Zoologischer Anzeiger 245 (2
of
riu


differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate
large proportion of the taxonomic diversity of zoo-
plankton in lentic and lotic systems (Nogrady et al.
1993; Ricci and Balsamo 2000). Most planktonic rotifers
contain any colonial species. All colonial rotifers are
restricted to the order Flosculariacea, a clade de?ned by
the structure of their pharyngeal hardparts, the jaw-like
ARTICLE IN PRESSmalleoramate trophi (Nogrady et al. 1993). The clade
has questionable relations to the Ploima, and to the
0044-5231/$ - see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jcz.2006.04.001

Tel.: +1 978 934 2885; fax: +1 978 934 3044.
E-mail address: Rick_Hochberg@uml.edu.rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed
serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in
nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta.
r 2006 Elsevier GmbH. All rights reserved.
Keywords: Rotifer; Nervous system; Immunohistochemistry; Serotonin
1. Introduction
Rotifers are some of the smallest free-living animals
in freshwater environments, and yet account for a
are solitary animals of the order Ploima, which
contribute substantially to the trophic dynamics of
freshwater communities (Wallace and Snell 2001).
Ploima is the largest and most diverse monophyletic
assemblage of monogonont rotifers, yet does notmost dorsal pair innervates a coronal nerve ring that encircles the apical ?eld. Within the apical ?eld is a second nerve
ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to
modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Speci?cDepartment of Biological Sciences, University of Massachusetts, One University Avenue, Lowell, MA 01854, USA
Received 21 November 2005; received in revised form 4 April 2006; accepted 10 April 2006
Corresponding editor: M. Schmitt
Abstract
The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the ?rst
immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species
of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epi?uorescence
and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and
C. dossuarius, the serotonergic nervous system is de?ned by a dorsal cerebral ganglion, apically directed cerebral
neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT-
immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical ?eld. TheRick HochbergOn the serotonergic nervous system
Conochilus coenobasis and C. dossua
Flosculariacea, Conochilidae)006) 53?62
two planktonic rotifers,
s (Monogononta,
www.elsevier.de/jcz
The purpose of the current analysis is to provide the
ARTICLE IN PRESS
er Aorder Collothecacea with which it is traditionally
aligned (S?rensen 2002).
Within the Flosculariacea are approximately 25
species of rotifers that form colonies (Wallace 1987).
Colony formation is generally described as autorecrui-
tive or allorecruitive, depending on whether partheno-
genetic embryos develop alongside adults, often within a
gelatinous matrix, or settle on adults, arriving from
conspeci?cs outside of the colony (Nogrady et al. 1993).
Since colonies are essentially aggregations of solitary
individuals, they lack any anatomical interconnections,
and so do not share any metabolic demands or trophic
resources. However, the organic matrix that binds a
colony together, whether it be made of a soft gelatinous
medium or hardened secretions, may serve as protection
from predators, and large colony size may itself be a
deterrent (Stemberger and Gilbert 1987; also reviewed in
Wallace 1987). The number of individuals that make up
a colony is highly variable and sometimes species-
speci?c. Species of Lacinularia have been reported to
comprise colonies of greater than 1000 individuals, while
some species of Conochilus form colonies of only a few
individuals (Wallace 1987). Many of these colonies are
sessile and highly selective of their substratum, but other
colonies form in the plankton, where they grow
presumably by autorecruitment. There is apparently
little morphological difference between congeners that
form sessile and planktonic colonies (Nogrady et al.
1993).
The planktonic lifestyle is prevalent among species of
Conochilidae, but coloniality is found in less than half
of the known species. The genus Conochilus contains
both colonial and solitary animals, the former desig-
nated to the subgenus Conochilus and the latter to the
subgenus Concohiloides. As noted in the cladistic
analysis of Segers and Wallace (2000), the colonial
lifestyle is hypothesized as the derived condition within
the Flosculariacea, and probably arose several times
independently. While the embryos of solitary species
may develop alongside their parents within a gelatinous
matrix, as is present in all species of Conochilidae and
Flosculariidae, adults generally lead a solitary lifestyle.
To date, our knowledge of the anatomical differences
between solitary and colonial species, or planktonic and
benthic species, is generally limited to differences in
optically visible characteristics such as body shape and
the organization of the corona. However, many such
characters are often highly malleable and therefore
ambiguous from a phylogenetic standpoint. Alterna-
tively, pharyngeal hardparts have provided a solid
foundation for making phylogenetic inferences because
of their conserved structure (Segers and Wallace 2000;
S?rensen 2002). Still, mastax structure alone provides
limited information on the evolution of lifestyle changes,
although it can presumably provide insights into habitat
R. Hochberg / Zoologisch54partitioning through trophic specialization. With this in?rst details on the organization of the nervous system in
planktonic species of Conochilus. Speci?cally, solitary
species of the subgenus Conochiloides are examined
using anti-serotonin (5-HT) immunohistochemistry,
epi?uorescence and confocal laser scanning microscopy,
and 3D imaging software. The distribution of seroto-
nergic neurons is currently known for three species of
Ploima (Kotikova et al. 2005) and provides a basis for
comparing neural topologies and the innnervation of
speci?c organ systems.
2. Material and methods
2.1. Collection and identi?cation
Specimens of Conochilus were collected with a 64 mm
plankton net from small lakes at the West Palm Beach
Airport, Florida in February and March 2005. Rotifers
were anaesthetized in 1% MgCl2 and photographed
alive as whole mounts on glass slides. Specimens were
identi?ed according to the taxonomic atlas of the Frank
J. Meyers Rotifera Collection CD (The Academy of
Natural Sciences).
2.2. Immunohistochemistry
Twenty-three rotifers (Conochilus coenobasis Skorikov,
1914, N ? 15; C. dossuarius (Hudson, 1885), N ? 8)
were anaesthetized in 1% MgCl2 and ?xed in 4%
paraformaldehye in 0.1M PBS for 24h at 4 1C.
Specimens were next rinsed (3
 ) over the course of 12hmind, recent attention has been directed at other organ
systems, namely the muscular system (Hochberg and
Litvaitis 2000; Kotikova et al. 2001, 2004; S?rensen
et al. 2003; Santo et al. 2005; S?rensen 2005a, b) and
nervous system (Kotikova 1995, 1997, 1998; Kotikova
et al. 2005), the latter of which generally shows a high
degree of conservation and may therefore provide clues
to the origins and evolution of different lifestyles. More
speci?cally, increasing attention has been directed at the
nervous systems of the most heterogeneous rotifers, the
ploimates (Kotikova, 1988, 1995, 1997, 1998; Kotikova
et al., 2005). While no ploimates are colonial, their
diverse lifestyles and body morphologies provide
an index against which to measure neuroanatomical
variation ? indeed, conservation within such diversity
suggests that a Bauplan for the construction of the
nervous system arose prior to the divergence in lifestyles.
Whether non-ploimate rotifers possess a fundamentally
different Bauplan or simply modi?cations to a common
pattern will require greater knowledge of species from
outside the Ploima.
nzeiger 245 (2006) 53?62in 0.1M PBS and then placed in IT Signal Enhancer
Sciences) on glass slides and refrigerated at 4 1C for 24h
before examination.
ARTICLE IN PRESS
her A2.3. Microscopical examination
Wholemount specimens were examined on two
microscopes: (1) a Zeiss Axioimager equipped with
epi?uorescence, digital Axiocam MRm, and Axiovision
software (Rel. 4.4) at the University of Massachusetts
Lowell; and (2) a Nikon Eclipse E800 compound
microscope equipped with a Biorad Radiance 2000 laser
system at the Smithsonian Marine Station in Fort
Pierce, Florida. The specimens observed on the Zeiss
microscope were kept refrigerated for 2 months prior to
examination. The specimens observed on the confocal
system were examined within 1 week of staining.
Lasersharp software was used to collect a series of
optical sections with maximum intensity projection
along the z-axis. Confocal images were imported into
Confocal Assistant and made into TIF ?les. Additional
digital ?les were imported into Volocity (Improvision)
to render 3-D images and create X?Y?Z rotations in
TIF and AVI formats. Movie ?les (AVI) are available
upon request. No manipulations of the original images
were made other than changes of color (false coloring or
grayscale) or cropping. The program Carnoy V 2.0
(r 2001 Peter Schols) was used to make measurements
of neurons in some digital images.
3. Results
Adults of Conochilus coenobasis Skorikov, 1914 are
enclosed in a gel-like matrix that often contains
developing embryos and juveniles (Fig. 1A and C). No
more than three developing young are present in any
one ??colony?? and adults are often solitary. Most
specimens are 170?250 mm long. Adults of C. dossuarius
(Hudson, 1885) are always solitary and enclosed in a gel-
like matrix. Maximum length of adults is 300 mm.
Anti-serotonin immuno?uorescence is prevalent
throughout the central and peripheral nervous systems(Ingenta) for 2h to minimize non-speci?c staining. Twenty
specimens were transferred to anti-rabbit serotonin anti-
body (dilution 1:200, Sigma-Aldrich) in 1.5ml centrifuge
tubes at 4 1C on an orbital shaker for 48h. The antibody
was diluted with PBT (0.1M PBS plus 1% Triton X-100).
Three specimens were used as controls and omitted from
the primary antibody. Specimens were then rinsed (3
 ) in
0.1M PBS over 48h and transferred to goat anti-rabbit
Alexa Fluor 546 (dilution 1:500, Sigma-Aldrich). Speci-
mens were stained in the dark at 4 1C on an orbital shaker
for 48h and then rinsed in 0.1M PBT for 24h. Specimens
were mounted in Fluoromount G (Electron Microscopy
R. Hochberg / Zoologiscof adult C. coenobasis and C. dossuarius. Immuno-?uorescence was never detected in any embryos.
Omission of the primary antibody did not produce any
immunoreactivity, although secondary antibody stain-
ing is present in small amounts on the coronal cilia and
the gel-like matrix that surrounds each individual rotifer
(not shown). Some auto?uorescence is also present in
the gut. The nervous systems of both species are similar
and are therefore described together.
The cerebral ganglion is located dorsally above the
mastax and contains at least four bilateral pairs of
serotonin-immunoreactive perikarya (Figs. 2A?C and
3?5). The cell bodies form a cluster that measures
approximately 10 mm wide by 10 mm long (Range:
8?12 mm long by 9?10 mm wide). Average perikaryon
diameter is 4.5 mm (range: 3.8?5.5 mm). Three pairs of
neurites project from the cerebral ganglion toward the
corona: a dorsal pair (dsn), a medial pair (msn), a
ventral pair (vsn) (Figs. 2?5). The dorsal pair of neurons
are unipolar and have neurites (0.6 mm wide by 20 mm
long) that project toward the coronal nerve ring
(cnr) (Figs. 4A?C and 5A). The cnr forms a strongly
immunoreactive loop perpendicular to the body axis,
encircling the entire apical region on the periphery of the
coronal ciliature (Figs. 3D?E, 4A?B and 5). The
diameter of the individual neurites that form the cnr is
0.7?0.9 mm. Average diameter of the entire cnr is 49 mm
(range: 47?51 mm). Within the borders of the cnr is an
inner nerve ring (inr) in the shape of a horseshoe (inr,
Fig. 4B). The inr is parallel to the cnr, perpendicular to
the longitudinal body axis, and abuts the inner margin
of the coronal ciliature (Figs. 4B and 5). There were no
obvious perikarya associated with the inr, nor did it
receive any detectable innervation from the cnr or any
apically directed neurites from the cerebral ganglion.
Projecting within the cnr are two pairs of neurites. One
pair of neurites (0.4 mm diameter) originates from the
base of the cerebral ganglion and projects towards
the center of the corona and through the cnr (vsn,
Figs. 2A?B, 4A?B and 5). Each neurite follows a slight
ventral angle until reaching the cnr where it bends
medially. The site of innervation is unknown but may be
close to the mouth. A second pair of weakly immunor-
eactive neurites (msn, Fig. 4A and B) originates from the
middle of the cerebral ganglion and innervates the apical
region, but the site of innervation was not determined.
A pair of neurites (nc) project from the posterior
region of the cerebral ganglion and are presumably part
of the nerve cords. Each neurite appears to originate
from a single perikaryon close to the posterior midline
of the cerebral ganglion (Fig. 4D). After exiting the
cerebral ganglion, the neurites bend ventrally for
approximately 20?25 mm before curving posteriorly
(Figs. 2?5). A pair of cell bodies is present along the
length of the nerve cords at approximately 50% body
length (Fig. 2D). The nerve cords coalesce in the foot
nzeiger 245 (2006) 53?62 55region.
ARTICLE IN PRESS
er AR. Hochberg / Zoologisch56Very few peripheral neurites are present. In C.
dossuarius, very ?ne neurites appear to innervate the
ventral antennae, while there is no such innervation
present in C. coenobasis. In the former species, the ventral
antennae are partially fused along their length for
approximately 20mm. Very thin immunoreactive ?bers
are present along the ventral side of the fused portion, but
do not enter either of the individual antennae (Fig. 3A).
Fig. 1. Live specimens of Conochilus coenobasis: (A) a colony consis
matrix; and (B) anterior end of an adult specimen. Compared to Fig
an adult specimen. Inset: cell bodies at mid-length of nerve cords
ju ? juvenile, ma ? gelatinous matrix, ms ? mastax, st ? stomach,nzeiger 245 (2006) 53?624. Discussion
The rotifer central nervous system can be broadly
described as comprising a dorsal cerebral ganglion that sits
atop the mastax and paired ventrolateral nerve cords that
innervate the trunk region (Remane 1929?1933). Neurites
from the cerebral ganglion project toward the apical region
within the coronal ?eld and innervate the ciliature and
ting of one adult, one juvenile, and one embryo in a gelatinous
. 5A; (C) developing embryo; and (D) stalk-like foot region of
. an ? anus, eg ? egg, em ? embryo, gv ? germovitellarium,
tr ? trophi, va ? ventral antennae.
ARTICLE IN PRESS
her AR. Hochberg / Zoologiscvarious sensory devices (Cle?ment and Wurdak 1991).
Peripheral neurites innervate the mastax and sometimes
form a complex neural network (Kotikova et al. 2005). In
the posterior trunk, neurites may arise off the major nerve
cords and innervate the toes (Kotikova 1995). While some
variation in this organization is present, the extent of our
knowledge is based almost exclusively on species of
Ploima, and to a large degree, the results of chemical
histo?uorescence (Nogrady and Alai 1983; Raineri 1984;
Fig. 2. Anti-serotonin immunohistochemistry in specimens of Cono
region of an adult specimen; ventral is to the left; (B) Z-projection o
optical sections; (C) Z-projection of the anterior end of a juvenile sp
cerebral neurons. 0.05mm
 100 optical sections; and (D) epi?uoresc
bodies on the nerve cords. af ? auto?uorescent material in gut, cg
apical ?eld.nzeiger 245 (2006) 53?62 57Keshmirian and Nogrady 1987, 1988; Kotikova 1988,
1995, 1997, 1998).
To date, only three ploimates have been studied using
anti-serotonin immunohistochemistry: Plationus patulus
(Mu?ller, 1786), Euchlanis dilatata (Ehrenberg, 1832),
and Asplanchna herricki (Guerne, 1888) (Kotikova
et al. 2005). The distribution of serotonergic perikarya
and neurites in these species is similar despite their
distant evolutionary relationships within the Ploima
chilus coenobasis: (A) epi?uorescence view of the lateral trunk
f a different adult specimen; ventral is to the left. 0.1 um
 150
ecimen revealing part of the cerebral ganglion and some of the
ence view of the nerve cords in the foot. Inset: Serotonergic cell
? cerebral ganglion, vsn ? ventral cerebral innervation of the
ARTICLE IN PRESS
er AR. Hochberg / Zoologisch58(see S?rensen 2002). In general, serotonergic perikarya
make up only a small proportion of the cell bodies in the
cerebral ganglion (compared to FMRFamidergic peri-
karya). Cerebral innervation is limited to the coronal
tufts of cilia and the nerve cords. The mastax is
innervated by serotonergic neurons, but a direct associa-
tion with the cerebral ganglion has not been demon-
strated; an indirect association, by way of the nerve
cords, is known in A. herricki (Kotikova et al. 2005).
Fig. 3. Anti-serotonin immunohistochemistry in specimens of Con
adult specimen. 0.1mm
 452 optical sections; (B) Z-projection of
sections; (C?E) 3D images rendered using Volocity software and con
end of an adult specimen; (D) Same specimen viewed with ?uoresc
from a postero-lateral view; (F) Same specimen viewed with ?uore
ventral antenna, cg ? cerebral ganglion, cnr ? coronal nerve ring,
nc ? cerebral innervation of the nerve cords, va ? ventral antennaenzeiger 245 (2006) 53?62A similar condition to that described above has now been
demonstrated for species of Conochilus (subgenus Con-
ochiloides). Both the cerebral ganglion and neurites that
presumably innervate the nerve cords display strong anti-
serotonin immunoreactivity. The cerebral ganglion is
composed of only a few serotonergic perikarya, all of
which produce neurites that innervate the apical region.
However, speci?c differences exist between species
of Conochilus and the ploimates, namely in cerebral
ochilus dossuarius: (A) Z-projection of the anterior end of an
same specimen, different focal plane. 0.35 mm
 206 optical
focal imaging; (C) gray-scale transmission image of the anterior
ence; (E) gray-scale transmission image of the same specimen
scence. arrow ? thin immunoreactive ?bers at the base of the
inr ? inner nerve ring (horseshoe shape) of the apical ?eld,
, vsn ? ventral cerebral innervation of the apical ?eld.
ARTICLE IN PRESS
her AR. Hochberg / Zoologiscinnervation of the apical ?eld and the distribution of
immunoreactive cell bodies in the mastax.
The cerebral ganglion of both C. coenobasis and
C. dossuarius, as delineated by anti-serotonin immuno-
reactivity, is relatively small and weakly de?ned. Speci?c
pathways between cerebral perikarya are therefore
dif?cult to map out. Still, pathways from perikarya to
regions outside the cerebral ganglion can be traced,
making comparative studies of innervation possible.
Speci?cally, there are at least three pairs of neurites that
innervate the apical ?eld. A pair of thin dorsal neurites
(dsn) innervate the coronal nerve ring (cnr) and may
function to control ciliary beat and therefore swimming
velocity. In the ploimates P. patulus and E. dilatata, a
pair of serotonergic neurites also innervates the corona,
but in neither species is there any neural structure
equivalent to the coronal nerve ring; instead, there is an
??anterior dorsal semi-ring?? close to the corona.
Kotikova (1998) also documents a catecholaminergic
Fig. 4. Three-dimensional images of the serotonergic nervous system
scale and inverted for contrast: (A) lateral view of head region s
specimen rotated to the left; (C) frontal view of the cerebral ganglion
ganglion, inr ? inner nerve ring (horseshoe shape) of the apical
nc ? cerebral innervation of the nerve cords, dsn ? dorsal cerebral
innervation of the apical ?eld.nzeiger 245 (2006) 53?62 59ring in species of Philodina, Brachionus, and Dicrano-
phorus, but whether such rings innervate the coronal
ciliature, and whether the chemical histo?uorescence
is due to serotonin (see Lindvall and Bjo?rklund 1974;
De La Torre and Surgeon 1976), is unknown.
Based on studies of serotonin distribution in other
invertebrates (Hay-Schmidt 2000) and its function in
modulating ciliary beat (reviewed in Weiger 1997), it is
not unexpected to ?nd direct innervation of the coronal
ciliature in rotifers. Most invertebrate larvae, of either
protostomian or deuterostomian ancestry, have brains
that innervate ciliary organs by way of serotonergic
neurons (Hay-Schmidt 2000). The same is true of adult
invertebrates without larval stages (Hochberg and
Litvaitis 2003). Perhaps what is most surprising is that
so few rotifers possess a similar innervation of the
corona as in species of Conochilus. All rotifers, despite
their speci?c form of locomotion (e.g., creeping or
swimming) or lifestyle (e.g., planktonic or sessile), rely
of Conochilus dossuarius. Volocity images are collected in gray
howing major neurons innervating the apical ?eld; (B) same
; and (D) posterior view of the cerebral ganglion. cg ? cerebral
?eld, msn ? medial cerebral innervation of the apical ?eld,
innervation of the coronal nerve ring, vsn ? ventral cerebral
ARTICLE IN PRESS
er AR. Hochberg / Zoologisch60on the coronal ciliature to capture food. In the absence
of complete serotonergic innervation, there may be
other neurotransmitters that have taken over the
stimulatory role (e.g., FMRFamide, see Kotikova
et al. 2005). Alternatively, the neural stimulus may be
propagated along the ciliated epithelium by other means
such as gap junctions, although such junctions are
unknown from the rotatory apparatus of other species
(see Cle?ment and Wurdak 1991).
Two additional pairs of cerebral perikarya send
neurites to a region around the corona in species of
Conochilus, although these neurites do not appear to
innervate the coronal ciliature. The largest diameter
neurites (vsn) innervate a region around the ventral
mouth margin, and a pair of extremely thin neurites
(msn) innervate a region around the dorsal apical ?eld.
It is unknown whether either of these neurites contribute
Fig. 5. Schematic illustrations of the serotonergic nervous system
showing the general nervous system architecture; (B) frontal view
perikarya and axons; and (C) posterior view of the cerebral gan
cnr ? coronal nerve ring, dsn ? dorsal cerebral innervation of the c
shape) of the apical ?eld, mo ? mouth, ms ? mastax, msn ? m
innervation of the nerve cords, st ? stomach, va ? ventral antennaenzeiger 245 (2006) 53?62directly or indirectly to the inr in the apical region. This
ring is especially interesting because it outlines the
horseshoe shape of the corona, but is located along the
corona?s inner margin. Unfortunately, immunoreactiv-
ity in the nerve ring was extremely weak making it
dif?cult to locate the source perikarya. It seems likely
that this nerve ring may also function in the control of
the coronal cilia, but without a more detailed knowledge
of its precise location and innervation, this remains
speculative.
Aside from direct innervation of the apical ?eld,
serotonin is often detected in the nerve cords and
pharynges of rotifers (Kotikova et al. 2005). In species
of Conochilus, a single pair of serotonergic perikarya in
the posterior region of the cerebral ganglion send
neurites into the presumptive nerve cords. Serotonergic
perikarya are not present in a similar position in any of
of species of Conochilus: (A) lateral view of the anterior end
of the cerebral ganglion showing the general distribution of
glion. an ? anus, cc ? coronal cilia, cg ? cerebral ganglion,
oronal nerve ring, eg ? egg, inr ? inner nerve ring (horseshoe
edial cerebral innervation of the apical ?eld, nc ? cerebral
, vsn ? ventral cerebral innervation of the apical ?eld.
M.K.S., 2004. Musculature of an illoricate rotifer As-
planchnopus multiceps as revealed by phalloidin ?uroes-
ARTICLE IN PRESS
her Athe previously examined ploimate rotifers (Kotikova et
al. 2005), nor in the rotifer Notommata copeus (Hoch-
berg, personal observations). However, in most ploi-
mates there is always a pair of cells in the pathway that
connects the nerve cords to the cerebral ganglion
(Kotikova et al. 2005). Anti-serotonin immunoreactivity
is present along the entire length of the nerve cords in
species of Conochilus, as well as N. copeus (Hochberg,
personal observation), but in other ploimates, immu-
noreactivity is lost in the posterior region of the nerve
cords (Kotikova et al. 2005).
Different neural patterns are also present in the
pharynges of P. patulus, E. dilatata, and A. herricki,
although no serotonergic innervation is detected in species
of Conochilus. Serotonergic control of muscles associated
with feeding structures is well documented in other
invertebrates such as Caenorhabditis elegans (Horvitz
et al. 1982) and Aplysia californica (Kupfermann and
Weiss 1981), where serotonin stimulates contractions of
the myoepithelial pharynx and buccal mass, respectively.
Similar control of mastax musculature has not been
analyzed in rotifers, although neuropharmacological
analysis is certainly possible as has been demonstrated
previously using neurotransmitters, hormones, and neuro-
transmitter antagonists (Nogrady and Keshmirian
1986a, b; Nogrady 1987; Gallardo et al. 1999, 2000). The
different levels of serotonergic innervation in rotifers may
be a function of the number of different muscles and/or
hardparts under neural control. How this relates to the
lack of immunoreactivity in species of Conochilus is
unknown.
The present study demonstrates that there are
common patterns in the distribution of serotonergic
neurons among rotifers that may prove useful in future
studies of rotifer phylogeny and evolution. Speci?cally,
species of Flosculariacea as demonstrated herein, as well
as species of Collothecacea, occupy phylogenetic posi-
tions outside of the Ploima and are therefore uniquely
suited to help evaluate trends in nervous system
organization within the most heterogeneous clade (see
also S?rensen 2002). While some similarities in seroto-
nergic innervation are present between ploimates and
species of Conochilus, the differences among all species
(even within Ploima) are large enough to require further
examination of increased rotifer biodiversity.
Acknowledgements
I thank Dr. Martin V. S?rensen and two anonymous
reviewers for their constructive criticism of this manu-
script. I am also grateful to the staff at the Smithsonian
Marine Station in Fort Pierce, Florida for the use of
their facilities. This research received ?nancial support
from the University of Massachusetts Lowell and from
the Sumner Gerard Foundation through a postdoctoral
R. Hochberg / Zoologiscfellowship to the Smithsonian Marine Station at Fortcence and confocal microscopy. Tissue Cell 36, 189?195.
Kotikova, E.A., Raikova, O.I., Reuter, M., Gustafsson,
M.K.S., 2005. Rotifer nervous system visualized byPierce, Florida. This is Smithsonian Marine Station at
Fort Pierce contribution # 638.
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