even of the two small species), are too large
for conventional microscopy and are still studied resorting mostly to
histological sectioning (e.g., Sabaliauskas et al., 2006). (See Table 1.)
Morphomics and a rekindled interest in detailed anatomical studies
have recently gained prominence in developm ental biology, after
mesoscopic imaging by Optical Projec tion Tomography or Light-
Sheet Microscopy, were introduced to embryology by Sha rpe et al.
(2002) and Huisken et al. (2004), respectively. Both techniques proved
valuable to study embryos of model organisms, in ways that were not
possible with conventional microscopy; for example, light-sheet
micro scopy is well suited for imaging the early develo pment of
live zebrash and drosophila embryos (Huisken et al., 2004; Keller
et al., 2008), and opti cal tomography for 3D imaging large embry os
(Bryson-Richardson and Currie, 2004; Ruparelia et al., 2014). The
open-source community has already provided DIY solutions based on
hardware and software which, for the most part, are already familiar to
developmental biologists (Pitrone et al., 2013; Gualda et al., 2013). A
question some labs are facing is whether these techniques, in simpler
open-source forms are worth the trouble, in other words, whether
they provide better results than those obtained with existing techniques,
and effectively solve the difcu lties of large non-model organisms.
Fishes from the Batrachoididae family, which include midshipman
and toadsh, are less familiar to developmental biologists, but widely
used in ecotoxicology and ethology (Caçador et al., 2012), in bioacous-
tics (Ric e et al., 2011; Vasconcelos et al., 2012) and neurophysiology
(Bass et al., 2008; Vasconcelos et al., 2011; Elemans et al., 2014).
Batrachoidids lay large benth ic eggs (N 5 mm) with a thick chorion
and adhesive disk, and the embryos develop rather slowly (Arora,
1948; Dovel, 1960; Britz and Toledo-Piza, 2012), making them less ame-
nable for ontogenetic studies. They are characteristic for their acoustic
communication, and although it is known that hearing and sound
Mechanisms of Development xxx (2016) xxxxxx
Corresponding author at: Instituto Gulbenkian de Ciência, R. Quinta Grande, 6, 2780-
156 Oeiras, Portugal.
E-mail addresses: (P.M. Felix), (G.G. Martins).
MOD-03395; No of Pages 6
0925-4773/© 2016 Elsevier Ireland Ltd. All rights reserved.
Contents lists available at ScienceDirect
Mechanisms of Development
journal homepage:
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
production develop early (Vasconcelos and Ladich, 2008; Alderks and
Sisneros, 2011; Vasconcelos et al., 2015) the details on the ontogeny
of the associa ted anatomical structures remain largely unknown.
Those structures are too minute and delicate for micro dissection, and
yet too large and deep inside the larvae to be accessible by conventional
micro scopy; furthermore, since some of the structures are cavities
(e.g., the contents of the otic capsule) they cannot be properly dissected
out, and are best studied intact in toto. Having obtained a collection of
Halobatrachus didactylus (the Lusitanian toadsh) embryos at several
stages with sizes ranging from 3 to 30 mm in length, we took advantage
of a classical tissue clearing technique and a custom-built OPenT -
optical tomography sc anner, based on the Open SpinMicroscopy
platform (Gualda et al., 2013), to gain insight into the anatomy and de-
velopment of the stato-acoustic organs, swimming bladder and associ-
ated sonic muscles, and highlight the potential of optical tomography
as a prime tool for developmental biologists.
2. Results & discussion
The H. didactylus embryos were rst visible only betwee n 10
12 days post-fertilization (dpf) as a 2.82. 9 mm lon g streak with an
engorged rostral end. After the second week, embryos reached N 3mm
in length, and showed a neural tube, otic and optic vesicles, pectoral
n buds and overt segmentation of paraxial mesoderm (Fig. 1C), with
1520 somites; none of the major organ systems were yet recognizable
at this stage. As a way of comparison, this was merely equivalent to a
zebrash 17 h post-fertilization (Kimmel et al., 1995). Up to this stage,
the embryos were too small and positioned far from the centre of the
large yolk mass, to allow optimal imaging with optical tomography.
They could be imaged with confocal microscopy (Fig. 1), but that re-
quired excising the embryo from the yolk sac and acquiring Z-stacks
in multiple adjacent elds (followed by 3D-image stitching) using a
10× objective, otherwise the embryo's natural curvature did not pro-
vide access to the limited working distance of high-quality objectives;
confocal imaging wit h low NA (e.g. 4× magnication) objectives did
not provide images properly resolved in depth.
After the rst two weeks, and throughout organogenesis, embryos
could no longer be imaged with confocal microscopy and only OPenT
provided images of the whole embryo and its internal anatomical
details (Figs. 1, 2), with magnications of 0.33 × at the detector
plane, a range of magnications not available on conventional confocal
microscopy setups. Large-scale (i.e., low magnication) laser-scanning
imaging with a macro confocal allowed imaging of a lateral
view closer to that of OPenT, but with signicantly limited axial eld-
of-view and resolution, when imaging 30 dpf embryos. Though th e
lateral resolution was high (at the em bryo surface), the 3D dataset
was highly anisotropic, showing low axial resolution and light penetra-
tion when compared to images obtained with OPenT (not shown).
After the rst four weeks of development, all H. didactylus embryos
had hatched and most organ systems were already visible. These larvae
were developmentally equivalent to a 60 h pec-n stage zebrash
(Kimmel et al., 1995), though almost 3× larger. Our observations of ex-
ternal anatomy were similar to those described for the oyster toadsh
Opsanus tau by Tracy (1959) and Dovel (1960). Our use of OPenT
allowed the reconstruction and analysis of both the external and inter-
nal 3D anatomy in situ of H. didactylus, without the need to dissect or
section embryos or larvae, up until free-living forms 3 months old
(N 20 mm long; Fig. 1), often allowing identication of organs earlier
than was detected using a dissecting stereoscope. This demonstrates
that OPenT is a useful technique to study large embryos and larvae,
allowing detailed morphological studies up to late stages, covering the
full mesoscopic range.
Measurements obtained from 3D datasets, allowed us to follow
the embryo's natural curvature and determine the full length of the
body and head. The body grew progressively from the second week on-
ward at a pace of 0.147 mm/day (~6 μm/h), slowing down at the end of
the second month (Fig. 2C). This rate of growth is comparable to that
previously reported for O. tau (Tracy, 1959), and considerably slower
than that reported for Danio rerio which grows at a rate of 125 μm/h
during embryogenesis and at 20 μm/h during larvagenesis (Kimmel
et al., 1995). By the end of the second month, the yolk mass had been
consumed (Fig. 1) and larvae begun feeding and swimming freely. In-
terestingly, the head of H. didactylus (measured as the distance from
tip of mouth to level of pectoral n bud) grew constantly throughout or-
ganogenesis and larvagenesis (Fig. 2C), which contrasts with zebrash
and medaka whose heads' length practically does not increase during
organogenesis and early larvagenesis (as per gures in Kimmel et al.,
1995; Iwamatsu, 2004). A disproportionately large head is a feature of
most vertebrate embryos and early larvae, and is lost during
larvagenesis as the body lengthens at a fast pace. Our observations
suggest that the disproportionately large head patent in juvenile and
adult Batrachoidids, may be a neotenic trait, instead of a morphological
characteristic that develops secondarily.
Because of the interest of H. didactylus as a model organism for stud-
ies of communication we then focused our observations on the anatom-
ical details of the stato-acoustic organs. We found that the semicircular
canals (SCCs) + sacullae and swim bladder were well visible before the
end of the rst month (Fig. 2). Using OPenT 3D reconstructions we were
able to identify and precisely measure these structures earlier than was
possible using a stereoscope and dissection of fresh larvae. Before the
Table 1
Comparison of optical techniques used to image H. didactylus embryos.
Advantages Disadvantages
Stereoscope Allows fresh/live embryos
(i.e., not xed)
Fast screening
Fluorescence not required
Natural colour images
Inexpensive and widely available
No 3D imaging
Limited internal anatomy
Low contrast/resolution
Confocal (micro & macro) 3D imaging at cell resolution
Widely available
Requires embryo xation + clearing
Requires 3D stitching of whole embryos
Impractical to image larvae whole
Anisometric resolution; especially limiting in macro variant
Limited imaging in depth, even with macro confocal
Expensive to buy and operate
Optical μTomography (OPenT) 3D imaging of larvae (330 mm)
3D in uorescence & brighteld modes
Isometric resolution
High contrast images
Fast screening of whole anatomy
(even considering image processing steps)
Inexpensive to build and operate
Requires embryo xation + clearing
Limited resolution in early stages (before organogenesis)
Requires more experience with image processing
Commercially unavailable (has to be custom built, e.g., OPenT) as of 2015
2 P.M. Felix et al. / Mechanisms of Development xxx (2016) xxxxxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
end of the rst month, the ovoid-shaped otic vesicle had transformed
into a complex assembly of cavities which, when reconstructed in 3D re-
sembled a typical inner ear of sh, with three yet incomplete SCCs, and
the saccule and lagena (Fig. 2A). At 50 dpf, the three SCCs were
completely formed (i.e., their lumen was continuous), and a utricule
was now well individualized (Fig. 2B). The length of the otic capsule
also increased linearly from the second week onwards until the end of
the second month (Fig. 2D). The swim bladder rst appeared as a min-
ute sac (collapsed dorso-ventrally) appended to the gastro-intestinal
tract dorsally (Fig. 2A), although the sonic muscles were not yet discern-
ible; this was not noticeable with stereoscopy, even after dissecting the
larva. Later, at 50 dpf the swim bladder had differentiated into two well
individualized sacs, each with an associating sonic muscle which ap-
peared as two cell masses positioned medially t o the swim b ladder
(Fig. 2B). The growth of the swim bladder also seems to be linear during
the rst 2 months of development.
The datasets obtained with this work will be made available publicly
through the Haeckaliens online database (
research/haeckaliens), and the segmented organs of two specimens
can be inspected in 3D interactively with the 3D gure is supplementary
materials using the standalone Acrobat reader application (Adobe).
Video 1 shows a sequence of coronal, sagittal and axial sections of 30
and 46 dpf embryos, and a 3 month-old larva. The 3D reconstructions
of these organs allowed measurements with a nominal resolution of
2.75 μm (empirical resolution, measured as half-width-at-half-
maximum of small features, was of 5 μm; for the largest larvae, nominal
resolution was 5 μm, and empirical 10 μm).
Compared to other techniques accessible to developmental biology
labs, OPenT provided clear advantages both in terms of image quality,
cost of operation and data throughput. Though images of confocal mi-
croscopy are better resolved when imaging early embryos (i.e., before
the onset of organogenesis), they require excision of embryos and
acquisition of multiple high-magnication Z-st acks + 3D-stitc hing,
which is impractical once organogenesis is underway and organ sys-
tems begin assembling.
Other contenders to OPenT are X-ray micro-Computed Tomography
(μCT) and micro-Magnet ic Res onance Imaging (μMRI), which we
did not test. However, OPenT image details and speed acquisitions
were one order of magnitude smaller than those typical of μMRI and
similar to μCT, and cost of installation/operation two orders of magni-
tude lower than μCT, its closer contender. One further advantage of
OPenT for developmental biologists, besides the access ibility of the
Fig. 1. A) 12 dpf H. didactylus embryo, xed, dechorionated and imaged with: uorescence stereoscopy (top inset), confocal microscopy (middle) and OPenT (bottom). The embryo was
excised from the egg before imaging with the stereoscope and confocal microscope. B) Similar embryo imagedfresh with side-illumination and a colour camera coupled to a stereoscope;
because of the curvature, the embryo cannot be imaged completely in a single eld-of-view. C) 3D reconstruction of a 16 dpf embryo imaged with optical tomography; because of their
volume and curvature around the yolk sac, these embryos could no longer be properly imaged in 3D with confocal microscopy. D) 3D reconstruction of a 26 dpf embryo imaged with
optical tomography. By this stage most organ systems had formed and internal anatomical details could easily be studied (see Fig. 2); these were not visible in images obtained with a
macro confocal. E) 42 dpf embryo. F) Larva at 3 months, already free-swimming and completely devoid of yolk. Total length = 24.76 mm. See Supp material Movie 1 for slices and
details of internal anatomy of embryos depicted in D), E) and F).
3P.M. Felix et al. / Mechanisms of Development xxx (2016) xxxxxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
Fig. 2. 3D reconstruction of H. didactylus embryos showing left and dorsal views, internal anatomy and segmented organs of interest: Semi-circular canals (opaque blue), swim-bladder
(opaque orange), central nervous system (CNS; transparent blue), and gastro-intestinal tract (GI; transparent orange), sonic muscles (red). A) 26 dfp. Embryo total length = 8.6 mm
B) 50 dfp embryo; total length = 20.6 mm. Embryos are drawn to scale. B) After hatching at 50 dpf, the swim-bladder (orange) now consists of two separate chambers, and the sonic
muscles (red) are already forming. C) Graph showing increase in length of whole body and head length. D) Graph showing growth of otic vesicle/capsule and swim bladder, measured
as the an tero-posterior length (see diagrams in A) and B)). CNS = central nervous system, SB = Swim bladd er, SM = sonic mus cle, SCC = s emi-circu lar canal. See also
Supplementary gure in the form of an interactive 3D pdf image.
4 P.M. Felix et al. / Mechanisms of Development xxx (2016) xxxxxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
technique, is the similarity of sampl e pr eparation and imaging
The accessibility to image organs deep inside embryos/larvae with-
out having to physically dissect them is important to create visual 3D
maps of the location and shape of these organs, and to understand
how they are formed during development. It is especially important in
the case of inner-body cavities, such as SCCs or the otic vesicles, which
cannot be easily dissected out and are best studied in toto. Following
with precision the development of hearing and sound organs allows
parallels with studies of ontogenesis of behaviour of acoustic communi-
cation and its role in social interactions. It is also useful for studies
involving the physiology of hearing and sound production, namely to
dene the ages at which these organs are formed and function and for
the proper positioning of electrodes for stimulation. It has potential
application for developing optogenetics methods which rely on precise
location of tissues/cells of interest in deep tissues. OPenT can easily cope
with large embryos, of which H. didactylus is a particularly challenging
example, but also large larval stages of more common species, which
are typically not used because of their size, especially zebrash larvae
beyond the rst days of development.
One particular aspect of the whole imaging and analysis workow
that remains a challenge for all these techn iques is the automatic
segmentation of anatomy. In our case, due to the isometric nature of re-
constructed tomograms, image stacks typically contained hundreds-to-
thousands of optical sections, and manual or semi-automatic segmenta-
tion, though cumbersome and with low through put, still was more
dependable than even machine learning tools such as those of WEKA
segmentation or Ilastik (Hall et al., 2009; Sommer et al., 2011). We facil-
itated the semi-automatic segmentation process by su btracting from
the reconstructed 3D tomogram a derivative (e.g. a Sobel edge detection
or Gr adien t Magnitude); interestingly this procedure also alleviated
some of the star-like and beam-hardening artefa cts that ca n occur
with reconstruction of optical CT images.
Another standing challenge to researchers is the difculty in pre-
senting real 3D imagery along the traditional manuscript format. For
that, we have explored accessible online tools which require only limit-
ed computer expertise from life-scientists. This included the prepara-
tion of interactive 3D-pdf illustrations as in Ruthensteiner and Heß
(2008), an example of which is presented in the supplementary mate-
rials, and the use of simple online tools for dissemination of OPenT 3D
datasets such as those used in the Haeckaliens online database.
3. Materials & methods
3.1. Egg/embryo collection
Fertilized eggs were colle cted in an intertidal zone of a sandy
beach in a public-restricted area (38° 41 41 N, 2 55 W, Montijo,
Portugal), and took place d uring the breeding season (JuneJuly
2013). Articial nests were placed 2 m apa rt and their inner surface
was coated with plasti c sheets to facilitate egg collection. Territorial
males spontaneously occupied these nests and the females, attracted
by the vocalizati on of males, entered the n est to spawn, after which
the eggs were fertilized. This semi-natural approach did not allow us
to determine the exact date and time of fertilization, so we also collected
males and females and kept them, in loco,inpoolswitharticial nests;
in this case eggs were collected immediately after fertilization and we
could record the rst stages of development. These eggs were treated
similarly to those of the intertidal area and used to determine the
exact age of the latter. Collected eggs were then transported to facilities
at Lisbon University and kept in aquaria under controlled conditions:
salinity of 22 and average temperature of 19 °C 1), with constant aer-
ation. To follow and register embryonic and larval development, a sam-
ple of four eggs was collected randomly from the batches at pre-set time
intervals: every 12 h for the rst four days; every 24 h for the following
ve days; every 48 h for the next 10 days; and every 96 h until the onset
of the juvenile stage. Embryos/larvae were anesthetized with MS222
and in vivo images were collected using a Leica DFC 280 digital camera,
coupl ed to a Leica MZ6 stereomicroscope and the Leica Application
Suite v4.1. Later, all larvae were exposed to a lethal dose of MS222, for
further sample preparation and 3D imaging.
3.2. Sample preparation, 3D image acquisition, analysis and presentation
For 3D imaging (optical tomography & confocal microscopy), em-
bryos were xed in 4% form alin for 24 h at 4 °C, washed extensively
with PBS and H
O at room temperature, and then slowly dehydrated
through a series of methanol soluti ons up to 100%. This dehydration
step, required for tissue clearing, was gradual to minimize tissue defor-
mations and anisometric shrinkage. When observed fresh under a ste-
reoscope, the eggs measured an average diameter of 6.19 ± 0.39 mm,
and after dehydration they had shrunk an avera ge 9.4% (5.66 ±
0.31 mm); all measurements presented herein do not account for this
Dehydrated embryos were then transferred to BABB (benzyl
alcohol:benzyl benzoate 1:2) until the tissues became completely trans-
parent, which required 15 days. Cleared embryos were glued with
cyanocrylate to the tip of a metal rod which was magnetically attached
to a stepper-motor axis, and imaged using a custom-built OPenT optical
tomography scanner as described in Gualda et al. (2013, 2014), which,
along with the ofcia l website includes all detai ls on how to build
the system ( inmicroscopy/). For
image acquisition, we used a DMK 2Mpixel camera (The Imaging Source
Inc.) mounted on an Innitube 1× lens + lens tube assembly (Innity
Optics), and captured the uorescence of whole embryos while rotating.
To obtain different magnications we changed the tube length in
innity space behind the objective, adjusting it so the embryo image
completely lled the CCD detector. Tissue auto-uorescence was ob-
tained by excitation with a 470 nm LED source (+470 ± 20 nm excita-
tion lter) , and collected after a LP510nm emission lter at innity
space in the lens tube. A total of 1600 projections were acquired for a
360° turn using micromanager and the OpenSpin plugin (Gualda et al.,
2013). The projection dataset was then pre-processed by noise ltering
and alignment of the rotation axis using FIJI (Schindelin et al., 2012;see
more details also in Gualda et al., 2013), and su bsequently, back-
projection reconstructed with the free software NRecon (Skyscan
Inc.). This produced an isometric 3D stack of sections from which the
internal anatomical details and 3D reconstructi ons could be studied
(see Supp Material movie1). For comparison purposes, Z-stacks were
also acquired with confocal imaging, either a Leica SP5 confocal using
a 10 × 0.3NA objecti ve (early stage embryos) or a zoom macro-
confocal Leica LSI (30 dpf).
The 3D stacks were then processed and segmented (internal organs
digitally labelled) using Fiji and Amira v5.3 (FEI Inc.). To facilitate
semi-automatic segmentation, in some cases we computed a derivative
of the original stack (gradient magnitude or Sobel edge detection),
which was then subtracted from the original stack. After segmentation,
each organ was turned into a 3D model, exported as Wavefront obj le
and later imported into Simulab composer V4 (Simlab soft) to prepare
the 3D PDF interactive illust ration (Fig. 2). Movie 1 was prepared
using FIJI. The whole body, head, inner ear and swim bladder were mea-
sured with both FIJI or Amira, using 3D reconstruction of the whole vol-
ume or of free-angle slices, and measured as antero-posterior lengths. In
SCCs we considered this to be the linear distance between the anterior-
most end of anterior SCC and the posterior-most end of the posterior
SCC. The full body length and head were measured from the tip of the
nostril to the posterior end of the tail; when the embryo presented a
curvature we followed the body's mid axis using a spline. The head-
body separation was limited at the level of insertion of the nbuds,or
in earlier stages at the caudal end of the otic vesicles. The measurements
presented are an average of 23 embryos at the stages mentioned.
5P.M. Felix et al. / Mechanisms of Development xxx (2016) xxxxxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
Supplementary data to this article can be found online at http://dx.
The authors acknowledge the help of the personnel of the Air Force
Base No. 6 in Montijo (Portugal) for allowing egg collection in their mil-
itary facility, Manuel Vieira, Joana Amado, and Daniel Alves for help dur-
ing the sampling ca mpaigns, Nuno Martins and Hugo Pereira of the
Advanced Imaging Unit of IGC for help with imaging and discussions,
and Leica for the use of a the LSI macro confocal for test purposes. This
study was funded by Science and Technology Foundation, Portugal
(project PTDC/MAR/118767/2010 and the strategic project UID/MAR/
04292/2013 granted to MARE, and PEst-OE/MAR/U10199/2014).
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6 P.M. Felix et al. / Mechanisms of Development xxx (2016) xxxxxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography OPenT allows the study of large toadsh Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016),
... Descriptions of the anatomical features of early stages of development are essential for understanding multiple biological aspects of organisms, as ontogenetic variation can reveal patterns of development relevant to evolutionary, phylogenetic, and taxonomic analyses (Ferreira Marinho, 2017, 2022Melo, 2020;Vaz and Hilton, 2022). Several aspects of larval morphology are known to date for a few species of Batrachoidiformes: the external morphology of larval development of Opsanus tau, P. notatus, and Aphos porosus (Gill, 1907;Arora, 1948;Dovel, 1960;Balbontín et al., 2018), skeletal changes during larval development of P. notatus (Vaz and Hilton, 2020;2023;Balbotin et al., 2018) and, more specifically, changes of stato-acoustic organs of the neurocranium of Halobatrachus didactylus (Felix et al., 2016). The development of the swim bladder has been extensively studied, particularly for O. tau (Fine, 1975;Fine et al. 1984;Fine and Pennypacker, 1986;Fine, 1989;Fine et al. 1990;Fine et al. 1993;Loesser et al. 1997;Fine and Waybright, 2015;Fine et al. 2016;Fine and Parmentier, 2022), and in a lesser extent, H. didactylus (Felix et al., 2016). ...
... Several aspects of larval morphology are known to date for a few species of Batrachoidiformes: the external morphology of larval development of Opsanus tau, P. notatus, and Aphos porosus (Gill, 1907;Arora, 1948;Dovel, 1960;Balbontín et al., 2018), skeletal changes during larval development of P. notatus (Vaz and Hilton, 2020;2023;Balbotin et al., 2018) and, more specifically, changes of stato-acoustic organs of the neurocranium of Halobatrachus didactylus (Felix et al., 2016). The development of the swim bladder has been extensively studied, particularly for O. tau (Fine, 1975;Fine et al. 1984;Fine and Pennypacker, 1986;Fine, 1989;Fine et al. 1990;Fine et al. 1993;Loesser et al. 1997;Fine and Waybright, 2015;Fine et al. 2016;Fine and Parmentier, 2022), and in a lesser extent, H. didactylus (Felix et al., 2016). Lindholm and Bass (1993) provided in-depth descriptions of the development of muscle fibers and innervation of sonic muscles of the swim bladder of P. notatus. ...
... The use of traditional histological methods, although offer great resolution and detail, allowing even cell counts, presents challenges and several caveats for manipulation and preparation of soft anatomy structures compromising three-dimensional reconstructions of the examined structures (Gignac and Kley, 2014;Bribiesca-Contreras and Sellers, 2017;Shu et al., 2018). Despite not having equivalent resolution to histological preparations, CT-scanning techniques, with enhanced contrast to visualize soft anatomy (Metscher, 2009;Konstantinidis et al., 2015), allow precise three-dimensional reconstructions, in addition to being a non-invasive (i.e., destructive) preparation (Felix et al., 2016;Vasconcelos-Filho et al., 2019;Scadeng et al., 2020;Wang et al., 2021). Considering the aforementioned gaps in the anatomical study of the Batrachoididae, our study described the three-dimensional ontogenetic changes of the swim bladder and other abdominal organs of P. notatus, from the early stages of post-hatching larvae to free-swimming juveniles. ...
The batracoidid Plainfin Midshipmen Porichthys notatus Girard has been extensively studied due to the sound production abilities and specializations of its swim bladder. The present study describes three-dimensional variations of the morphology of the swim bladder and sonic muscles of P. notatus during its post-hatch larval development, with the use of three-dimensional computed tomography. This study also includes descriptions of the relative position of the swim bladder to other visceral organs. The swim bladder, digestive tract, and liver were already present in the smallest examined specimens (5.9 mm; newly hatched larvae) along with the yolk sac. In the smallest specimens, the digestive tract is straight, but from 7.1 mm TL, the digestive tract forms the first intestinal loops, and at 25.5 mm TL, a second intestinal loop. In smallest specimens, the swim bladder is oval, but at 7.1 mm TL, the anterior margin starts invaginating, forming a pair of anterior lobes. The first appearance of the intrinsic sonic muscles in swim bladder occurs at 13.1 mm TL. Additionally, we provide comparisons between the shape of the swim bladder of P. notatus and other species. The shape of the swim bladder of P. notatus and other members of Porichthyinae have an ovoid posterior region with two anterior lobes and differs from the cordiform or semiconected/bilobed the swim bladders observed in the other Batrachoididae.
... Reproductive behavior and early life history are unknown for most species of Batrachoidiformes. Collette (2005) summarized most of the information known for the order, and the most detailed accounts comes from Poricthys notatus (Arora, 1948), Aphos porosus (Balbontín et al., 2018), Opsanus tau (Dovel, 1960), and, a lesser extent, Halobratrachus didactylus (Felix et al., 2016). ...
... Male toadfishes are nest builders and vocalize to attract females during the spawning season (Arora, 1948;Balbontín et al., 2018;Dovel, 1960;Felix et al., 2016;Rice & Bass, 2009). Females lay large eggs (>5 mm diameter) on the roof of nests that are formed by rocks or other hard substrates (Arora, 1948;Britz & Toledo-Piza, 2012;Dovel, 1960). ...
... Balbontín et al. (2018) offered a generalized skeletal description of larval specimens of Aphos porosus, however, they did not provide accounts of individual bones and cartilages. Felix et al. (2016) focused their ontogenetic descriptions on the stato-acoustic organs and swimbladder of larval stages of Halobatrachus didactylus. ...
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Batrachoidiformes are benthic fishes that utilize the undersides of rocks as spawning nests. Their larvae are attached to the nest and nourished by a large yolk sac. The evolutionary shift from feeding, free-swimming larvae to sedentary larvae that are reliant on their yolk sac for nutrition can lead to changes in skeletal development. Batrachoidiformes also have many morphological specializations, such as five pectoral-fin radials (versus four in other acanthomorphs) that are of uncertain homology, the determination of which may have phylogenetic implications. A larval series of Porichthys notatus was collected and its skeletal ontogeny is described. In P. notatus the ossification of the pharyngeal toothplates occurs relatively later than in percomorphs with free-swimming larvae. The posterior basibranchial copula cartilage (= fourth basibranchial) in Porichthys notatus has a unique development among fishes: it initially develops as a paired element at 6.8–7.1 mm NL before fusing posteriorly and forming single median cartilage at 7.4 mm SL. Cartilages of hypobranchial four are transitory, being observed in two specimens of 6.8 and 7.3 mm NL before fusing with ceratobranchial four. The previously identified dorsalmost pectoral radial is a bone formed by a hypertrophied propterygium that ossifies later in development. The earliest stages of P. notatus have three dorsal spines, but during late larval development, the growth of the third dorsal spine is interrupted. The development of P. notatus is compared and discussed in context to that of other acanthomorph.
... Note that Lusitanian toadfish larvae stay attached until nearly all the yolk sac has been absorbed (Collette 2005). This means that larvae cannot escape from noise exposure for up to 2-3 months, depending on temperature (Felix et al. 2016; MCP Amorim, personal observation). Recently, Amorim et al. (2022) and Faria et al. (2022) have revealed some insights on the impact of boat noise in eggs' survival and early life stages development. ...
Most marine soundscapes have changed due to the massive presence of anthropogenic noise. Lusitanian toadfish (Halobatrachus didactylus) is a vocal fish species that has been recurrently used as a model in both behavioral and physiological studies, making it an excellent species also to understand the effects of aquatic noise. This chapter aims to review what is known about the effects of boat noise on this species and its possible implications. Vocal behavior, hearing, reproduction, and early stages development of the Lusitanian toadfish are summarized, including several studies that observed effects of boat noise on this species in these different topics. Boat noise can disrupt and decrease calling activity, mask environmental and conspecific signals, reduce reproduction success, induce stress, affect parental care, and even affect larvae development. These results warn of the possible severe effects of noise pollution on fish and warrant the need of further studies addressing the consequences of noise at the population level.
... Dehydrated toadfish embryos and larvae have been cleared in BABB for subsequent imaging in optical tomography and confocal microscopy. This has provided new insights into the embryonic development of Batrachoidids, a fish family unknown in developmental biology but widely studied in other fields [27]. Moreover, in mammals, transparent mouse brains and embryos have been accomplished by Scale clearing, providing a 3D reconstruction and imaging of the brain structures and the neuronal network. ...
Full-text available
Light scattering is a challenge for imaging three-dimensional organisms. A number of new tissue clearing methodologies have been described in recent years, increasing the utilities of clearing techniques to obtain transparent samples. Here, we describe the optimization of a suitable and novel protocol for clearing Galleria mellonella larvae, an alternative infection animal model with a promising potential for the toxicological evaluation of different molecules and materials. This has allowed the visualization of internalised fluorescent nanoparticles using confocal microscopy, opening the door to a wide range of different applications.
... Although the focus was on soft tissues, otoliths are clearly visible in scanned images of paddlefish (Polyodon spathula) and pike (Esox lucius) hatchlings (figures 2 & 5 in Metscher 2009). Otoliths in situ are also visible in the recent, high-resolution X-ray, CT-based re-description of the tuvirão, Gymnotus inaequilabiatus (Maxime & Albert 2014), and in a range of other studies employing CT imagery (e.g., Bignami et al. 2013, Edds-Walton et al. 2015, Felix et al. 2016, Fisher & Hunter 2018, Schulz-Mirbach et al. 2018. Although no attempt was made to age otoliths in any of these studies, these results demonstrate that in situ observation of otoliths using CT scans is possible. ...
Technical Report
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Knowledge of the age of fish is an integral part of fisheries science, being a key requirement for estimating growth, age at recruitment and sexual maturity, longevity, mortality rates, population age structure, and age-dependent fishing gear selectivity, all of which are important components of age-based stock assessments. The current method for determining ages of most fish species relies on manually extracting, preparing (embedding, sectioning), and reading otoliths. This process is expensive, time-consuming, and can be subject to biases such as variations in age estimations between readers and within readers over time. Recent advances in imaging technologies and machine learning suggest that automation of at least some aspects of these processes may be possible. This study examined the feasibility of automating otolith ageing using CT scanning and machine learning, through a review of published work in these areas and trials on New Zealand fish species. The utility of CT scanning technologies for imaging otolith annular structure was trialled using the MARS Bioimaging Ltd X-ray scanning machine housed at the University of Canterbury. Trials were undertaken on three species: snapper (Pagrus auratus); hoki (Macruronus novaezelandiae); and ling (Genypterus blacodes). Ages of individuals used in the trial were estimated prior to scanning through standard ageing techniques. The MARS CT scanner was able to resolve banding patterns, with best results generated from sections taken parallel to the distal surface, similar to the process used when otoliths are read whole. Spectral analyses revealed varying concentrations of calcium and other minerals across the otoliths. Transverse sections through the otolith core showed banding potentially indicative of annual growth bands, but the resulting images generally lacked sufficient resolution and contrast to detect outermost bands for most individuals. The first growth band was also often difficult to identify. Accordingly, ageing trials using systems capable of achieving higher resolutions are warranted. To investigate the feasibility of using machine learning to estimate age in New Zealand fish species, we adapted a pre-trained convolutional neural network (CNN) designed for object recognition to estimate age using otolith images obtained via microscopy for snapper and hoki. For each species, the model was trained on a collection of images of fish previously aged by human readers (n = 687 and 882 for snapper and hoki, respectively). After training, the model gave the same age as the human reader for 47% of snapper in a test dataset, with a further 35% of ages estimated within 1 year of the human reader estimate of age. For hoki, the model gave the same age as the human reader for 41% of individuals. Our preliminary examination suggests there is significant potential for imaging of otoliths for ageing purposes using a variant of CT scanning technology, for automating age estimation from otolith images, and for the potential to combine both of these techniques to form a fully automated ageing system. The key future research directions to the development of such a system are: 1. optimise and further evaluate the CNN-based approach used here to automate age estimation of snapper and hoki; 2. investigate the potential for the CNN-based model to automate age estimation of additional species from photographic images of sectioned otoliths; 3. further identify the potential to use otolith scans from CT scanning technologies and, in particular micro-CT systems, for imaging otolith annular structure; and 4. identify the potential to automate ageing from images generated from micro-CT scanning using deep learning approaches.
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Tissue clearing increases the transparency of late developmental stages and enables deep imaging in fixed organisms. Successful implementation of these methodologies requires a good grasp of sample processing, imaging and the possibilities offered by image analysis. In this Primer, we highlight how tissue clearing can revolutionize the histological analysis of developmental processes and we advise on how to implement effective clearing protocols, imaging strategies and analysis methods for developmental biology.
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Background: The Portable Document Format (PDF) is the standard file format for the communication of biomedical information via the internet and for electronic scholarly publishing. Although PDF allows for the embedding of three-dimensional (3D) objects and although this technology has great potential for the communication of such data, it is not broadly used by the scientific community or by clinicians. Objective: The objective of this review was to provide an overview of existing publications that apply 3D PDF technology and the protocols and tools for the creation of model files and 3D PDFs for scholarly purposes to demonstrate the possibilities and the ways to use this technology. Methods: A systematic literature review was performed using PubMed and Google Scholar. Articles searched for were in English, peer-reviewed with biomedical reference, published since 2005 in a journal or presented at a conference or scientific meeting. Ineligible articles were removed after screening. The found literature was categorized into articles that (1) applied 3D PDF for visualization, (2) showed ways to use 3D PDF, and (3) provided tools or protocols for the creation of 3D PDFs or necessary models. Finally, the latter category was analyzed in detail to provide an overview of the state of the art. Results: The search retrieved a total of 902 items. Screening identified 200 in-scope publications, 13 covering the use of 3D PDF for medical purposes. Only one article described a clinical routine use case; all others were pure research articles. The disciplines that were covered beside medicine were many. In most cases, either animal or human anatomies were visualized. A method, protocol, software, library, or other tool for the creation of 3D PDFs or model files was described in 19 articles. Most of these tools required advanced programming skills and/or the installation of further software packages. Only one software application presented an all-in-one solution with a graphical user interface. Conclusions: The use of 3D PDF for visualization purposes in clinical communication and in biomedical publications is still not in common use, although both the necessary technique and suitable tools are available, and there are many arguments in favor of this technique. The potential of 3D PDF usage should be disseminated in the clinical and biomedical community. Furthermore, easy-to-use, standalone, and free-of-charge software tools for the creation of 3D PDFs should be developed.
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A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
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Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.
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The egg surface structure of Thalassophryne amazonica, a freshwater toadfish from the Amazon basin is described. Eggs of this species show a remarkable, highly unusual system of parallel ridges and intermittent grooves that originate at the equator of the egg and lead to the micropylar pit, at which they end in a spiralling pattern. A similar egg surface structure has so far been described only from a group of Asian anabantoid percomorphs, obviously not closely related to Thalassophryne. This egg surface pattern may enhance fertilization success by guiding sperm to the micropyle. We review museum records for T. amazonica, present an updated map of its occurrence in the Amazon basin, and provide information on its habitat.
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The relation between acoustic signaling and reproductive success is important to understand the evolution of vocal communi-cation systems and has been well studied in several taxa but never clearly shown in fish. This study aims to investigate whether vocal behavior affects the reproductive success in the Lusitanian toadfish (Halobatrachus didactylus) that relies on acoustic communication to attract mates. We recorded 56 nest-holding (type I) males during the breeding season and analyzed the calling performance and acoustic features of the mate advertising sounds (boatwhistles) exhibited over circa 2 weeks. Hormonal levels of the subjects and the number of eggs (reproductive success) present in the respective nests were quantified. Nesting males attracted both females and other males, namely smaller type I males with significantly lower total length (TL), body condition, sonic muscle mass, gonad mass, and accessory glands mass. Calling rate (CR), calling effort (CE) (% time spent calling), and sound dominant frequency were significantly higher in nesting males with clutches than in those without clutches. Sex steroids (11-ketotestosterone and testosterone) were not correlated with vocal parameters or number of eggs. Maximum CR and CE were the best predictors of the number of eggs. In addition, these vocal variables were best explained by male's TL, condition, and sonic muscle mass. We provide first evidence that vocal behavior significantly determines reproductive success in a vocal fish and show that acoustic signaling at higher and constant rates can operate as an indicator of the male's size and body condition and probably of elevated motivation for reproduction. Key words: acoustic communication, Batrachoididae, mate attraction, reproductive success, toadfish. [Behav Ecol 23:375–383 (2012)] INTRODUCTION
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The Japanese quail is a widely used model organism for the study of embryonic development; however, anatomical resources are lacking. The Quail Anatomy Portal (QAP) provides 22 detailed three-dimensional (3D) models of quail embryos during development from embryonic day (E)1 to E15 generated using optical projection tomography. The 3D models provided can be virtually sectioned to investigate anatomy. Furthermore, using the 3D nature of the models, we have generated a tool to assist in the staging of quail samples. Volume renderings of each stage are provided and can be rotated to allow visualization from multiple angles allowing easy comparison of features both between stages in the database and between images or samples in the laboratory. The use of JavaScript, PHP and HTML ensure the database is accessible to users across different operating systems, including mobile devices, facilitating its use in the laboratory.The QAP provides a unique resource for researchers using the quail model. The ability to virtually section anatomical models throughout development provides the opportunity for researchers to virtually dissect the quail and also provides a valuable tool for the education of students and researchers new to the field. Database URL: (For review username: demo, password: quail123)
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Sound communication is fundamental to many social interactions and essential to courtship and agonistic behaviours in many vertebrates. The swimbladder and associated muscles in batrachoidid fishes (midshipman and toadfish) is a unique vertebrate sound production system, wherein fundamental frequencies are determined directly by the firing rate of a vocal-acoustic neural network that drives the contraction frequency of superfast swimbladder muscles. The oyster toadfish boatwhistle call starts with an irregular sound waveform that could be an emergent property of the peripheral nonlinear sound-producing system or reflect complex encoding in the CNS. Here, we demonstrate that the start of the boatwhistle is indicative of a chaotic strange attractor and tested whether its origin lies in the peripheral sound-producing system or in the vocal motor network. We recorded sound and swimbladder muscle activity in awake, freely-behaving toadfish during motor nerve stimulation, and recorded sound, motor nerve and muscle activity during spontaneous grunts. The results show that rhythmic motor volleys do not cause complex sound signals. However arrhythmic recruitment of swimbladder muscle during spontaneous grunts correlates with complex sounds. This supports the hypothesis that the irregular start of the boatwhistle is encoded in the vocal pre-motor neural network, and not caused by peripheral interactions with the sound-producing system. We suggest that sound production system demands across vocal tetrapods have selected for muscles and motorneurons adapted for speed, which can execute complex neural instructions into equivalently complex vocalizations.
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OpenSPIM and OpenSpinMicroscopy emerged as open access platforms for Light Sheet and Optical Projection Imaging, often called as optical mesoscopy techniques. Both projects can be easily reproduced using comprehensive online instructions that should foster the implementation and further development of optical imaging techniques with sample rotation control. This additional dimension in an open system offers the possibility to make multi-view microscopy easily modified and will complement the emerging commercial solutions. Furthermore, it is deeply based on other open platforms such as MicroManager and Arduino, enabling development of tailored setups for very specific biological questions. In our perspective, the open access principle of OpenSPIM and OpenSpinMicroscopy is a game-changer, helping the concepts of light sheet and optical projection tomography (OPT) to enter the mainstream of biological imaging.
Detailed illustrations of the early larval development of the oyster toadfish,Opsanus tau (Linnaeus), are presented for the first time to show variation in size and morphology, sequence of fin formation and yolk absorption. Eggs, 5 mm in diameter, were attached to a substrate by an adhesive disk and hatched into larvae about, 7.0 mm T.L. They were studied through essentially prolarval growth until about 19.0 mm T.L. and 20 days old. Prominent features were the large stalk-like yolk sac and a pectoral-pelvic fin apparatus position shift. The color pattern was apparent at 16.4 mm. Head, snout-anus and eye lengths compared with total length showed proportional growth. Body depth-total length showed non-proportional growth. The full complement of various fin rays was apparently attained at these total lengths: pectoral—17.1 mm; spinous dorsal—10.8 mm; soft dorsal—12.8 mm; anal—12.8 mm; and caudal—probably at 18.0 mm.