The brain in three crustaceans from cavernous darkness
Note on terminology
We widely apply the neuroanatomical terminology suggested for the decapod brain by
Sandeman et al. 55], and extend it only where necessary. In comparison to the many soma clusters in Decapoda,
for which Sandeman et al.55] suggested a labeling that is widely accepted, the studied representatives of MST
feature considerably less soma clusters that are separated from each other by soma-free
regions. A soma cluster in this sense may comprise somata from one or more brain regions.
We refer to somata in six different brain regions, applying a simple nomenclature
schematically explained in Figure 2A: anterolateral somata (AlS) surround the optic lobes and the lateral protocerebrum;
anterodorsal somata (AdS) lie dorsally in the median protocerebrum; anteroventral
somata (AvS) lie ventrally in the median protocerebrum; lateral somata (LS) lie laterally
in the deutocerebrum; ventromedial somata (VmS) lie ventrally in the deutocerebrum;
posterior somata (PS) lie laterally and dorsally in the tritocerebrum.
Figure 2. Morphology of the brain inMictocaris halope. Overview and semi-thin sections. A: Schematic depiction of our simplified nomenclature for soma clusters. B–E: Neuropil and nerves without somata. B, C: 3D-reconstructions in (B): anterior and (C): dorsal view. D, E: Schematic drawing in (D): posterior and (E): lateral view. F–L: Transverse semi-thin sections, ordered from anterior to posterior. F–H: Arrow points at a large dorsal extension of anterodorsal somata (AdS). G, H: Rocket points at the lateral root of the antenna 1 nerve (A1Nv). H: Points mark the olfactory glomeruli in the olfactory lobes (ON). H–J: Double arrowheads point at the large lateral extensions of lateral somata (LS).
I–J: Simple arrowhead points at a large ventral extension of anteroventral somata (AvS).
Scale bars: 50 ?m.
The morphological description of spatially extended structures often requires giving
border points and using directional terms, such as ‘run into’ or ‘project anteriorly’.
In our work, this does not imply any physiological, ontogenetic, or evolutionary direction.
All positions given (such as anterior or dorsal) refer to the body axis. The term
‘medial’ is used, when two paired structures lie near the midline of the animal, whereas
the term ‘median’ is used, when an unpaired structure lies directly in the midline
of the animal. The identification of blood vessels was based on Wirkner and Richter
35] for M. halope and S. lepidops, and on Wirkner and Richter 36] for T. argentarii.
Sullivan and Beltz 23] pointed out that what is generally referred to as the ‘terminal medulla’ in the malacostracan
protocerebrum is rather an assembly of several neuropils than one coherent neuropil.
We use ‘terminal medulla’ as an inclusive term for all that neuropil of the lateral
protocerebrum which is not clearly specified from its surroundings by shape, texture, or spatial separation.
The described ‘small lateral neuropils’ of our study are specified by their dense
texture and clear spatial separation from the terminal medulla in the periphery of
the lateral protocerebrum. In contrast to the terminal medulla, our criteria for the
identification of a hemiellipsoid body are its distally convex shape, its dense texture,
and its anteromedial position within the lateral protocerebrum, as found in other
Malacostraca 22],55].
The brain comprises a number of tracts, which we here divided into vertical (VT),
longitudinal (LT), and transverse tracts (TT), and numbered consecutively. If tracts
in different species occur in the corresponding position, they received the same label
(e.g., LT1 was found in M. halope and S. lepidops). All vertical and longitudinal tracts described occur in pairs. Transverse tracts
are unpaired and midline-spanning if not otherwise noted.
Major results
As in other Peracarida, there are no signs of a nauplius eye or nauplius eye-related
neural structures in MST. In all three species studied, the brain is a syncerebrum
composed of protocerebrum, deutocerebrum, and tritocerebrum. The protocerebrum is
divided into a distal protocerebral neuropil (which is here labeled ‘Ia’ due to its
unresolved homology relationships), the lateral protocerebrum (Ib), and the unpaired
median protocerebrum (Ic). A distinctive hemiellipsoid body occurs only in the lateral
protocerebrum of T. argentarii. The deutocerebrum (II) gives rise to the antenna 1 nerve and includes the olfactory
lobe. The tritocerebrum (III) gives rise to the antenna 2 nerve. Additional nerves
arise mainly from the deuto- and tritocerebrum. One post-esophageal commissure interconnects
the paired halves of the tritocerebrum transversely. On each side, the tritocerebrum
is connected to the posterior-next ganglion by a soma-free connective. S. lepidops features a separate mandibular ganglion, while in M. halope and T. argentarii, a subesophageal ganglion is composed of several neuromeres. Somata are arranged in
only a few, often large clusters around the brain’s neuropil. Soma clusters differ
interspecifically in size and shape (see above for terminology of these soma clusters).
Each species features a number of neuropils, the most prominent of which are the central
body and protocerebral bridge. Proto-, deuto-, and tritocerebrum are arranged along
a neuraxis which deflects from the body axis anteriorly and shows a different course
in each species.
Mictocaris halope (Mictocarididae)
General aspects
M. halope lacks compound eyes. The pyriform eye stalk inserts anteriorly in the cephalon (position
indicated in Figure 1C). Antenna 1 exhibits a prominent 3-segmental peduncle bearing a 4-segmented inner
and an 8-segmented outer flagellum (37]; see also Figure 1C). Each of the five distalmost segments of the outer flagellum bears one very long
aesthetasc (37]; see also Figure 1C). Antenna 2 exhibits a prominent 4-segmented peduncle, which bears a large scale
on its second segment and, distally, a 35-segmented flagellum 37]. Bowman and Iliffe 37] described several loosely arranged setae both on antenna 1 and 2.
The distal protocerebral neuropil (Ia) is situated directly lateral to the lateral
protocerebrum (Ib, Figures 2B-E, 3H, 4A), which is adjoined by the median protocerebrum (Ic) ventromedially (Figures 2B-H, 3A,C, 4A). In comparison to the other species, the olfactory lobe is relatively large and
ellipsoid, protruding laterally from the rest of the deutocerebrum (Figures 2B-E,H-J, 3A,C, 4E,G,I). In lateral aspect, the lateral protocerebrum is situated posterodorsal to
the median protocerebrum, and the latter anterodorsal to the deutocerebrum (Figures 2E, 3C). The neuraxis flattens posteriorly to take on the anteroposterior course of the
ventral nerve cord (III, SG; Figures 2E, 3C). The unpaired brain artery (BA) enters the median protocerebrum from the posterior
direction (Figure 2D). The artery first splits into an upper and a lower branch (Figure 2G), each of which bifurcates into one pair of sub-branches leaving the median protocerebrum
laterally (Figure 2H).
Figure 3. Morphology of the brain inMictocaris halope. Soma clusters, neuropil, and internal structure – click on A and H for interactive
3D models. A,B: Soma clusters (grey) in posterior view (A): with neuropil (yellow) and (B): without neuropil. C, D: Soma clusters in lateral view (C): with and (D): without neuropil. A–D: Arrows mark a dorsal extension of anterodorsal somata (AdS). Double arrowheads mark
a lateral extension of lateral somata (LS). Simple arrowhead marks a ventral extension
of anteroventral somata. E: Schematic drawing of neuropils (dark yellow) and tracts (grey) in posterior view.
F–H: 3D-reconstructions of neuropils and tracts in (F): lateral, (G): dorsal, and (H): anterior view. Neuropils are in yellow; tracts are in grey. F: Olfactory lobe (ON) is shown semitransparent. Brain width about 200 ?m (perspective
view).
Figure 4. Morphology of the brain inMictocaris halope. Acetylated ?-tubulin immunoreactivity and nuclear counterstaining. A–I: Dorsal view on confocal laser-scans of horizontal vibratome sections (50 ?m) labeled
for acetylated ?-tubulin immunoreactivity (green) and nuclear marker (blue). A: Overview of distal protocerebral neuropil (Ia) and lateral protocerebrum (Ib). B: The neuropilar subunits of the protocerebral bridge (PB) are visible as a negative
imprint at the anterior part of the median protocerebrum (Ic). C, D: The central body (CB) is subdivided into several elongated or spheroidal compartments
(asterisks), which are connected posteriorly via numerous fine neurites (arrowheads
in C) to the 3rd transverse tract (TT3, red arrows). The 2nd transverse tract (TT2, yellow arrows) passes the CB anteriorly, sending fine processes
into the neuropil (arrowheads in D). The 1st transverse tract (TT1) lies dorsal to the CB. E, F: The paired branches of the OGT form a characteristic chiasm (X in E) in the center
of the median protocerebrum (Ic) and connect the olfactory neuropil (ON) to the lateral
protocerebrum (Ib). Before reaching the target structure in the medial part of the
lateral protocerebrum, the OGT splits into two neurite bundles (arrows in F). G: Overview of the deutocerebrum (II). H: Higher magnification of the medial part of the deutocerebrum (II). The 4th transverse tract (TT4) seems to originate in the nerves of the antenna 1 (A1Nv),
forming a deutocerebral commissure (arrows). I: The antenna 1 nerves (A1Nv) enter the deutocerebrum from the anterior direction,
each splitting into a thick medial and a slender lateral branch. Several neurite bundles
from the medial branch project into the lateral deutocerebral neuropil (arrowheads),
while others constitute to the 4th transverse tract (TT4) (arrows in H). The lateral branch innervates the olfactory neuropil (arrows). Scale bars: 50 ?m.
Soma clusters
The brain in M. halope features three pairs of soma clusters and one unpaired soma cluster (i.e., altogether
seven clusters).
The first paired soma cluster is composed of anterolateral somata (AlS), which cover
the distal protocerebral neuropil and lateral protocerebrum dorsally, ventrolaterally,
and posteriorly (Figures 2F,H,J, 3A,C, 4A,F). It is dorsally extended (AlS; Figures 2J, 3A-D).
The second paired soma cluster consists both of lateral somata (LS) covering the olfactory
lobes anteriorly, dorsolaterally, and posteriorly (Figures 2H-K, 3A-D, 4E,G,I) and of posterior somata (PS) which are situated dorsolaterally in the tritocerebrum
(Figures 2K, 3A-D). The posterior somata form a short lobe-like extension pointing posterolaterally
(Figures 2K, 3A-D).
The third paired soma cluster is minute, only comprising a few ventromedial somata
(VmS) that lie ventrally in the deutocerebrum between the olfactory lobe and the root
of the antenna 1 nerve (A1Nv; Figures 2H, 3B-D).
The unpaired soma cluster comprises anterodorsal somata (AdS) and anteroventral somata
(AvS) and extends from the dorsal (Figures 2F-H, 3A,B) over the anterior (Figure 3B-D, 4A,E) to the ventral region of the median protocerebrum (Figures 2F-J, 3B-D). On each side, the soma cluster shows a long dorsal extension (AdS, arrows; Figures 2F-H, 3A,B), a lateral extension (AvS, double arrowheads; Figures 2H-J, 3A,B,D), and a ventral extension (AvS, simple arrowheads; Figures 2I,J, 3B-D). Each dorsal extension contains a small globular cavity reminiscent of the Bellonci
organ in other crustaceans (GC; Figure 2F). The lateral extension extends posteriorly along the lateral side of the median
protocerebrum as far as the olfactory lobe (Figures 2H-J, 3A). The ventral extension extends posteriorly along the ventromedial side of
the deutocerebrum (II) as far as the tritocerebrum (III; Figures 2I,J, 3B-D).
Neuropils
Distal protocerebral neuropil (Ia)
The distal protocerebral neuropil (Ia) has a spheroidal shape and appears as a uniformly
structured neuropil that is in part confluent with the terminal medulla (Figures 2B-E,H,J, 3E-H, 4A). In our histological and immunocytochemical preparations, we detected neither any
nerve nor tract extending from the distal protocerebral neuropil into the eyestalk.
Lateral protocerebrum (Ib)
On each side, the lateral protocerebrum comprises, on the one hand, the large terminal
medulla (TM; Figures 2B-H,J, 3A,C,E-H, 4A,F) and, on the other hand, three small lateral neuropils (SLN1-3 in Figure 3E-H; see also SLN3 in Figure 2J). The terminal medulla is evenly textured, with subcompartments that are hard to
distinguish. We could not identify a hemiellipsoid body. The 1st small lateral neuropil lies dorsomedially, the 2nd dorsally (Figure 3E-H), and the 3rd posteriorly in the periphery of the lateral protocerebrum (Figures 2J, 3E-H).
Median protocerebrum (Ic)
The unpaired anteromedian protocerebral neuropil (AmnPN) lies anteriorly in the median
protocerebrum (Figures 2F, 3E-H). Posteriorly, it is confluent with its surroundings. The unpaired cigar-shaped
central body (CB) lies horizontally across the center of the median protocerebrum
and has a comparatively dense texture (Figures 2G, 3E,F,H, 4C,D). Acetylated ?-tubulin immunoreactivity reveals several spheroidal subcompartments
within the central body (asterisks; Figure 4C). The unpaired protocerebral bridge (PB) is situated dorsally in the median protocerebrum
(Ic) and consists of at least four spheroidal subunits (here counting both body sides
together) with a comparatively dense texture (Figures 2G, 3E-H). These compartments are visible as a negative imprint in the immunocytochemical
preparations (Figure 4B). On each side, the posterodorsal protocerebral neuropil (PdPN) extends from the
dorsal to the posterior region of the median protocerebrum (Figures 2C,H, 3E-H). The lateral region of the median protocerebrum shows a comparatively dense texture
and is here interpreted as the lateral accessory lobe (LAL; Figures 2H, 3E-H). Medially, the lateral accessory lobe is confluent with its surroundings.
Deutocerebrum (II)
The lateral antenna 1 neuropil (LAN) lies laterally in the deutocerebrum and medial
to the olfactory lobe (Figures 2J, 3E-H, 4G-I). The large and ellipsoid olfactory lobe (ON; Figures 2H-J, 3A,C,E-H, 4E,G,I) is composed of a number of nearly spheroidal olfactory glomeruli (points; Figure 2H,J). The median antenna 1 neuropil (MAN) spans horizontally across the median region
of the deutocerebrum (Figures 2I, 3E-H, 4H). Since its halves are only connected by a tract, the median antenna 1 neuropil
in M. halope has to be considered as paired.
Tritocerebrum (III)
The lateral tritocerebral neuropil (LTN) lies laterally in the tritocerebrum (Figures 2K, 3E-H). Posteriorly, it is confluent with its surroundings. The ventral tritocerebral
neuropil (VTN) extends longitudinally along the ventral side of the tritocerebrum
(III; Figures 2K, 3E-H). It is situated directly posterior to the nerve root of the labral nerve (Figures 2K, 3E-H). Posteriorly, it is confluent with its surroundings. The posterior region of
the tritocerebrum is dorsoventrally flattened (III; Figure 2L) and confluent with the post-tritocerebral connectives.
Tracts
The olfactory globular tract (OGT) connects the center of the olfactory lobe with
the terminal medulla (Figures 2H, 3E,F,H, 4E,F). Within the terminal medulla, the olfactory globular tract splits into two conspicuous
branches, one terminating medially and the other anterolaterally in the undifferentiated
terminal medulla (arrows; Figure 4F). The olfactory globular tracts of both body sides decussate posterior to the central
body (Figures 2H, 3E, 4E). The 1st vertical tract (VT1) extends from the posteroventral region of the terminal medulla
to the deutocerebrum in the medioventral direction (Figure 3E,F,H). The 1st transverse tract (TT1) starts near the distal protocerebral neuropil in the ventral
region of the terminal medulla, from there extending to the other side of the body
(Figures 3E-H, 4A,C). On its way, it passes the central body dorsally, but we could not identify whether
it is associated with it (Figures 3E,F,H, 4C). The second and third transverse tracts (TT2, TT3), identified by immunocytochemical
staining, span across the median protocerebrum. Each of these tracts is associated
with the central body, one passing it anteriorly (TT2) and one posteriorly (TT3, Figure 4C,D). The anterior of these tracts sends neurites into the central body where the
neurites form a conspicuous chiasm with their counterparts from the other body side.
The posterior tract contributes neurites to the subcompartments of the central body
(arrowheads in Figure 4C). The 4th transverse tract (TT4) interconnects the anteromedial region of the deutocerebrum
of both body sides and is slightly bent up in the middle (Figures 2I, 3E,G, 4H). Based on acetylated ?-tubulin staining, this tract originates near the root of
the antenna 1 nerve (arrows; Figure 4H). On each side, the 1st longitudinal tract (LT1) extends along the ventral side of the median protocerebrum
(Ic) in the posteroventral direction (Figures 2G,H, 3E,F,H). For a short section, the tract leaves the surrounding neuropil of the median
protocerebrum and is bordered only by a lateral branch of the brain artery and anteroventral
somata (Figure 2G,H). The tritocerebral commissure (TC) interconnects the opposite halves of the tritocerebrum
transversely (Figures 2B-E,L, 3A,C,E-G). It is slightly bent up in the middle. On each side, the dorsoventrally flattened
post-tritocerebral connective (PTC) connects the tritocerebrum to the subesophageal
ganglion (Figures 2C-E,L, 3C).
Nerves
On each side, the antenna 1 nerve (A1Nv) enters the deutocerebrum from the anterior
direction (Figures 2B,E-H, 3C,F, 4E,G-I), thereby bifurcating into a thick medial and a thin lateral root (Figure 4I). The medial root of the antenna 1 nerve (Figures 2H, 4I) enters the lateral antenna 1 neuropil (Figures 2J, 3F). Neurites in the lateral root are arranged more densely (rocket in Figure 2G,H). Immunolabeling reveals that the lateral root splits again into two neurite bundles
(arrows in Figure 4I). The more lateral bundle proceeds directly into the center of the olfactory lobe
(ON; Figures 2H, 4I), while the more medial bundle forms a decussation with the olfactory globular tract
directly medial to the olfactory lobe (Figure 4I). Distally, each antenna 1 nerve soon splits into several small branches (e.g.,
Figure 2B, one branch shown by arrow). The prominent antenna 2 nerve (A2Nv) enters the tritocerebrum
from the anteroventral direction (Figures 2B,D,E,J, 3A,C,F,H, 4G). In its proximal region, each antenna 2 nerve is entered by a lateral tritocerebral
nerve (LTNv; Figures 2B,D,E, 3A,C,E). The tegumentary nerve (TNv) enters the tritocerebrum laterally (Figures 2B-E, 3A,C). Each tegumentary nerve is distally split into three branches (Figures 2B,D, 3A), all of which extend to the body wall. The posterodorsal nerve (PdNv) enters the
tritocerebrum from the posterodorsal direction (Figures 2C-E, 3A,C). On its way, it penetrates the described soma cluster which is composed of lateral
and posterior somata (Figure 3A,C). The labral nerve (LbNv) enters the posteroventral region of the tritocerebrum
from the anteroventral direction (Figures 2B,E,K, 3C). The stomatogastric nerve (StNv) enters the ventromedial region of the tritocerebrum
from the anteromedial direction (Figure 2B,C). The stomatogastric nerves from both body sides unite in the midline to form
a single unpaired nerve in front of the esophagus (Figures 2B-D, 3A,C). This single nerve extends dorsally (Figures 2D,E, 3A,C) along the frontal side of the gut.
Anatomy of the ventral nerve cord
The ventral nerve cord of Mictocaris halope is composed of segmental ganglia which are interconnected by pairs of soma-free longitudinal
connectives (Figure 5). The mandibular, maxillular, and maxillar neuromeres together form a subesophageal
ganglion, while each thoracic and pleonic segment features a separate ganglion. The
somata of each ganglion are arranged in one midline-spanning cortex, with most somata
situated ventrolaterally in the ganglion. The segmental neuropil of both sides is
confluent in the midline of each ganglion. Thus, true (free) commissures do not exist
in M. halope (Figure 5).
Figure 5. Morphology of the ventral nerve cord inMictocaris halope. Acetylated ?-tubulin immunoreactivity and nuclear counterstaining. A–G: Dorsal view on confocal laser-scans of horizontal vibratome sections (50 ?m) labeled
for acetylated ?-tubulin immunoreactivity (green) and nuclear marker (blue). A: The tritocerebrum (III) is connected posteriorly to the subesophageal ganglion (SG)
via the posttritocerebral connectives (PTC). The SG comprises the mandibular, maxillular,
and maxillar neuromeres. B: Close-up of the maxillular neuromere. Two thick commissure-like neurite bundles
(arrows) link the hemiganglia. C: The maxillular nerves project laterally into the appendage. The anterior nerve splits
into several branches (arrows), whereas the more posterior nerve remains unbranched
(arrowheads). D: The 2nd-8th thoracic ganglia (ThG2-8) are associated with two segmental nerves (SNv), which project
laterally into the thoracic limbs (TL) (exemplarily shown for ThG4-5). Furthermore,
an intersegmental nerve (IsNv) leaves the connectives laterally. Paired longitudinal
lateral neurite bundles are visible parallel to the connectives (arrows). E: As in the thorax, an intersegmental nerve (IsNv) leaves each connective (Con) between
pleonic ganglia (PlG) laterally and contributes neurites to the lateral longitudinal
neurite bundle (arrows). The main branch of the IsNv projects further posterolaterally.
One segmental nerve arises from each PlG. F: Five nerves (numbers 1-5) extend from the terminal ganglion (TG). Nerves 1 and 2
project in the lateral direction, whereas nerves 3 to 5 extend more posterolaterally.
Two commissure-like neurite bundles connect both hemispheres of the TG (red and yellow
arrows). The anterior commissure-like neurite bundle (red arrows) is associated with
nerves 1 and 2, the posterior commissure-like neurite bundle (yellow arrows) with
the nerves 3 and 4. An intersegmental nerve (IsNv) leaves the connectives anterior
to the TG and splits into several fine branches innervating the periphery (white arrows).
G: Nerve 4 extends into the uropods, where it splits into several branches. Scale bars:
100 ?m.
Subesophageal ganglion
The subesophageal ganglion features one large soma cortex, so that soma-free connectives
between its three neuromeres are missing (Figure 5A). A pair of connectives links the subesophageal ganglion anteriorly to the tritocerebrum
and posteriorly to the (first thoracic) maxillipedal ganglion (Figure 5A), respectively. Laterally, each of the three hemineuromeres of the subesophageal
ganglion gives rise to an anterior and a posterior appendage nerve; both nerves projecting
straight laterally into their appendage (Figure 5A). The anterior nerve soon splits into several branches (arrows; Figure 5C), whereas the more posterior nerve remains unbranched (arrowheads; Figure 5C). Other (e.g., intersegmental) nerves in the subesophageal region could not be detected.
The mandibular and maxillular neuropils are fused. The maxillar neuropil is set off
from the latter by a very short connective-like longitudinal neurite bundle. Both
the mandibular/maxillular and the maxillar neuropil are fused with their counterpart
on the other body side and embedded in the soma cortex (Figure 5A). Amongst the numerous neurites crossing the midline, a distinct commissure-like
anterior and posterior ‘tract’ (sensu56]) are discernible in each subesophageal neuromere (see e.g., in the maxillular neuromere,
Figure 5B).
Thoracic ganglia 1 to 8
The first thoracic segment is associated with the maxillipeds. The anatomy of the
corresponding ganglion is more similar to the other thoracic ganglia than to the maxillar
ganglion in terms of size and segmental nerve arrangement. Two segmental nerves leave
each thoracic hemiganglion laterally projecting into the appendages (Figure 5D). These segmental nerves are thicker than the segmental nerves of the subesophageal
ganglion. An intersegmental nerve projects from each connective laterally into the
periphery, starting with the connective between the (first thoracic) maxillipedal
segment and the second thoracic segment (Figure 5D). These intersegmental nerves split into three branches. The main branch extends
further in the lateral direction; two more slender branches extend anteriorly and
posteriorly, respectively (arrows Figure 5D). Both latter branches are connected to the segmental nerves of the anterior and
posterior adjacent segments, forming a lateral longitudinal neurite bundle. The laterally
extending main branch of the intersegmental nerve could not be traced farther. In
contrast to the subesophageal neuromeres, no distinct transverse tracts could be identified
within thoracic ganglia.
Pleonic ganglia 1 to 5
Pleonic ganglia are smaller than thoracic ganglia (Figure 5E). Each hemiganglion gives rise to one slender pleopod nerve (Figure 5E). An intersegmental nerve arises laterally from each connective of the pleon and
splits immediately into three branches similar to the situation in the thoracic ganglia
(Figure 5E), one branch extending posterolaterally, one anteriorly, and one posteriorly. The
anterior and posterior branches form, together with the corresponding branches of
other segments, a lateral longitudinal neurite bundle (arrows, Figure 5E). The posterolateral branch could be traced into the posteriorly adjacent segment
until the lateral border of the body. Thus, we suggest, that this branch of the intersegmental
nerve innervates the tegument and/or musculature of the lateral body wall. As in the
thorax, distinct transverse tracts within the ganglia could not be observed.
Terminal ganglion
The terminal ganglion, situated in the sixth pleomere, is larger than the pleonic
ganglia 1 to 5 (Figure 5F). Five nerves arise from each hemiganglion (labeled 1-5; see Figure 5F,G). Nerves 1 and 2 extend laterally. These nerves could not be traced to their destination
in the periphery. As they do not project towards the uropod or telson, they may innervate
the tegument or musculature of the sixth pleomere. Nerves 3, 4 and 5 extend posterolaterally
(Figure 5F). While nerve 4 could be traced into the uropod (Figure 5G), nerves 3 and 5 proceed towards the telson. Besides several neurites that cross
the midline, the terminal ganglion features two commissure-like transverse tracts
(red and yellow arrows; Figure 5F). The more anterior transverse tract (red arrows; Figure 5F) is closely associated with the nerves 1 and 2, the more posterior transverse tract
(yellow arrows; Figure 5F) is closely associated with the nerves 3 and 4. An intersegmental nerve arises from
the connective between pleonic ganglion 5 and the terminal ganglion. The main branch
gives rise to several fine sub-branches that extend anteriorly and posteriorly to
innervate the periphery (white arrows; Figure 5F).
Lateral neurite bundle
A pair of lateral longitudinal neurite bundles (arrows, Figure 5D, E) is situated lateral to the ventral nerve cord, extending from the first thoracic
through the sixth pleonic segment. As described above, these lateral neurite bundles
are supplied by neurites arising anteriorly and posteriorly from the intersegmental
nerves. While the lateral neurite bundles in the thorax are additionally associated
with the segmental nerves, this is not the case in the pleon. No lateral longitudinal
neurite bundles have been observed in the subesophageal region.
Spelaeogriphus lepidops (Spelaeogriphacea)
General aspects
S. lepidops lacks compound eyes. The elongate, ellipsoid eye stalk inserts anteriorly in the
cephalon (position indicated in Figure 1A). Antenna 1 exhibits a prominent 3-segmental peduncle bearing an up to 40-segmented
inner and an up to 36-segmented outer flagellum (38]; see also Figure 1A,B). The distal three-fourths of the outer flagellum exhibit a series of short aesthetascs
(38]; see also Figure 1A,B). Antenna 2 exhibits a prominent 4-segmented peduncle, which bears a small scale
on its second segment and, distally, an about 70-segmented flagellum 38]. Gordon 38] described fields of conical papilla on antenna 1 and rows of setae both on antenna
1 and 2, but found no statocysts.
The distal protocerebral neuropil (Ia) and the lateral protocerebrum (Ib; Figure 6B,C,D) are located in the eyestalk (St; Figure 6E) and connected to the median protocerebrum (Ic) via the protocerebral tract (PT;
Figures 6A-D, 7C,E,F). Two unpaired blood vessels enter the median protocerebrum from the posteroventral
direction, uniting into a single blood vessel which leaves the median protocerebrum
dorsally (not shown). The olfactory lobe (ON) protrudes laterally from the rest of
the deutocerebrum (II; Figures 6A-D,H,I, 7C,E-G). The median protocerebrum (Ic) lies anterodorsal to the deutocerebrum (II;
Figures 6D, 7C). The neuraxis flattens posterior to the deutocerebrum (II) and takes on the anteroposterior
course of the ventral nerve cord (III, MdG; Figures 6D, 7C).
Figure 6. Morphology of the brain inSpelaeogriphus lepidops. Overview and semi-thin sections. A–D: Neuropil and nerves without somata. A, B: 3D-reconstructions in (A): dorsal and (B): anterior view. C, D: Schematic drawing in (C): posterior and (D): lateral view. E–L: Transverse semi-thin sections, ordered from anterior to posterior. E: Arrow points at the large cluster of anterolateral somata (AlS). F: Rocket points at the lateral root of the antenna 1 nerve (A1Nv). H, I: Points mark the olfactory glomeruli in the olfactory lobes (ON). Scale bars: 50
?m.
Figure 7. Morphology of the brain inSpelaeogriphus lepidops. Soma clusters, neuropil, and internal structure – click on A and F for interactive
3D models. A, B: Soma clusters (grey) in posterior view (A): with neuropil (yellow) and (B): without neuropil. C, D: Soma clusters in lateral view (C): with and (D): without neuropil. A–D: Arrow points at a large cluster and arrowhead at a small cluster of anterolateral
somata (AlS). E: Schematic drawing of neuropils (dark yellow) and tracts (grey) in posterior view.
F–H: 3D-reconstructions of neuropils and tracts in (F): anterior, (G): dorsal, and (H): lateral view. Neuropils are in yellow; tracts are in grey. H: Lateral accessory lobe (LAL) and olfactory lobe (ON) is shown semitransparent. Brain
width about 300 ?m.
Soma clusters
The brain of S. lepidops features five pairs of soma clusters and one unpaired soma cluster (i.e., altogether
eleven soma clusters).
The unpaired soma cluster consists of anteroventral somata (AvS) that lie ventrally
in the median protocerebrum (Ic; Figures 6F-I, 7A-D). On each side, it has a posterior extension reaching along the ventral and medial
side of the deutocerebrum (II; Figures 6H,I, 7A-D). The anterior and posterior regions of the median protocerebrum lack somata.
The first paired soma cluster consists of anterolateral somata (AlS, arrowheads),
surrounding the neuropil of the distal protocerebral neuropil (Ia; Figures 6E, 7A,C) and covering the anterior and lateral region of the lateral protocerebrum (Ib;
Figure 7A-D). Distally, this soma cluster extends far into the eyestalks (St; Figure 6E).
Also the second paired soma cluster consists of anterolateral somata (AlS, arrows),
but it lies apart from the previous one, dorsally in the lateral protocerebrum (Ib;
Figure 7A-D). The anterodorsal and posterior regions of the lateral protocerebrum as well
as the protocerebral tracts are not covered by somata.
The third paired soma cluster consists of anterodorsal somata (AdS) that are situated
dorsally and dorsolaterally in the median protocerebrum (Ic; Figures 6F-H, 7A-D). Each cluster shows a long dorsal extension and may be connected to its counterpart
by a thin midline-spanning bridge consisting of only a few somata (AdS; Figures 6F, 7A,B).
The fourth paired soma cluster consists of lateral somata (LS) situated dorsally,
laterally, and posteriorly in the olfactory lobes (ON; Figures 6H,I, 7A-D) and dorsolaterally in the rest of the deutocerebrum (II; Figure 7A-D). Also this cluster has a long dorsal extension (LS; Figures 6H, 7A-D). Although it is here described as a separate soma cluster, thin extensions of
only a few somata may connect it to neighboring soma clusters, respectively. The medial
sides of the deutocerebrum lack somata.
The fifth paired soma cluster consists of posterior somata (PS) that lie posterodorsally
in the tritocerebrum (III), opposite to the root of the antenna 2 nerve (A2Nv; Figures 6J-L, 7A,C). It has a long extension projecting posterodorsally (PS; Figures 6L, 7A-D).
In addition to the eleven large soma clusters in the periphery of the brain, several
single somata are scattered within the neuropil of the median protocerebrum (Ic; see
Figure 6F,G).
Neuropils
Distal protocerebral neuropil (Ia)
The distal protocerebral neuropil is a small and distinct spheroidal neuropil, which
is uniformly structured and directly borders the lateral protocerebrum (Ib; Figures 6B-E, 7F-H). It is directed towards the tip of the eyestalk (St; Figure 6E).
Lateral protocerebrum (Ib)
The terminal medulla is half as long as the eyestalk, shows an ellipsoid shape, and
constitutes the only neuropil in the lateral protocerebrum of S. lepidops (TM; Figures 6A-E, 7A,C,E-H).
Median protocerebrum (Ic)
The central body (CB) appears as a densely textured, spindle-shaped neuropil that
lies horizontally across the center of the median protocerebrum (Ic; Figures 6F,G, 7E-H). It is anteriorly subdivided into several small vertical lamellae (Figure 6F). The protocerebral bridge (PB) is composed of a pair of elongated neuropils lying
horizontally at the dorsal end of the median protocerebrum that are connected across
the midline via a thin tract (Ic; Figures 6F, 7E-H). On each body side, an elongated ventromedial protocerebral neuropil (VmPN) extends
diagonally through the ventromedial region of the median protocerebrum (Ic; Figures 6F,G, 7E,F,H). Anteriorly, the neuropil fuses with its surroundings. Its posterior end lies
anterior to the root of the antenna 1 nerve (A1Nv; Figure 7H). The lateral accessory lobe (LAL) lies laterally in the median protocerebrum, lateral
to the central body (Ic; Figures 6F,G, 7E-H). Its texture is denser than the surrounding neuropil.
Deutocerebrum (II)
With respect to the size of the brain, the olfactory lobe (ON) in S. lepidops is comparably smaller than that in M. halope. The olfactory lobe in S. lepidops shows a nearly spheroidal shape and is composed of numerous spheroidal olfactory
glomeruli (ON, points; Figure 6H,I). The unpaired median antenna 1 neuropil (MAN) spans across the midline of the
deutocerebrum (II; Figures 6H, 7E-H). Its lateral end is slightly bent posteriorly (MAN; Figure 7G). The lateral antenna 1 neuropil (LAN) lies in the dorsal region of the deutocerebrum
(II; Figures 6I, 7E,G,H), close to the olfactory lobe (ON; Figure 6I).
Tritocerebrum (III)
The ventral tritocerebral neuropil (VTN) lies ventrally in the tritocerebrum (III;
Figures 6K, 7E-H), directly posterior to the root of the labral nerve (LbNv; Figures 6K, 7H).
Tracts
The protocerebral tract (PT) extends from the dorsal region of the lateral protocerebrum
(Ib) to the anterolateral region of the median protocerebrum (Ic; Figures 6A-D, 7C,E,F). The olfactory globular tract (OGT) connects the olfactory lobe (ON) to the
lateral protocerebrum (Ib), traveling on its way through the median protocerebrum
(Ic; Figure 7E-G) and the protocerebral tract (PT). The olfactory globular tracts of both sides
form a chiasm posterior to the central body (CB; Figures 6G, 7E,G). The ventral portion of each olfactory globular tract pervades the ventromedial
protocerebral neuropil (VmPN; Figures 6G, 7E). One soma-free tritocerebral commissure (TC) arises from the tritocerebrum dorsomedially.
The commissure performs a posterodorsal arc when interconnecting both halves of the
tritocerebrum transversely (Figures 6A,C,D,L, 7A,E,G,H). On each side, a posttritocerebral connective connects the tritocerebrum
(III) to the mandibular ganglion (MdG; Figures 6A,C,D, 7A,C). It is dorsoventrally flattened like the tritocerebrum (Figures 6L, 7E).
The 1st vertical tract (VT1) connects the median protocerebrum with the deutocerebrum and
is situated posterior to the lateral accessory lobe (LAL; Figure 7E,G,H). The 2nd vertical tract (VT2) spans between the dorsal and ventral region of the median protocerebrum
(Ic), and lies anterior to the central body (CB; Figure 7F,H). The (vertical) Y/Z, the X, and W tracts arise from the dorsal side of the central
body and extend dorsally towards the protocerebral bridge. The Y/Z and X tracts pass
the olfactory globular tract anteriorly and were traced as far as the protocerebral
bridge. In contrast, the W tract passes the olfactory globular tract posteriorly,
and is not associated with the protocerebral bridge (Figure 7E-G). One neurite bundle from the dorsal portion of the W tract was traced into the
anterodorsal somata (not shown). The 4th transverse tract (TT4) interconnects the anterodorsal regions of the deutocerebrum
(II; Figures 6H, 7E-G). The 5th transverse tract (TT5) extends across the whole median protocerebrum, thereby forming
a ventral arc and passing the central body ventrally (CB; Figure 7E-H). The end of the 3rd transverse tract lies close to the arising protocerebral tract. The 6th transverse tract (TT6) lies posteroventral to the 3rd transverse tract and interconnects the lateral accessory lobes (LAL; Figures 6G, 7E,H). The 7th transverse tract (TT7) interconnects the anterior regions of the deutocerebrum and
is situated directly anterior to the median antenna 1 neuropil (MAN; Figure 7F). The 1st longitudinal tract (LT1) connects the anterior region of the median protocerebrum
with the anterior region of the deutocerebrum. At the midpoint of its way through
the ventral region of the median protocerebrum, the 1st longitudinal tract (LT1) leaves the surrounding neuropil, so that its middle portion
is only surrounded by anteroventral somata (Figure 6G). On each side, an 8th transverse tract (TT8) connects the nerve root of the tegumentary nerve to the medial
region of the tritocerebrum. On its way, the tract remains near the surface of the
tritocerebrum, before turning ventrally inside (III; Figure 6J).
Nerves
The antenna 1 nerves (A1Nv) enter the deutocerebrum (II) from the anteroventral direction
(Figures 6B,D,F,G, 7C). Proximally, each nerve splits into a thick medial and a thin lateral root. The
lateral root (A1Nv, rocket) proceeds into the center of the olfactory lobe (ON; Figure 6F), while the medial root enters the rest of the deutocerebrum. Distally, each antenna
1 nerve gives rise to three smaller branches (A1Nv; Figure 6B). The prominent antenna 2 nerve (A2Nv) enters the tritocerebrum (III) from the ventrolateral
direction (Figures 6B,C,D,J, 7C,E). After giving rise to the lateral tritocerebral nerve (LTNv; Figures 6A-D, 7C) and tegumentary nerve, the antenna 2 nerve splits distally into two branches that
extend into the antenna 2. The tegumentary nerve splits distally into two branches
which extend to the inner body wall (Figures 6A-D, 7A,C). The posterodorsal nerve (PdNv) enters the dorsal region of the tritocerebrum
(III) from the posterodorsal direction (Figures 6A,C,D, 7A,C). The labral nerve (LbNv) enters the anteroventral side of the tritocerebrum (III)
from the anterior direction (Figures 6A,B,D,K, 7C). Distally, it extends into the labrum which lies anteroventral to the tritocerebrum
in S. lepidops. On each side, a stomatogastric nerve (StNv) arises from the tritocerebrum, anteromedially
(Figure 6A-D). The stomatogastric nerves from both body sides (StNv) unite forming a single
unpaired nerve in front of the esophagus. This single nerve extends dorsally along
the upper side of the gut (Figures 6A,C, 7A).
Tethysbaena argentarii (Thermosbaenacea)
General aspects
T. argentarii lacks compound eyes and eyestalks (Figure 1D, E). Antenna 1 exhibits a 3-segmented peduncle bearing an up to 5-segmented outer
and an up to 10-segmented inner flagellum (57]; see also Figure 1D, E). Unlike in the other species, the up to nine-segmented antenna 2 of T. argentarii is considerably less prominent than antenna 1 (57]; see also Figure 1D, E). Simple setae are loosely distributed over antenna 1 and 2. In addition, the
peduncle of antenna 1 is equipped with feather-like (setulated) setae 57].
The distal protocerebral neuropil (Ia) is situated directly lateral to the lateral
protocerebrum (Ib; Figure 8A-F). The lateral protocerebrum is directly connected to the median protocerebrum
(Ic) ventromedially (Figure 8A-D,F,G). On each side, the neuropil of the median protocerebrum shows a dorsal, an
anterior, and a lateral bulge (Figure 8C,F). The median protocerebrum (Ic) lies dorsal to the deutocerebrum (II; Figure 8A-D). The olfactory lobe (ON) protrudes laterally from the rest of the deutocerebrum
(II; Figures 8A,C,D,I, 9A,E,F). T. argentarii features a posterior accessory neuropil on each side (PAN), which is connected to
the deutocerebrum via a thin tract whose target is unclear (Figures 8A,C,D,I, 9B,E,F). The deutocerebrum (II) lies anterodorsal to the tritocerebrum (III; Figure 8A-D). Posterior to the latter, the neuraxis takes on the anteroposterior course of
the ventral nerve cord, parallel to the body axis (Figures 8D, 9E).
Figure 8. Morphology of the brain inTethysbaena argentarii. Overview and semi-thin sections. A-D: Neuropil and nerves without somata. A: 3D-reconstruction in dorsal view. Anterior directed towards the right. B: 3D-reconstruction in anterior view. C: Schematic drawing in posterior view. Double arrowhead marks a dorsal extension,
simple arrowhead marks a lateral extension of the median protocerebrum (Ic). D: Schematic drawing in lateral view. Rocket marks the lateral root of the antenna
1 nerve (A1Nv). E–G, I–K: Horizontal semi-thin sections, ordered from dorsal to ventral. F: Double arrowheads as in A. G: Arrow points at a condensation of neuropil within the anteromedial protocerebral
neuropil (AmlPN) that was observed on each body side. H: Virtual transverse section. Dotted lines mark the protocerebral bridge (PB) and
the central body (CB). The latter is divided into spheroidal subunits (asterisks).
Arrowheads as in A. I: Rocket points at the lateral root of the antenna 1 nerve (A1Nv). Points mark the
(only faintly recognizable) olfactory glomeruli in the olfactory lobes (ON). Arrows
mark the large posterolateral extensions of lateral somata (LS), which embrace the
posterior accessory neuropils (PAN). Scale bars: 50 ?m.
Figure 9. Morphology of the brain inTethysbaena argentarii. Soma clusters, neuropil, and internal structure – click on A and G for interactive
3D models. A: Soma clusters (grey) and neuropil (yellow) in anteroventral view. B, C: Soma clusters in posterodorsal view (B): with and (C): without neuropil. D, E: Soma clusters in lateral view (D): without and (E): with neuropil. A–E: Arrows mark the large posterolateral extensions of lateral somata (LS), which embrace
the posterior accessory neuropils (PAN, only depicted in B and E). F: Schematic drawing of neuropils (dark yellow) and tracts (grey) in posterior view.
G: 3D-reconstructions of neuropils and tracts in lateral view. Neuropils are in yellow;
tracts are in grey. Brain width about 200 ?m.
Soma clusters
T. argentarii features one pair of soma clusters and one unpaired soma cluster (i.e., altogether
three soma clusters).
The paired soma cluster is composed of all anterolateral somata (AlS), covering the
distal protocerebral neuropil (Ia) and the lateral protocerebrum (Ib) almost completely
(Figure 9A-E).
All other somata of the brain are included in one large unpaired soma cluster. It
is situated dorsally (AdS; Figures 8E-H, 9A-C), anteriorly (AvS; Figures 8H, 9A,D,E), and posterolaterally (LS; Figures 8H, 9B-E) in the median protocerebrum (Ic); anteriorly (VmS; Figure 9A,D,E), laterally, and posterolaterally (LS; Figures 8H,I, 9B-E) in the deutocerebrum (II); and laterally and ventrally (PS) in the tritocerebrum
(III; Figures 8J,K, 9A-E). A thin row along each posttritocerebral connective even connects the unpaired
soma cluster of the brain with the somata of the mandibular ganglion (MdGS; Figure 9D,E). On each body side, the unpaired soma cluster of the brain shows one posterior
extension pointing posterolaterally from the median protocerebrum (LS; Ic; Figure 8G) and one posterolateral extension (arrows, LS) pointing posterolaterally and lying
adjacent to the posterior accessory neuropil (PAN; Figures 8I, 9B-E). The posterior side of the median protocerebrum (Figures 8F,G, 9B), the lateral side of the olfactory lobe (ON; Figures 8I, 9E), the anterolateral and medial side of the deutocerebrum (II; Figures 8I, 9A,B), and the medial and posterior side of the tritocerebrum (III; Figures 8J,K, 9B) are soma-free.
Neuropils
Distal protocerebral neuropil (Ia)
The distal protocerebral neuropil (Ia) is a uniformly structured neuropil with a spheroidal
shape (Figure 8A-G) and in part confluent with the lateral protocerebrum (Ib; Figure 8E).
Lateral protocerebrum (Ib)
The largest region of the lateral protocerebrum is constituted by the terminal medulla
(TM; Figures 8E-G, 9F,G). The hemiellipsoid body (HE) is situated anteromedially in the lateral protocerebrum
(Ib; Figures 8E, 9F,G). The anteromedial (distal) region of each hemiellipsoid body has a convex shape
and is more densely textured than the rest of the hemiellipsoid body. Posterolaterally
(proximally), the hemiellipsoid body (HE) is confluent with the terminal medulla (TM;
Figure 8E). Furthermore, a glomerular small lateral neuropil (SLN4) is distinguishable directly
anterolateral to each terminal medulla (TM; Figures 8E, 9F,G).
Median protocerebrum (Ic)
An anteromedial protocerebral neuropil (AmlPN) protrudes anteriorly from the median
protocerebrum on each body side (Ic; Figures 8A,G, 9F,G). The medial region (arrow) of each anteromedial protocerebral neuropil (AmlPN)
shows a significantly higher neuropil density than the surrounding neuropil (Figure 8G). The unpaired central body (CB) lies horizontally across the center of the median
protocerebrum (Ic; Figures 8H, 9F,G). The central body (CB) of T. argentarii is comparatively thick and shows a compartmentalization into altogether five spheroidal
subunits (asterisks, Figure 8H). The protocerebral bridge (PB; Figure 8G,H) lies dorsally in the median protocerebrum (Ic), adjacent and directly dorsal
to the central body (CB; Figures 8H, 9F,G). It is composed of two elongated subunits which are identifiable due to their
comparatively dense neuropil texture (Figure 8G,H); these subunits contact one another in the midline. Unlike in M. halope and S. lepidops, both of which feature a lateral accessory lobe, the median protocerebrum in T. argentarii (arrowheads in Figure 8C,H) does not show any compartmentalization or condensation of neuropil in the lateral
region.
Deutocerebrum (II)
The olfactory lobe (ON) lies lateral to the rest of the deutocerebrum (II), receiving
the lateral root of the antenna 1 nerve (A1Nv, rocket) from the anterior direction
(Figure 8D,I). It is composed of densely packed olfactory glomeruli (ON, points) whose exact
shape could not be identified (Figure 8I). A posterior accessory neuropil is situated posterolateral but slightly distant
to each olfactory lobe (PAN; Figures 8A,D,I, 9F,G). The posterior accessory neuropil is embedded within lateral somata that are
arranged in a long extension in this region (arrows, LS; Figures 8I, 9B-E). Since the tracts connecting the posterior accessory neuropil to the rest of
the brain could not be traced through this group of lateral somata, it remains unclear
whether the posterior accessory neuropil is associated with the olfactory lobe or
with another region of the deutocerebrum. The texture of the posterolateral accessory
neuropil is unstructured (PAN; Figure 8I). Further neuropils could not be distinguished within the deutocerebrum.
Tritocerebrum (III)
Distinct neuropils could not be identified in the tritocerebrum in T. argentarii.
Tracts
The 3rd transverse tract (TT3) is situated posterior to the central body and interconnects
the lateral regions of the median protocerebrum (Ic; Figure 9F,G). The tritocerebral commissure (TC) interconnects the halves of the tritocerebrum
(III) horizontally (Figures 8A-C,K, 9A,B,F,G). It is anteroposteriorly flattened and has a minute anteroposterior hole
in the midline. On each body side, a posttritocerebral connective (PTC) connects the
tritocerebrum (III) to the mandibular ganglion (MdG; Figure 8A,D,K). Each posttritocerebral connective is dorsoventrally flattened and medially
engraved by a large unpaired transverse apodeme that is associated to several mandible
muscles and passes on the dorsal side of the connective.
Nerves
The prominent antenna 1 nerve (A1Nv) enters the deutocerebrum (II) from the anterior
direction (Figures 8B,D,I, 9A,E), splitting proximally into a thick medial and a thin lateral root. The lateral
root (rocket, A1Nv) proceeds into the center of the olfactory lobe (ON), while the
medial root proceeds into the rest of the deutocerebrum (Figure 8I). Distally, two smaller branches diverge from each antenna 1 nerve (A1Nv; Figure 8D). A lateral deutocerebral nerve (LDNv) enters the posterior side of each olfactory
lobe (ON) from the lateral direction (Figures 8A,C,D, 9A,B,E). The antenna 2 nerve (A2Nv), which is thinner than the antenna 1 nerve in T. argentarii, enters the tritocerebrum (III) from the lateral direction (Figures 8B,D,J, 9A,E,G). A stomatogastric nerve (StNv) enters each half of the tritocerebrum (III)
from the anteromedial direction (Figure 8B,D,K). Distally, stomatogastric nerves from both body sides unite forming a single
unpaired nerve (StNv) in front of the esophagus; this single nerve extends dorsally
along the upper side of the gut (E, Figures 8A-D,J, 9A,B). The labral nerve (LbNv) enters the ventral side of the tritocerebrum (III) from
the anterior direction (Figure 8B,D).