Genome-Wide Analyses Reveal a Role for Peptide
Hormones in Planarian Germline Development
James J. Collins III1,2, Xiaowen Hou3, Elena V. Romanova4, Bramwell G. Lambrus1, Claire M. Miller2, Amir
Saberi1, Jonathan V. Sweedler2,4, Phillip A. Newmark1,2*
1Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of
America, 2Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 3Center for Biophysics and Computational
Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 4Department of Chemistry, University of Illinois at Urbana-Champaign,
Urbana, Illinois, United States of America
Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in
metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral
role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these
molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide
hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on
our observation that defective neuropeptide processing results in defects in reproductive system development, we
employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement,
identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses
of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a
prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of
the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ
cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the
parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and
validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone
complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in
flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for
understanding the reproductive biology of flatworm parasites.
Citation: Collins JJ III, Hou X, Romanova EV, Lambrus BG, Miller CM, et al. (2010) Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian
Germline Development. PLoS Biol 8(10): e1000509. doi:10.1371/journal.pbio.1000509
Academic Editor: Volker Hartenstein, UCLA, United States of America
Received April 20, 2010; Accepted August 25, 2010; Published October 12, 2010
Copyright: ? 2010 Collins III et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (www.nih.gov) F32 HD062124 and T32 HD007333 (JJC), P30 DA018310 and R01 NS031609
(JVS), R01 HD043403, and the National Science Foundation (www.nsf.gov) IOS-0744689 (PAN). PAN is an Investigator of the Howard Hughes Medical Institute
(www.hhmi.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: CNS, central nervous system; FISH, fluorescence in situ hybridization; gH4, germinal histone H4; GSCs, germline stem cells; LC, liquid
chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; NPY, neuropeptide Y; OVP, ovary maturing parsin;
PTMs, Post-Translational Modifications; RNAi, RNA interference; Smed-pc2, Smed-prohormone convertase 2; sNPF, short Neuropeptide F; VNC, ventral nerve cord.
* E-mail: firstname.lastname@example.org
Platyhelminthes (flatworms) inhabit a variety of aquatic and
terrestrial environments and members of the phylum are thought
to parasitize most vertebrate species . The remarkable ability of
flatworms to maintain plasticity in their reproductive cycles is a
key to their success. As an example, free-living planarian flatworms
are capable of reproducing sexually as cross-fertilizing hermaph-
rodites or asexually by transverse fission . Some planarian
species even maintain the ability to switch between modes of
sexual and asexual reproduction, resorbing and regenerating their
reproductive organs, depending on the environmental context .
This dynamic regulation of reproductive development is not
limited to free-living platyhelminths; parasitic flatworms can also
undergo dramatic changes in their reproductive development in
response to external stimuli. In dioecious parasites of the genus
Schistosoma, female reproductive development requires pairing with
a male worm [4–8]. Thus, female schistosomes derived from
single-sex infections have underdeveloped ovaries and accessory
reproductive organs when compared to females from mixed sex
infections. Interestingly, the reproductive organs of mature females
deprived of their male counterpart regress and are capable of
regrowing once male-female pairing is reestablished . Because
flatworms, including schistosomes, are responsible for causing
important neglected tropical diseases, understanding the mecha-
nisms that coordinate the reproduction of both free-living and
parasitic members of the phylum is of fundamental importance.
Peptide hormones (i.e. neuropeptides) are among the most
structurally and functionally diverse class of metazoan signaling
molecules . In vertebrates, a neuroendocrine axis involv-
ing peptide hormone signaling between the brain and the
gonads controls the maturation and long-term maintenance of
PLoS Biology | www.plosbiology.org1October 2010 | Volume 8 | Issue 10 | e1000509
reproductive development and function [10–13]. A similar role for
neuroendocrine signals in controlling flatworm reproduction is
suggested by studies exploiting the well-known regeneration
abilities of planarians. Head amputation (i.e. removal of the
brain/cephalic ganglia) of sexually reproducing planarians results
in regression of the testes [14,15] to their germ cell primordia ,
which re-grow only when cephalic ganglia regeneration is
complete. These observations suggest that neural signals control
the dynamics of planarian reproduction. Thus, flatworms may
employ peptide-based mechanisms, similar to vertebrates, to
synchronize their reproductive development.
To date only limited data exist to support a ‘‘vertebrate-like’’
role for peptide hormones in invertebrate reproductive matura-
tion. Insulin-like peptides influence germline stem cell proliferation
in Drosophila [17,18] and C. elegans  and promote oocyte
maturation in the starfish Asterina pectinifera  and the mosquito
Aedes aegypti . In locusts, treatment with the peptide hormones
ovary maturing parsin (OVP)  or short Neuropeptide F (sNPF)
[23,24] can stimulate ovarian development and vitellogenesis.
Because of this paucity of data linking neuroendocrine function to
invertebrate reproductive development, additional studies are
required to determine how invertebrates modulate their repro-
ductive output in response to external and metabolic cues.
Peptide hormones are processed proteolytically from longer
secretory prohormone precursors and often require covalent
modifications before becoming biologically active [10,25]. As a
result of this extensive processing, and because the biologically
relevant signaling units are encoded by short stretches of amino
acid sequence (usually 3–40 amino acids), predicting genes
encoding these molecules represents a major challenge for
bioinformatics-driven genome annotation. The recent application
of bioinformatic approaches coupled to mass spectrometry-based
peptide characterization techniques (an approach called peptido-
mics [26–28]) has revolutionized discovery efforts, uncovering
hundreds of new genes encoding metazoan bioactive peptides.
Among invertebrates, however, much of this recent progress has
been focused on genome-wide studies of nematodes [29–31],
arthropods [32–36], and mollusks [37,38]. Thus, little is known of
the peptide hormones present in phyla such as Platyhelminthes.
Despite recent bioinformatic efforts to characterize flatworm
peptide-encoding genes [39,40], only three distinct peptides have
been characterized extensively at the biochemical level in all
Owing to a wealth of functional genomic tools  and a
sequenced genome , the planarian S. mediterranea represents an
ideal model to characterize flatworm neuropeptides. Furthermore,
this species exists as two distinct strains: an asexual strain that lacks
reproductive organs and propagates exclusively by fission and a
sexual strain that reproduces as cross-fertilizing hermaphrodites
. This dichotomy presents a unique opportunity to explore the
extent to which peptide hormones are associated with distinct
reproductive states. To address the possibility that peptide signals
influence planarian reproductive development, we began by
disrupting a gene encoding a prohormone processing enzyme,
Smed-prohormone convertase 2 (Smed-pc2, GB: BK007043), in sexual
planarians. Consistent with a role for peptide hormones in
controlling planarian reproduction, knockdown of Smed-pc2 led
to a depletion of differentiated germ cells in the planarian testes.
To identify potential peptide mediators of this effect, we used
peptidomic approaches to characterize the peptide hormone
complement of S. mediterranea. This analysis identified 51 genes
predicted to encode more than 200 peptides, 142 of which we
characterized biochemically by mass spectrometry. Global analysis
of the expression of these genes by whole mount in situ
hybridization revealed a distinct distribution of some peptide
prohormones between sexual and asexual strains of S. mediterranea.
We find one prohormone gene, npy-8, to be enriched in the
nervous system of sexual planarians and show that this gene is
required for the proper development and maintenance of
reproductive tissues. These results demonstrate the utility of S.
mediterranea as a model to characterize metazoan peptides and
suggest that flatworm reproductive development is controlled by
A Peptide Hormone-Processing Enzyme Is Required for
the Maintenance of Differentiated Germ Cells
To explore potential roles for peptide signaling in regulating
planarian reproductive physiology, we characterized Smed-pc2
(Figure S1), whose orthologues are required in both vertebrate and
invertebrate models for the proteolytic processing of prohormones
to mature neuropeptides (in the interest of brevity, we will drop the
prefix ‘‘Smed’’ from the remainder of the genes described below)
[30,45,46]. A large-scale RNA interference (RNAi) screen
determined that this gene was essential for coordinated movement
and normal regeneration in asexual planarians . Whole-mount
in situ hybridization in sexual planarians revealed expression of pc2
in the central nervous system , the pharynx, sub-muscular
cells, the photoreceptors, the copulatory apparatus, and the testes
To determine if peptide signals are likely to play a functional
role in coordinating reproductive development, we monitored the
effects of pc2 RNAi on the dynamics of germ cells within the
planarian testes. Individual testis lobes consist of an outer
spermatogonial layer in which cells divide to form cysts of eight
spermatocytes that, after meiosis, give rise to spermatids and,
ultimately, sperm [44,49]. After 17 d of RNAi treatment,
pc2(RNAi) animals displayed a decrease in both testis size
(Figure 1E) and the number of animals producing mature sperm
(28/29 for controls versus 2/36 for pc2 RNAi; p,0.0001, Student’s
t test). To establish which cell types are affected by pc2 RNAi, we
performed fluorescence in situ hybridization (FISH) to detect
Flatworms cause diseases affecting hundreds of millions of
people, so understanding what influences their reproduc-
tive activity is of fundamental importance. Neurally derived
signals have been suggested to coordinate sexual
reproduction in free-living flatworms, yet the neuroendo-
crine signaling repertoire has not been characterized
comprehensively for any flatworm. Neuropeptides are a
large diverse group of cell-cell signaling molecules and
play many roles in vertebrate reproductive development;
however, little is known about their function in reproduc-
tive development among invertebrates. Here we use
biochemical and bioinformatic techniques to identify
bioactive peptides in the genome of the planarian
flatworm Schmidtea mediterranea and identify 51 genes
encoding .200 peptides. Analysis of these genes in both
sexual and asexual strains of S. mediterranea identified a
neuropeptide Y superfamily member as important for the
normal development and maintenance of the planarian
reproductive system. We suggest that understanding
peptide hormone function in planarian reproduction could
have practical implications in the treatment of parasitic
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org2 October 2010 | Volume 8 | Issue 10 | e1000509
germinal histone H4 (gH4) (GB: DN306099) and nanos (GB:
EF035555) mRNAs, which are expressed in spermatogonia and
germline stem cells (GSCs), respectively [16,50,51]. In developed
testes of control animals, relatively few cells within the outer
spermatogonial layer are identifiable as nanos-positive GSCs
(Figure 1F). However in pc2(RNAi) animals, regressed testes
clustersalmost always co-expressed
(Figure 1G) (n=16/17 animals). These results suggest that pc2 is
Figure 1. pc2 is essential for the maintenance of the planarian testes. (A–C) Whole-mount in situ hybridization to detect pc2 mRNA in sexual
animals. (A) Ventral view, expression in CNS, pharynx, and copulatory apparatus. (B and C) Dorsal view, expression in testes. (D, E) DAPI staining
showing the distribution of testes in (D) control and (E) pc2(RNAi) animals fixed 17 d after the initiation of RNAi treatment. (F and G) Single confocal
sections showing expression of nanos (magenta) and gH4 (green) in testes of (F) control and (G) pc2(RNAi) animals. DAPI staining is shown in grey.
Orange and yellow arrows indicate spermatids and mature sperm, respectively. White arrows indicate germ line stem cells expressing both gH4 and
nanos. Scale bars: (A–C) 300 mm; (D and E) 500 mm; (F and G) 50 mm. Abbreviations: CG, Cephalic Ganglia; VNC, ventral nerve cord; PH, pharynx; CA,
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org3 October 2010 | Volume 8 | Issue 10 | e1000509
required for proper germ cell differentiation and/or for the
maintenance of differentiated germ cells in the testes.
Genomic Identification of Peptide Hormones and Their
Since our analysis of pc2 implicated peptide signaling in
regulating planarian reproductive development, we characterized
the peptide hormone complement of S. mediterranea. We employed
bioinformatic and mass spectrometry (MS)-based methodologies to
identify peptide prohormone genes from the S. mediterranea genome
 and predict their processing into bioactive peptides (Figure 2A)
. With these approaches, we identified 51 prohormone genes
in S. mediterranea, with peptides from 40 prohormones detected by
MS (Tables S1–S5, gene names and abbreviations are shown in
Table 1). In most cases, MS confirmed multiple distinct peptides
from a single prohormone, and in five prohormones we detected
every predicted peptide by MS (Figure 2B). In total, we
characterized 142 peptides biochemically, corresponding to
,45% of the distinct peptides predicted from our collection of
51 prohormone genes (Table S5). This analysis identified genes
encoding relatives of all previously characterized flatworm
neuropeptides (YIRFamide , spp-11; FRFamide , npp-4;
and neuropeptide Y-like , npy-1 to npy-11) and provided
biochemical validation for 10 prohormones previously predicted
from the S. mediterranea genome .
The neuropeptide Y (NPY)-superfamily represents a large
family of neuropeptides that influence diverse processes in both
vertebrate and invertebrate taxa [10,41,56]. This family is
considered to consist of two types of peptides: the NPY-like
peptides that possess a C-terminal amidated tyrosine (Y) residue
and the NPF peptides that possess a C-terminal amidated
phenylalanine (F) residue . Vertebrate genomes typically
encode NPY-like peptides , whereas invertebrate genomes
encode NPF peptides [55,58,59]. Our studies found that the
planarian genome possesses an expanded family of npy genes
predicted to encode both NPY-like and NPF-like peptides
(Figure 2C). Prohormones NPY-5, -7, -9, and -10 possess a C-
terminal tyrosine residue, similar to vertebrate NPY peptides, and
prohormones SMED-NPY-1, -2, -3, -4, -6, and -8 contain a C-
terminal phenylalanine residue, similar to invertebrate NPF
peptides. Three of these planarian npy genes (npy-1, -4, and -9)
have been described previously [39,60]. Additionally, our studies,
and those of others [39,61], find evidence of conservation in the
genomic organization of flatworm NPY genes. NPY genes from
vertebrates possess an intron that separates the exon encoding the
RXR motif from the penultimate amidated amino acid residue
(Figure 2D) . We found an identical architecture for S.
mediterranea genes npy-1, -2, -3, -4, -5, -6, -8, -9, -10, and -11,
indicating a close evolutionary relationship between chordate and
platyhelminth npy genes (Figure 2C,D).
The planarian genome also encodes peptides with sequence
similarities to those from other invertebrate taxa, including
mollusks (ppp-1, GB:BK007041; ppp-2, GB:BK007018; mpl-1,
GB: BK007017; mpl-2, GB: BK007016; and, cpp-1, GB:
BK007012) and arthropods (ppl-1, GB: BK007007). Furthermore,
our analysis found that previously characterized, novel planarian
genes encode peptide prohormones. Homologues of prohormones
eye53-1,2 (GB: BK007033 and GB: BK007024, respectively) and
1020-1,2 (GB: GU295180 and GB:BK007025, respectively) from
the planarian Dugesia japonica are required for proper visual system
function following amputation; knockdown animals show no
morphological defects after injury yet are unable to respond
properly to light . These previous observations, together with
our findings that these genes encode neuropeptides, suggest a role
for peptide signaling in the functional recovery of the planarian
nervous system following injury.
pc2 Is Required for Proper Prohormone Processing
To examine if pc2 is required for prohormone processing in
planarians, we disrupted pc2 expression using RNAi and
performed MS to analyze the peptide complement of pc2(RNAi)
animals. Consistent with pc2 encoding a genuine prohormone
convertase, analysis of peptide profiles in planarian tissue extracts
by MALDI-TOF MS analysis demonstrated that pc2 RNAi
resulted in a significant decrease in the signal intensity of a
specific set of peptides in sexual animals (Figure 2E,F and Table
S6). Interestingly, the levels of some peptides were increased
following pc2(RNAi); whether this alteration reflects a compensa-
tory mechanism for regulating peptide levels or an altered
threshold of detection for certain peptides caused by a global
reduction in neuropeptide levels remains to be determined.
However, these data parallel studies of pc2 knockout mice, in
which the abundance of some peptides was either increased or
decreased . Given that the S. mediterranea genome is predicted
to encode at least three additional proteins with similarity to
prohormone convertases (Figure S2), it is possible that compen-
satory mechanisms are responsible for the observed elevation in
the levels of some peptides. This redundancy among prohormone
convertases is also likely to explain why we only observed changes
in a subset of peptides following pc2 RNAi. These data suggest that
the reproductive defects observed in pc2(RNAi) animals may be
due to altered levels of specific peptides.
In Situ Hybridization Analyses Reveal the Complexity of
the Flatworm Nervous System
To determine the extent to which peptides may regulate
flatworm reproduction, we took advantage of the fact that S.
mediterranea exists as both sexually and asexually reproducing
strains. By comparing prohormone gene expression between these
strains we sought to uncover expression patterns specific to
sexually or asexually reproducing animals. Thus, we began by
performing comprehensive whole-mount in situ hybridization
analyses of prohormone genes in asexual planarians (Figure 3).
Our studies indicate that in asexual planarians ,85% (44/51) of
prohormone genes are expressed in the central nervous system
(CNS) (Table S5), which consists of bi-lobed cephalic ganglia and
two ventral nerve cords (VNCs) that run the length of the body
. Of the prohormones expressed in the CNS, 20% (10/51)
were detected only in the cephalic ganglia. Notably, the expression
of individual prohormones was often enriched in specific cell types
or regions within the CNS. For example, the expression of some
prohormones was enriched in either lateral (e.g. npp-4, GB:
BK007037; npp-8, GB: GU295189; spp-4, GB: GU295179; and
1020HH-2), medial (e.g. spp-2, GB: BK007032; and spp-6, GB:
GU295177) or posterior (e.g. npy-1, GB: GU295175) regions of the
cephalic ganglia (Figure 3). Strikingly, a large fraction of
prohormone mRNAs were detected in restricted cell populations
within the CNS (e.g. npy-1; npy-2, GB: BK007019; cpp-1; spp-6; spp-
9, GB: BK007026; spp-10, GB: BK007028; grh-1, GB: GU295185;
and ilp-1, GB: BK007034) (Figure 3).
Consistent with peptide signaling having a role in processes
outside the CNS, we also detected prohormone expression in: the
pharynx (e.g. npp-1, GB: BK007036; npp-22, GB: BK007038; npy-
11, BK007021; and ppp-1); photoreceptors (e.g. eye53-1,-2; npp-12,
GB: GU295182; and mpl-2); sub-epidermal marginal adhesive
glands (e.g. mpl-2); an anterior domain between the VNCs (e.g.
spp-6; spp-7, GB: GU295178; spp-8, GB: GU295181; spp-9; cpp-1;
and spp-10, GB: BK007028); cells surrounding the ventral midline
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org4October 2010 | Volume 8 | Issue 10 | e1000509
Figure 2. Overview of the peptidomic approach to characterize peptide-encoding genes from S. mediterranea. (A) Schematic
representation of the methodology used for the identification and confirmation of planarian prohormones and their respective peptides. We
performed homology and pattern searches for preliminary annotation of peptide prohormone genes and subsequently verified these predictions
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org5 October 2010 | Volume 8 | Issue 10 | e1000509
(e.g. npp-5, BK007015); the intestine (e.g. npp-8, GB: GU295189;
and npy-10, GB: BK007011); and various sub-epidermal cell types
(e.g. npp-18, GB: BK007027; spp-4; spp-16, GB: BK007042; and
npy-4, BK007039) (Figure 3).
To investigate the extent to which prohormones are expressed
in overlapping or distinct cell types in the CNS, we compared the
expression of prohormone genes using triple FISH. Prohormone
genes spp-1 (GB: GU295176), npp-2 (GB: BK007035), and ppp-1
encode unrelated peptides (Tables S1 and S5) that appear to be
expressed ubiquitously in the CNS (Figure 3). Comparison of the
expression domains of these prohormone genes revealed that spp-
1, npp-2, and ppp-1 are expressed in largely non-overlapping
populations of cells of the cephalic ganglia and VNCs (Figure 4A–
C). We also analyzed the expression of a family of paralogous
prohormone genes (spp-6; spp-7; spp-8; spp-9; and spp-17, GB:
GU295183) that encode similar neuropeptides (Figure S3).
Because this gene family has been expanded in the S. mediterranea
genome, we refer to these prohormones as the Planarins.
Examination of Planarins spp-6, -7, and -9 expression by FISH
demonstrated that these genes are expressed in a common set of
cells distributed between the VNCs and surrounding the pharynx
(Figure 4D). Despite being co-expressed in cells outside the CNS,
spp-6 and spp-9 transcripts were detected in distinct groups of cells
within the cephalic ganglia (Figure 4E,F). These findings, with
earlier observations [48,64], suggest a level of complexity not
previously appreciated for the patterning of the flatworm nervous
system (see Figure S4).
Prohormone Expression Identifies Anterior-Posterior and
Dorsal-Ventral Compartments Within the Planarian
We also examined four prohormone genes (eye53-1,-2; npp-12,
and mpl-2) expressed within the photoreceptors. The planarian
Table 1. Abbreviations of gene names for S. mediterranea neuropeptide prohormones.
Gene NameAbbreviationGene Subfamily
cerebral peptide prohormone-1cpp-1—
gonadotropin releasing hormone like-1 grh-1—
neuropeptide precursor-1-5,8,12,18,22 npp-1-5*,8*, 12*,18*,22*—
neuropeptide y superfamily-1-11npy-1*neuropeptide F
npy-2 neuropeptide F
npy-3 neuropeptide F
npy-5 neuropeptide Y
npy-6 neuropeptide F
npy-8 neuropeptide F
npy-9* neuropeptide Y
npy-11atypical neuropeptide Y
pyrokinin prohormone like-1 ppl-1—
pedal peptide prohormone like-1,2ppp-1,2—
secreted peptide prohormone-1-19spp-1-19—
*Genes previously predicted from the S. mediterranea genome .
with molecular techniques. The post-translational processing of verified prohormones to bioactive peptides was then predicted in silico using
Neuropred  and the sequences of mature peptides were then confirmed in whole animal tissue extracts from sexual and asexual planarians by LC-
MS/MS and/or MALDI-TOF MS. This approach is depicted in blue ovals. To complement the bioinformatics-driven discovery, de novo sequencing of
unassigned MS peaks was used to characterize novel neuropeptides (red ovals). The sequences of such peptides were then mapped to the S.
mediterranea genome and new prohormone genes were annotated. These prohormone genes were then analyzed further, leading to the
characterization of additional peptides. (B) Full sequence coverage of prohormones SPP-1B, SPP-3, SPP-4, NPP-18, and PPP-1 by mass spectrometry.
Underlined sequences indicate peptides identified by MS/MS sequencing and the shaded sequence indicates a peptide detected by MS mass match.
Signal peptides for each prohormone are italicized. (C and D) S. mediterranea possesses an expanded NPY family. (C) ClustalW alignment of two
vertebrate NPY-family peptides, Pancreatic Polypeptide (PP), with a variety of invertebrate NPY family members. Matching residues are shown in
yellow and a conserved a-amidation site is shown in green. C-terminal tyrosine and phenylalanine are highlighted in magenta and blue, respectively.
(D) Gene structure of vertebrate and S. mediterranea npy genes. These prohormone genes have an intron within the arginine codon preceding the
aromatic amino acid residue (blue), the a-amidation site (green), and the dibasic cleavage site (magenta). npy-11 lacks a C-terminal aromatic residue
but also shares this gene organization. (E and F) MALDI-MS analysis of pc2 RNAi in sexual animals. (E) Comparison of peptide profiles for control and
pc2(RNAi)-treated sexual animals 16 d after the initiation of RNAi treatment. MALDI-TOF MS spectra (limited to m/z 1150–1450) comparing control
and pc2(RNAi) groups (n=7 for each group); stars indicate peaks that were significantly different (p,0.05). (F) Characterized peptides and their
respective prohormones that were detected at significantly different levels (p,0.05) following pc2 RNAi. The pc2 RNAi/control column reports the
ratio of peak intensities of pc2 RNAi relative to control.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org6 October 2010 | Volume 8 | Issue 10 | e1000509
photoreceptors are comprised of two distinct cell types: neuronal
photoreceptive cells and pigment cells that envelop the rhabdo-
meric projections of the photoreceptor neurons [2,65,66]. Analysis
of prohormone gene expression within the photoreceptors
revealed that the planarian photoreceptor neurons are patterned
along the anterior-posterior axis. Specifically, prohormone genes
npp-12 and eye53-1 were expressed exclusively in the anterior
photoreceptor neurons, whereas mpl-2 and eye53-2 were expressed
exclusively in posterior neurons (Figure 5A). These findings are
consistent with dye-tracing studies demonstrating that anterior and
posterior photoreceptor neurons project to distinct anatomical
regions . In addition, we detected mpl-2 expression in a ventral
population of cells that was separate from the expression of eye53-2
(Figure 5B); this result suggests that the photoreceptors are also
patterned along the dorsal-ventral axis. Together, these data
indicate that at least three chemically and anatomically distinct
sets of neurons are present in the planarian photoreceptors.
Peptide Hormones Are Expressed Differentially in Sexual
To determine if peptide expression is correlated with reproduc-
tive state, we next analyzed the expression of a subset of
prohormones in the sexual strain of S. mediterranea. The
reproductive system of this animal is comprised of a pair of
ovaries located posterior to the cephalic ganglia, numerous
dorsolateral testes lobes, as well as a variety of accessory
reproductive organs (i.e. oviducts, sperm ducts, copulatory
apparatus, and accessory glands) (Figure 6A). We found several
prohormones expressed in sexual reproductive organs, including
the oviducts (Figure 6B,C), the copulatory apparatus (Figure 6B,C,
and D), gland cells surrounding the copulatory apparatus
(Figure 6E,F), and the testes (Figure 6G,H). These expression
patterns implicate peptide signaling in reproductive processes such
as copulation, fertilization, egg-laying, and gonadal function.
Our expression analyses also found evidence of differential
prohormone expression within the nervous system of sexual S.
mediterranea. ppl-1 encodes peptides related to the pyrokinin
peptides originally isolated from arthropods [68,69]. In contrast
to asexual planarians in which ppl-1 expression was detected
almost exclusively in the cephalic ganglia and the distal region of
the pharynx (Figure 3), ppl-1 was expressed widely in the VNCs
and surrounding the copulatory apparatus of mature sexual
animals (Figure 7A). To explore if ppl-1 expression was linked to
sexual maturation, we determined the distribution of ppl-1 in
immature sexual animals. In sexual animals analyzed within one
week of hatching from the egg capsule, ppl-1 was expressed in a
pattern similar to that of asexual animals (Figure 7A); thus, ppl-1
expression undergoes a change in spatial distribution during the
process of maturation.
The prohormone gene npy-8 (GB: BK007010) is predicted to
encode a 29 AA NPF-like peptide (NPY-8A) and a novel C-
terminal peptide (NPY-8B) (Figure 8A). By in situ hybridization we
failed to detect npy-8 expression in asexual animals (Figures 3 and
7B). In mature sexual animals, however, npy-8 RNA was detected
in a variety of cells within the central and peripheral nervous
systems including the cephalic ganglia, the VNCs, the sub-
muscular plexus, and the pharyngeal nervous system (Figure 7B).
Additionally, in a majority of animals (13/18) we detected npy-8
RNA in a dorsal population of cells (Figure 6C). Analysis of this
dorsal cell population by FISH localized npy-8 expression to cells
often, but not exclusively, found in association with testes lobes
(Figure 7D). To determine if npy-8 levels changed with sexual
Figure 3. Whole-mount in situ hybridization to detect neuropeptide prohormone gene expression in asexual planarians.
Prohormone genes are displayed alphabetically. Full gene names are provided in Table 1. No expression was detected for npy-8 in asexual animals.
Arrow for npy-11 indicates expression at the distal region of the pharynx. Gene names in bold indicate prohormones with at least one peptide
confirmed by MS analysis. Ventral views, anterior towards top. Scale bars (to right of images), 300 mm.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org7October 2010 | Volume 8 | Issue 10 | e1000509
maturation we examined npy-8 expression in sexual hatchlings. In
recently hatched animals npy-8 was detected in tissues similar to
those of mature sexual animals including the cephalic ganglia, the
VNCs, the sub-muscular plexus, and the pharyngeal nervous
system (Figure 7B). Furthermore, we observed dorsal cells
expressing npy-8 in a majority of animals (8/13) (Figure 7C).
The lack of observable expression of npy-8 in asexual animals by
in situ hybridization suggested a relationship between npy-8
expression and the ability to reproduce sexually. Because we
initially cloned the npy-8 gene by 39 RACE with cDNA derived
from asexual animals (Table S5), we wished to confirm our in situ
hybridization results using an alternative approach. Therefore, we
performed northern blot analyses to detect npy-8 transcript in
asexual, recently hatched sexual, juvenile sexual, and mature
sexual animals (Figure 7E). Consistent with our in situ hybridiza-
tion results, we detected high levels of npy-8 in sexual animals of all
developmental stages but not in asexual animals, suggesting that
npy-8 is expressed at negligible levels in asexual planarians.
npy-8 Is Required for the Maintenance of Reproductive
Because npy-8 was expressed at high levels only in sexually
reproducing planarians, we reasoned that peptides encoded from
this gene may be important for reproduction. Therefore, we
determined the knockdown phenotype of npy-8 using RNAi. For
this analysis we employed two distinct RNAi feeding regimens.
First, we measured the effect of npy-8 depletion on the
maintenance of the reproductive system by feeding mature sexual
animals bacterially expressed npy-8 dsRNA and observing the
structure of the reproductive system at 4- and 7-wk time points. As
a complementary approach, we fed juvenile sexual planarians in
vitro synthesized dsRNA and observed the development of the
reproductive system after 1 mo of feeding. Mature sexual animals
fed npy-8 dsRNA over the course of 4–7 wk displayed a range of
phenotypes consistent with loss of sexual maturity (data are
summarized in Table 2). Specifically, in comparison to controls, a
majority of npy-8(RNAi) animals had regressed testes and failed to
produce mature sperm (1/18 for controls versus 14/21 for npy-8
RNAi) (Figure 8B). In addition to testes defects, npy-8(RNAi)
treatment resulted in regression of the copulatory organs (0/18 for
controls versus 13/20 for npy-8 RNAi) (Figure 8B,C) and a
decrease in the size (or complete disappearance) of the gonopore
(unpublished data). Similar to mature sexual animals, juvenile
planarians fed npy-8 dsRNA for 1 mo displayed stunted testes
growth, failed to produce mature sperm (0/8 for controls and 6/8
for npy-8(RNAi)), and had shrunken or absent gonopores (0/20 for
controls and 16/20 for npy-8(RNAi), Figure 8D). Importantly, these
effects on reproductive maturation were not due to an overall
defect in growth since npy-8(RNAi) and control animals grew to
similar sizes over this time period (Figure S5A).
Since npy-8 is a member of an expanded family of NPY-like genes
in S. mediterranea (Figure 2C), we examined both the effectiveness
and the specificity of our npy-8 knockdowns. We fed juvenile
planarians dsRNA specific to npy-8 and monitored the transcript
levels of npy-8 and its closest relative, npy-1, by quantitative RT-
PCR. This analysis found that npy-8 RNAi treatment resulted in a
statistically significant decrease in npy-8 transcript levels while
having no effect on npy-1 mRNA levels (Figure S5B). To further
explore the specificity of the npy-8(RNAi) phenotype, we performed
Figure 4. Prohormone gene expression reveals morphological complexity of the planarian nervous system. (A) Three-color FISH for
ppp-1, npp-2, and spp-1. (B and C) Merged images, colors indicated in panel A. Prohormone genes ppp-1, npp-2, and spp-1 are not predicted to
encode any related peptides and do not appear to have overlapping distributions within the CNS. (D) Three-color FISH for prohormone genes spp-6,
spp-7, and spp-9. (E and F) Merged images, colors indicated in panel D. Prohormones genes spp-6, spp-7, and spp-9 encode related prohormones that
are co-expressed in cells between the VNCs; the expression of these genes is not co-localized in the CNS. Images from (A–F) are confocal projections;
whole animal views in (A) and (D) are derived from tiled stacks. Ventral views, anterior towards left. Scale bars, 100 mm.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org8 October 2010 | Volume 8 | Issue 10 | e1000509
Figure 5. Prohormone gene expression reveals distinct photoreceptor neuron domains. (A) Double FISH showing expression of
prohormone genes in anterior and posterior photoreceptor neurons. Top left, DAPI staining (magenta) and immunofluorescence with VC-1 antibody
that recognizes arrestin (green)  to show the photoreceptor cell bodies (magenta surrounded by green) and their projections (green); image also
indicates orientation for the other images in the panel. Remaining images are a matrix showing FISH for each prohormone gene expressed in the
photoreceptors in comparison to the other three genes. All panels are shown overlaid with differential interference contrast optics. Dorsal view,
anterior towards top. (B) Prohormones mpl-2 and eye53-2 are expressed differentially along the dorsal-ventral (D-V) axis of the photoreceptors. Shown
is a maximum projection of a confocal XZ-series through the photoreceptors. Left, staining with the VC-1 antibody showing the photoreceptor cell
bodies (pseudocolored red) and their rhabdomeric projections (pseudocolored yellow). Lateral (L) and medial (M) domains are indicated. Middle three
panels, FISH with mpl-2 and eye53-2; colors are indicated at bottom. Right, FISH and immunofluorescence with the VC-1 antibody (grey). Posterior
view, dorsal towards top, medial towards right. Scale bars, 25 mm.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org9October 2010 | Volume 8 | Issue 10 | e1000509
a long-term feeding experiment in which we fed juvenile animals
dsRNA specific to npy-8 or either of its two closest relatives, npy-1
or npy-2. In contrast to npy-8 RNAi, neither npy-1 nor npy-2 RNAi
treatments produced observable defects in the maturation of the
planarian reproductive organs (Figure S5C). Collectively, these
studies suggest that the effects of npy-8(RNAi) on reproductive
development are due to specific disruption of npy-8 function and
suggest that off-target effects are unlikely.
To examine the regressed testes of npy-8(RNAi) animals, we
performed FISH to detect nanos and gH4 expression. This analysis
uncovered a range of phenotypes associated with npy-8 RNAi
(Figure 8E). Some npy-8(RNAi) animals had clusters of gH4-positive
cells that were also nanos-positive; these testes clusters are similar to
those observed in pc2(RNAi) animals (Figure 1G). In other animals
we found gH4-positive clusters in which a subset of cells expressed
nanos. We interpret the former to represent a ‘‘severe’’ npy-8
knockdown phenotype, whereas we suggest that the latter
represents an ‘‘intermediate’’ phenotype resulting from incomplete
npy-8 knockdown and/or perdurance of the peptide.
In the most severe cases, the testes regression phenotypes seen in
pc2(RNAi) or npy-8(RNAi) animals were similar. One model to
explain this observation is that PC2 is required for proteolytic
processing of the NPY-8 prohormone, and loss of a mature peptide
(or peptides) encoded by npy-8 results in loss of the ability to
achieve or maintain sexual maturity. Since our MS analysis did
not identify any peptides encoded by npy-8 in extracts from either
asexual or sexual animals (Tables S1–S3), we used FISH to
determine if npy-8 and pc2 transcripts are localized to similar cell
types in the planarian nervous system. We found that npy-8-
expressing cells within the cephalic ganglia, the VNCs, the
pharynx, and the sub-muscular plexus also express high levels of
pc2 (Figure 8F; and unpublished data). This observation is
consistent with PC2 being required for the processing of peptides
encoded by the npy-8 gene.
Comparative Genomics Identifies Novel Peptide
Hormones Encoded in the Genomes of Parasitic
Related flatworms of the genus Schistosoma currently infect over
200 million people worldwide . Because of their complicated
life cycles, schistosomes are not readily amenable to the types of
Figure6.Severalprohormonegenesareexpresseddifferentiallyinsexuallyreproducingplanarians. (A)Diagramdepictingthelocation of
various organs in sexual S. mediterranea. Right, enlarged view of the copulatory apparatus. Abbreviations: SV, Seminal vesicles; CB, copulatory bursa; BC,
bursa canal; PP, penis papilla; GP, gonopore; G, cement glands. (B–H) Genes are listed with their sexual-specific expression pattern; (B–F) expression in
asexual(As)andsexual(S)animalsisshown.(B)cpp-1;oviductsandpenispapilla.(C)npp-22;oviductsandpenispapilla.(D) npp-2; penispapilla.(E) npy-9;
penis papilla and cement glands. (F) npp-18; gland cells surrounding copulatory apparatus. (G) spp-10; testes, (H) ilp-1; testes. Scale bars, 300 mm.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org 10October 2010 | Volume 8 | Issue 10 | e1000509
large-scale biochemical analyses that we have employed to
characterize the planarian peptidome. As an indirect means of
biochemically validating peptide sequences from these animals, we
compared our MS-validated prohormones with predicted proteins
from the genomes of the trematodes Schistosoma mansoni  and
Schistosoma japonicum . With this approach we validated the
sequences of peptides from eight previously characterized
schistosome prohormone genes (Tables 3 and S7) [39,40].
Furthermore, we identified eight additional Schistosoma genes not
previously annotated as peptide prohormones (Tables 3 and S7).
Among these newly annotated prohormones are schistosome genes
that encode the peptide YIRFamide, a well-conserved flatworm
peptide that has potent stimulatory effects on schistosome muscle
fibers  that was not identified in previous bioinformatic efforts
[39,40]. Together, these data provide biochemical validation for
roughly half of the predicted prohormones in Schistosoma and
demonstrate the utility of using planarians to understand flatworm
Traditional studies of neuropeptides have relied on the
biochemical purification of individual peptides possessing interest-
ing biological activities . However, with the application of
genomic and peptidomic technologies, a major bottleneck has
been the characterization of this expanded collection of neuro-
peptide-encoding genes (and their encoded peptides) in vivo. Here
we characterized peptide hormones in S. mediterranea using
genomic, molecular, and biochemical approaches and determined
the tissue-specific expression patterns for the entire collection of
prohormone genes. Comparing the distribution of prohormone
expression between asexual and sexual planarians, we identified a
Figure 7. Some prohormone genes are expressed differentially in the CNS of sexual and asexual planarians. Comparison of the ventral
expression of (A) ppl-1 or (B) npy-8 between asexual, immature sexual hatchlings, and mature sexual animals. (C) Dorsal expression of npy-8 in
immature sexual hatchlings (left) and mature sexual animals (right). (D) Transparency rendering showing expression of npy-8 in a cell in close
proximity to testes lobes. Inset shows higher magnification of npy-8-expressing cell. (E) Northern blot comparing expression of npy-8 in asexual ‘‘As,’’
immature sexual hatchlings ‘‘H,’’ juvenile sexual animals ‘‘J,’’ and mature sexual animals ‘‘M.’’ grb-2 (GB: DN305385) is expressed at similar levels in
asexual and sexual animals (J. Stary and P. Newmark, unpublished observations) and is shown as a loading control. Scale bars: (A–C) 300 mm; (D)
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org11 October 2010 | Volume 8 | Issue 10 | e1000509
Figure 8. npy-8 is required to maintain features of sexually mature planarians. (A) Sequence of the prohormone and predicted peptides
encoded by npy-8. Following removal of the signal peptide (italics) the NPY-8 prohormone is predicted to be processed at two consensus
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org12 October 2010 | Volume 8 | Issue 10 | e1000509
single prohormone gene, npy-8, as important for the maintenance
of reproductive function. While our main focus was to understand
the role of peptide hormones in planarian reproductive develop-
ment, these studies lay the groundwork for using S. mediterranea as
an experimental model for studies aimed at understanding the
diverse functions of metazoan bioactive peptides.
Diverse Prohormone Gene Expression Patterns Reveal
Novel Biological Insights About Planarian Biology
Although previous studies have characterized the expression of
subsets of prohormones or their corresponding peptides [73–76], a
comprehensive accounting of the expression of these genes at the
level of the whole animal has not yet been performed. Here we
describe the distribution of all known neuropeptide-encoding
genes in the planarian S. mediterranea by whole mount in situ
hybridization. One surprising finding from these studies was the
complexity of prohormone expression within the planarian CNS,
which is considered to be among the most primitive centralized
nervous systems in the animal kingdom . We find that
prohormone gene expression is localized to distinct regions of the
cephalic ganglia and that many individual prohormones are
expressed in unique CNS cell types. These results parallel
observations in the planarian D. japonica in which small molecule
neurotransmitters (e.g. serotonin and dopamine) are found in sepa-
rate CNS cell populations . The expression of prohormone
genes in distinct regions/cell-types in the CNS suggests that
processing centers for different neural functions (e.g. sensory,
motor, and neuroendocrine) may be localized to chemically and
spatially distinct domains of the flatworm CNS. In support of this
idea, a ‘‘visual center’’ has been proposed to exist at the medial
regions of the cephalic ganglia to which visual axons send their
projections . Elucidation of the functions of peptides expressed
in these discrete CNS foci may help relate specific anatomical
positions to distinct neural functionalities and allow for the
dissection of planarian neural circuits.
Our analysis of prohormone expression also revealed that many
prohormone genes are expressed in tissues of the reproductive
tract. Expression of peptide prohormones has also been observed
in the somatic reproductive organs of C. elegans . Interestingly,
the expression pattern of some planarian prohormones parallels
the immunohistochemical localization of similar gene products in
other invertebrates. The NPY family member Smed-npy-9 was
expressed in the cement glands (or shell glands) surrounding the
copulatory apparatus that are thought to be involved in egg
capsule synthesis and deposition [2,79]. Studies of S. mansoni
observed NPY-like immunoreactivity in the region of Mehlis’
gland , which is morphologically, and likely functionally ,
similar to the glands labeled by npy-9. cpp-1 encodes VPGWamide
and TPGWamide, peptides that are related to the APGWamide
peptides first described in molluscs . We found cpp-1 to be
prohormone convertase cleavage sites (red). This cleavage would result in two peptides: the C-terminally amidated peptide NPY-8A (potential
amidation site is shown in purple) and the 15–16 AA peptide NPY-8B. (B) DAPI staining showing distribution of testes in control and npy-8(RNAi)
animals at 4 wk after the first RNAi treatment. Arrows show region of the copulatory organs. (C) Penis papilla of control and npy-8(RNAi) animals
visualized by DAPI staining (red) and differential interference contrast microscopy. Anterior towards top. (D) Ventral view of live control and npy-
8(RNAi) animals showing the pharyngeal opening (PH) and the gonopore (GP). Anterior towards left. (E) Single confocal sections showing expression
of nanos (magenta) and gH4 (green) RNAs in testes of control (top) and npy-8(RNAi) animals that display either an intermediate or severe level of
testes regression. DAPI staining is shown in gray. Animals were fixed ,7 wk after the initiation of RNAi treatment. (F) Maximum confocal projection
showing the localization of the npy-8 and pc2 transcripts surrounding the nucleus (gray) of a neuron at the level of the ventral sub-muscular neural
plexus in a mature sexual animal. Similar co-localization was seen in other parts of the central and peripheral nervous systems (unpublished data).
Scale bars: (B) 500 mm; (C–D) 300 mm; (E) 20 mm; (F) 10 mm.
Table 2. Summary of npy-8(RNAi) experiments with mature sexual animals.
Days Since First
Animals With Testes
Animals With a Complete
Set of Copulatory Organsa
Distribution of nanos+ +
and gH4+ +Cellsb
control28 7/7 7/7 Not determined
npy-8(RNAi) 28 4/12 4/11Not determined
control 52 7/77/7 7/7 normal
npy-8(RNAi)52 2/51/5 1/5 normal
control 543/4 4/4 4/4 normal
npy-8(RNAi)541/4 2/4 1/4 normal
Control (Cumulative)— 17/18 18/18 10/11 normal
npy-8(RNAi) (Cumulative)— 7/217/20 2/9 normal
aAnimals were considered to have a full set of copulatory organs if a copulatory bursa, bursa canal, and penis papilla could be detected by DAPI staining.
b‘‘Normal’’ describes animals in which nanos expression was detected in a subset of gH4-expressing cells in the spermatogonial layer of mature testes lobes.
‘‘Intermediate’’ describes animals with regressed testes that label almost exclusively with gH4; a subset of these gH4-positive cells express nanos. ‘‘Severe’’ describes
animals with regressed testes that label almost exclusively with both gH4 and nanos.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org 13October 2010 | Volume 8 | Issue 10 | e1000509
expressed around the penis papillia and the oviducts of sexual
planarians, which mirrors APGWamide localization in the
molluscan oviducts and male copulatory organs [82,83]. While
specific functions for any of these peptides in planarian
reproductive function remain to be elucidated, these results
suggest evolutionarily conserved roles for peptides in several
Two prohormone genes (ppl-1 and npy-8) were expressed
differentially in the nervous systems of mature sexual versus
asexual planarians. The expression of ppl-1 was similar in asexual
and immature sexual animals but underwent a dramatic change in
distribution during sexual maturation. Conversely, npy-8 expres-
sion was detected at similar levels and distribution in sexual
animals yet was not detected in asexual animals. Interestingly, our
biochemical analyses detected a number of peptides uniquely in
either mature sexual or asexual planarians (Tables S1–S3). Taken
together, these results indicate that sexually mature planarians
possess unique signatures in both the composition and spatial
distribution of peptide hormones relative to asexual and immature
Peptide Hormone Signals Promote Planarian Sexual
Maturation and Germ Cell Development
To address the role of peptide signaling in planarian
reproductive physiology we first examined the planarian prohormone
convertase 2 orthologue, pc2. This analysis suggested that prohor-
mone processing is required for regulating the dynamics of germ
cell differentiation. A similar requirement for prohormone
processing in germ cell development has not been described in
other animal models. Loss-of-function mutations in the C. elegans
pc2 orthologue egl-3 result in a range of neuromuscular defects
[84,85], but mutant animals are capable of germ cell development
since they produce viable progeny. The role of the Drosophila pc2
orthologue Amontillado has not been assessed in adult reproductive
development due to a requirement for this gene at multiple points
during embryonic and larval development [86,87]. Despite the
fact that peptide hormones are known to regulate vertebrate germ
cells [11,12], extensive studies of prohormone convertase knockout
mice have also not revealed roles for prohormone processing in
germ cell development . Therefore, it is likely that functional
redundancies exist among the enzymes responsible for processing
hormones involved in vertebrate reproduction. Given this
possibility of genetic redundancy in vertebrates, we suggest
systematic characterization of prohormone processing in other
invertebrate models (e.g. C. elegans and Drosophila) may help address
the extent to which peptide signaling regulates reproductive
development in other animals.
Our studies suggest that NPY-8 may be among the prohor-
mones processed by PC2 that are required for normal sexual
development. At present it is not known which of the two
predicted peptides encoded by NPY-8 influence planarian
reproductive physiology. Prohormones that encode NPY-like
peptides, including NPY-8, often also encode a C-terminal peptide
or CPON (C-flanking peptide of NPY) [39,58,88,89]. Because the
Table 3. Peptides detected in S. mediterranea that are conserved in Schistosoma.
S. mediterranea Gene Predicted Schistosoma Peptidec
MS-Confirmed S. mediterranea Peptidec
Sma-npp-23/Sja-npp-23 spp-11 YIRFGYIRFG
Sma-npp-26 spp-15EHFDPIIY FDPIMFa
Sja-npp-26 spp-15SYFDPIAF FDPIMFa
Sma-npp-28/Sja-npp-28 spp-18, -19
mpl-1, -2 AVRLMRLa AVRLMRLa
aPrefixes Sma and Sja are for genes from S. mansoni and S. japonicum, respectively.
bProhormone genes described previously .
cIdentical residues are shown in bold; similar residues are underlined. Lower case ‘‘a’’ indicates C-terminal amidation. All peptides except YIRFG were confirmed by
tandem MS sequencing.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org14 October 2010 | Volume 8 | Issue 10 | e1000509
functions of both vertebrate and invertebrate CPON peptides
remain elusive, we speculate that the NPY-related peptide NPY-
8A is the functional unit of this prohormone. In vertebrates, NPY
signaling is thought to elicit diverse effects on the neuroendocrine
axis regulating reproduction. Depending on the hormonal milieu,
NPY administration can either promote or inhibit surges of
luteinizing hormone , a gonadotropin that regulates multiple
functions in the male and female reproductive systems [10,11,13].
The hypothalamic gonadotropin-releasing hormone, which pro-
motes luteinizing hormone release from the pituitary, can also be
influenced by NPY [91,92]. Additionally, NPY may influence the
timing of sexual maturation in mammals since it has been
suggested to either induce or inhibit the onset of puberty .
Since NPY is a well-known regulator of energy homeostasis, NPY
has been suggested to coordinate reproductive function with
nutrient status . Studies of Drosophila and Aplysia indicate
similar roles for NPY-like peptides in processes related to nutrient
homeostasis, such as feeding behavior [56,95]. However, func-
tional analyses in vertebrate  and invertebrate models 
have not described obvious reproductive deficits in animals
deficient for NPY-like peptides. Given the fact that S. mediterranea
possesses an expanded collection of NPY-like peptides relative to
other animals, additional work will be required to determine
whether the function of NPY-8 represents an ancestral or derived
function for NPY-like peptides.
Coordinated signaling between the hypothalamus, the pituitary,
and the gonads controls vertebrate reproduction. Although our
initial observation with pc2 RNAi implicated prohormone
processing in planarian germ cell development, the site of action
of this effect was difficult to interpret since pc2 expression was
detected in both the nervous system and the testes. Our studies of
npy-8 have clarified the role of the nervous system in planarian
reproduction. npy-8 is expressed in both the central and peripheral
nervous systems, and its transcripts are not detected in tissues
affected by npy-8 RNAi, such as the testes. Therefore, peptides
from NPY-8 are likely to act in a neuroendocrine fashion to
influence reproductive development. Since amputation studies
suggest that signals from the cephalic ganglia are essential for the
maintenance of mature gonads in planarians [14,15], one possible
source of NPY-8 is from the cephalic ganglia.
The function of pc2 within the testes is presently not known, but
testes are likely to be a site of prohormone processing since we
detect the expression of multiple peptide prohormones (ilp-1 and
spp-10) in this organ. Because peptide hormones can act as
endocrine and paracrine signaling molecules in the vertebrate
testes , it is possible that peptides play similar roles in
planarians. Therefore, we propose that peptides (e.g. NPY-8
peptides) from the nervous system promote events associated with
reproductive maturation (i.e. the production of differentiated germ
cells) and peptides produced in the testes may provide feedback to
the CNS and other organ systems about the physiological state of
the gonads. Additionally, peptides expressed within the testes may
serve as paracrine factors that regulate germ cell maturation. This
possibility of coordinated signaling between CNS and the gonads
may explain why the effects of pc2 RNAi on the reproductive
system are more severe than those of npy-8 RNAi. Due to a lack of
sufficient markers our studies have not examined the effects of
neuropeptide signaling on ovarian development; future efforts will
be directed at examining this question.
Although a chromosomal translocation distinguishes sexual and
asexual S. mediterranea [42,97], the strain-specific differences that
account for their divergent modes of reproduction remain
uncharacterized. With the exception of genes expressed in the
reproductive system , little is known about the transcriptional
differences between these strains. Here we identify npy-8 as
enriched in sexual animals and show an important role for this
gene in sexual development. Interestingly, the regressed testes of
mature sexual animals treated with either pc2 RNAi or npy-8 RNAi
resemble the primordial germ cell clusters of asexual planarians
that also label exclusively with gH4 and nanos . These
observations, together with the loss of somatic reproductive
structures in npy-8(RNAi) animals, suggest that lack of NPY-8
expression in asexual planarians may, in part, account for their
inability to promote germ cell differentiation and initiate sexual
maturation. However, because the phenotypes observed with
pc2(RNAi) were more severe than those observed with npy-8(RNAi),
we anticipate future studies may uncover additional factors that
act in concert with npy-8 to influence planarian reproductive
Studies of Planarians Will Help Us Inform the Biology of
According to one estimate, schistosomiasis (infection by Schisto-
soma) can be directly attributed to as many as 280,000 deaths per
year in sub-Saharan Africa alone . Despite the medical and
economic impact of schistosomiasis, only a single chemotherapeutic
agent (praziquantel) is currently used in treatment of this disease
. Therefore, identifying novel anthelmintic agents is an
important goal of flatworm research. Schistosome eggs can become
lodged in host tissues, such as the liver and bladder, forming
granulomas that are the major cause of the pathology associated
with schistosomiasis . Thus, targeting reproductive function in
adult animals represents a promising means by which to treat and
control schistosome infection. The S. mansoni genome is predicted to
encode two NPY-like prohormone genes: Sm-npp-20a and Sm-npp-
20b [39,101]. Comparison of the predicted peptides from these
prohormones with NPY-like peptides from S. mediterranea found that
the NPY-like peptide encoded from Sm-npp-20a shares its closest
similarity to NPY-8A (,48% identity, ClustalW) (Figure 2C). Given
this observation, and the similarities in the reproductive anatomy
between planarians and trematodes , it is possible that these
animals employ similar mechanisms to control their reproductive
output. Therefore, our results justify efforts aimed at understanding
the role of peptide hormones in flatworm reproductive physiology
and suggest that neuropeptide signaling may represent a viable
target for the treatment and eradication of flatworm parasites.
Materials and Methods
Sexual and asexual S. mediterranea were maintained at 20uC in
0.756 and 1.06 Montjuı ¨c salts, respectively . To minimize
non-specific background from gut contents after feeding, animals
were starved at least 1 wk prior to use. For all experiments with
sexual S. mediterranea, sexually mature animals (,1 cm in length,
unless otherwise specified) with a well-developed gonopore were
used, unless otherwise specified.
All chemicals were obtained from Sigma-Aldrich (St. Louis,
MO) unless otherwise stated. The peptide standards for Matrix-
assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF MS) calibration were purchased from Bruker
Daltonics (Billerica, MA).
Extraction of Peptides
For LC/MS analysis, peptide extracts were prepared from 80–
100 sexual or asexual planarians. Whole animals were mechanically
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org15October 2010 | Volume 8 | Issue 10 | e1000509
homogenized in 8–10 mL of acidified acetone (40:6:1 acetone/
water/HCl) or acidified methanol (90:9:1 methanol/acetic acid/
water). After sonication, vortexing, and centrifugation of the
homogenate, the supernatant was collected and the organic solvent
was removed by evaporation in a SpeedVac concentrator (Thermo
Scientific, San Jose, CA). The supernatant was then filtered through
a Microcon centrifugal filter with a 10 kDa cutoff (Millipore,
Billerica, MA), evaluated for peptide content by MALDI-TOF MS
sampling of 0.5 mL and subjected to sequential separations by
HPLC prior to tandem MS for peptide identification.
Peptide Separation and Measurement
Peptide extracts were fractionated using a microbore HPLC
system Magic 2000 (Michrom BioResources, Inc., Aubum, CA)
with a C18 reverse phase column (Dionex, 1,000 mm i.d., particle
size 3 mm, and pore size 100 A˚) at a 20 mL/min flow rate over a
70 min run. A four-step linear solvent gradient was generated by
mixing mobile phases A (95% water and 5% acetonitrile (ACN),
0.1% formic acid (FA) and 0.01% trifluoroacetic acid (TFA), and B
(95% ACN, 5% water, 0.1% FA, and 0.01% TFA) as follows: 5%–
10%B in20 min, 10%–50%B innext30 min, 50%–80%B innext
10 min, isocratic 80% B for 5 min, 80%–5% B in 4 min. Fractions
were manually collected, evaluated for peptide content by MALDI-
TOF MS, and subjected to 2nd stage separation using a Micromass
HPLC system (Manchester, U.K.) equipped with a C18 reverse
phase column (Dionex, 300 mm i.d., particle size 3 mm, and pore
size100 A˚)andcoupledtoa HCT Ultraion-trapmassspectrometer
via an electrospray ionization source (ESI) (Bruker Daltonics,
Bremen, Germany). Second stage separation parameters were
optimized individually for each fraction using either the same
water/ACN solvent system or water/methanol with 0.1% FA as a
counter-ion. Mass spectrometric detection of eluting peptides was
controlled by the Esquire software (Bruker Daltonics, Bremen,
Germany) in a data-dependent manner. Tandem MS ion precursor
selection was limited to 3 ions per min sorted by signal intensity,
preferred charge state was set to +2, and the active dynamic
exclusion of previously fragmented precursor ions limited to 2
spectra per minute. The scan m/z ranges for MS and MS/MS were
300–1,800 and 50–3,000, respectively.
For peptide identification, tandem mass spectra were converted
to the .mgf file format (Mascot generic file) and processed for
sequencing automatically using the PEAKS Studio 4.5 software
(Bioinformatics Solutions, Inc., Waterloo, CA). PEAKS generated
data were manually inspected and verified. Automatic sequencing
was performed against an in-house planarian prohormone
database using the following search parameters: cleavage sites,
variable Post-Translational Modifications (PTMs) (including N-
terminal pyro-Glu and pyro-Gln, C-terminal amidation, and
disulfide bond; the maximum number of PTMs on a single peptide
was set to four), mass tolerance equal 0.3 Da for the precursor ion,
and 0.5 Da for fragments.
Criteria for peptide assignments and prohormone confirmation
were based on confidence scores generated by PEAKS for each
sequenced peptide and detection mass error. A PEAKS confidence
score is given as a percentage value from 1% to 99% and
represents the statistical likelihood that an amino acid sequence
matches a given MS fragmentation spectrum. The PEAKS
statistical algorithm considers factors such as signal to noise, total
intensity, and spectrum tagging (PEAKS Studio Manual 4.5
PEAKSStudioManual4.5.pdf). Our results are based on the current
database of 51 prohormones. Our criteria for the validation of a
prohormone include the identification of at least one peptide from
the prohormone with a PEAKS score .80% and a mass accuracy
#300 ppm, or with a score of .50% and a mass accuracy within
150 ppm. In addition, we manually verified automatic sequencing
results, examined prohormone cleavage sites, and evaluated the
possible PTMs of the identified peptides. A match of at least three
consecutive fragments in an ion series from manual sequencing to
an automatically generated peptide sequence was considered
sufficient to validate the peptide assignment. As prohormone
identification increases with the number of detected encoded
peptides, we employed high identification criteria for the first
peptide but allowed lower standards for assignment of additional
peptides from the same prohormone (PEAKS score .20%, mass
accuracy #500 ppm) provided the fragmentation spectrum satisfied
In cases in which a prohormone had already been confirmed by
tandem MS, occasionally we assigned peptides by mass match
with MALDI-TOF-MS data. Such assignments were based on a
mass-match within 200 ppm to protonated molecular ions of
peptides predicted by NeuroPred (http://neuroproteomics.scs.
uiuc.edu/neuropred.html) . These assignments are tentative
since they are not accompanied by sequencing data.
Gene Prediction and Annotation
Two distinct bioinformatic approaches were used to identify
prohormone genes in the S. mediterranea genome. First, similarity
searches were performed with collections of peptides or prohor-
mones from invertebrate species such as Drosophila melanogaster,
Aplysia californica, Apis mellifera , Caenorhabditis elegans , and
various Platyhelminthes  with stand-alone BLAST (BLOS-
SUM62 or PAM30 matrices and Expect values $10). Peptides
and prohormones were obtained from genome databases (i.e.
Wormbase, http://www.wormbase.org), from NCBI, or from an
online catalog of bioactive peptides (http://www.peptides.be,
). Additionally, sequence tags generated by de novo MS
sequencing of unassigned peptides were also used as queries for
genomic BLAST searches (BLOSSUM62 or PAM30 matrices and
Expect values $10). As an alternative to similarity searching we
analyzed translated S. mediterranea EST [98,104] and 454 (Roche,
Mannheim, Germany) sequence data (Y. Wang and P.A. New-
mark, unpublished) for sequences that possessed characteristics of
prohormone genes including multiple dibasic cleavage sites and a
signal sequence (www.cbs.dtu.dk/services/SignalP). Translations
of nucleotide sequences were performed with longorf.pl, a script
that translates the longest open reading frame in a nucleotide
sequence (www.bioperl.org/wiki/Bioperl_scripts). Putative pro-
hormone genes identified using these two approaches were used as
queries to search the S. mediterranea genome to determine if
additional related prohormones existed in the genome. The full-
length coding sequences of prohormone genes were predicted
using a variety of gene and splice-site prediction tools, including
NetGene2 (http://www.cbs.dtu.dk/services/NetGene2), FSPLICE
(http://www.softberry.com), GENSCAN (http://genes.mit.edu/
GENESCAN.html), and GeneQuest (v8.0.2, DNASTAR, Madi-
son, WI). Where full-length sequences could not be predicted in
silico, 59 and 39 Rapid Amplification of cDNA Ends (RACE)
(FirstChoice RLM-Race Kit, Ambion, Austin, TX) analyses were
performed following the manufacturer’s protocol. The predictions
of all genes reported here were independently verified by cDNA
analysis (see below). Once verified, genes were considered to be
genuine prohormone genes if they (1) possessed a signal sequence,
(2) possessed basic cleavage sites that flanked predicted or MS-
confirmed peptides, and (3) were less than 200 amino acids in
length. Sequences were excluded if they shared similarity with genes
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org16 October 2010 | Volume 8 | Issue 10 | e1000509
previously annotated to be other than neuropeptide prohormones.
All genes were named according to the S. mediterranea genome
nomenclature guidelines .
Comparison of Prohormones from S. mediterranea and
Translated nucleotide sequences were downloaded either from
the Schistosoma mansoni FTP server (ftp.sanger.ac.ik/pub/patho-
gens/Schistosoma/mansoni) or from the NCBI taxonomy browser
were then compared to the sequences of MS-confirmed S.
mediterranea prohormones using BLASTP. NPY-family members
were not included in this analysis, although three NPY-like
proteins have been previously described in Schistosoma [39,101].
Additionally we analyzed EST sequences in the NCBI database to
identify schistosome prohormone genes. Newly annotated schisto-
some prohormones were analyzed further with SignalP and
Neuropred to predict final gene products. These genes were
named as described previously .
To facilitate efficient analyses of prohormone genes, we
constructed a plasmid vector that permits TA-mediated cloning
of PCR-amplified cDNAs. To generate a suitable vector
backbone, oligonucleotide primers 59-GATCACGCGTCGATT-
TCGGCCTATTGGTTA-39 and 59-GATCACGCGTGCTT-
CCTCGCTCACTGACTC-39 were used to amplify the kanamy-
cin and ampicillin resistance markers and the origin of replication
of plasmid pCRII (Invitrogen, Carlsbad, CA); this PCR product
was digested with MluI and ligated to generate a circular plasmid.
Following circularization, an Eam1105I restriction site was
removed from the b-lactamase gene of this plasmid by introduction
of a silent mutation using site-directed mutagenesis (Quickchange
II, Statagene, La Jolla, CA). For the functional elements of the
vector, two mini genes were synthesized (Integrated DNA
Technologies, Coralville, IA): T7TermSP6 and T7TermT3.
T7TermSP6 included (59 to 39) KpnI, MluI, T7-terminator, AscI,
T7 Promoter, SP6 promoter, GACCTTAGGCT (an Eam1105I
site), and XhoI. T7TermT3 included (59 to 39) SacI, MluI, T7
terminator, T7 promoter, T3 promoter, GACCTTAGGCT (an
Eam1105I site), and NotI. T7TermSP6 and T7TermT3 were
shuttled to pBluescript SK II+ using the KpnI and XhoI sites from
T7TermSP6 or the SacI and NotI sites from T7TermT3. These
plasmids were digested with MluI and EcoRI and ligated with the
MluI site of the vector backbone. A XhoI and NotI-digested PCR
fragment including the ccdB and camR genes from plasmid pPR244
 were inserted to generate the final plasmid-pJC53.2.
Eam1105I (Fermentas, Burlington, Ontario) restriction digest of
this plasmid generates 39 T overhangs that can be ligated to an A-
tailed Taq polymerase-amplified PCR product . The ccdB
gene prevents any undigested plasmid from giving rise to viable
clones . Once cDNAs have been inserted into pJC53.2,
riboprobes for in situ hybridization analysis can be generated by in
vitro transcription with SP6 or T3 RNA polymerases and dsRNA
for RNAi knockdowns can be generated by in vitro transcription
with T7 RNA polymerase, or by transformation of E. coli
To generate riboprobes for in situ hybridization, prohormone
genes not represented by EST clones  were PCR amplified
(Platinum Taq, Invitrogen, Carlsbad, CA) from cDNA generated
from total RNA (iScript cDNA Synthesis Kit, Bio-Rad, Hercules,
CA) or 39 RACE cDNA (RLM-RACE Kit, Ambion, Austin, TX)
generated from either total or poly-(A)+RNA (Poly-A Purist,
Ambion, Austin, TX). For cDNA preparations, RNA was
extracted using Trizol Reagent (Invitrogen, Carlsbad, CA). For
cloning, 2–3 mL of PCR product was ligated with 70 ng of
Eam1105I-digested pJC53.2 (Rapid DNA Ligation Kit, Roche,
Mannheim, Germany) and used to transform DH5a. In vitro
transcriptions with the appropriate RNA polymerase were
performed using standard approaches with the addition of
Digoxigenin-12-UTP (Roche, Mannheim, Germany), Fluoresce-
in-12-UTP (Roche, Mannheim, Germany), or Dinitrophenol-11-
UTP (Perkin Elmer, Waltham, MA).
In situ hybridizations were performed using the formaldehyde-
based fixation procedure essentially as described previously .
However, due to their large size, sexual animals were killed in 10%
N-Acetyl Cysteine, fixed for 20–30 min in 4% Formaldehyde in
PBSTx (PBS+0.3% Triton X-100), permeabilized with 1% SDS
(10 min at RT) prior to reduction (10 min at RT), and treated with
10 mg/mL Proteinase K (10–20 min at RT) after bleaching. Some
samples were processed in either a BioLane HTI (Ho ¨lle & Hu ¨ttner,
Tu ¨bingen, Germany)  or an Insitu Pro (Intavis, Koeln,
Germany) hybridization robot . Sexual animals were imaged
with either a Microfire digital camera (Optronics, Goleta, CA)
mounted on a Leica MZ12.5 stereomicroscope or a Leica DFC420
camera mounted on a Leica M205A stereomicroscope (Leica,
Wetzlar, Germany). Both microscopes were equipped with a Leica
TL RC base. Asexual animals were imaged over a piece of white
filter paper and illuminated from above with an LED light source.
For FISH, following post-hybridization washes and blocking,
animals were incubated in a-Digoxigenin-POD (1:1000, Roche,
Mannheim, Germany), or a-Dinitrophenol-HRP (1:100, Perkin
Elmer, Waltham, MA) overnight at 4uC, washed in MABT,
equilibrated in TNT (100 mM Tris pH 7.5, 150 mM NaCl, and
0.05% Tween-20), and developed in Amplification Diluent
containing a fluorescent-tyramide conjugate (Cy3-tyramide, Cy5-
tyramide, or Fluorescein-tyramide; TSA-Plus, Perkin Elmer,
Waltham, MA). Following development, animals were washed in
TNT and HRP activity was quenched by a 1 h incubation in
1.5%–2.0% H2O2dissolved in TNT. Following HRP inactivation,
animals were washed in MABT, incubated in a different a-hapten-
HRP antibody, and the process was repeated with a different
fluorescent-tyramide conjugate. Samples were mounted in Vecta-
shield (Vector Laboratories, Burlingame, CA) and imaged on a
Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany) (Plan-
Apochromat 206/0.8, C-Apochromat 406/1.2 W korr UV-VIS-
IR, or Plan-Apochromat 636/1.4 Oil DIC objectives). Fluores-
cein, Cy3, and Cy5 were excited with 488 nm, 561 nm, and
633 nm lasers, respectively. Images were processed using either
Zen 2008 (Carl Zeiss, Germany) or ImageJ .
Northern blot procedures were performed essentially as
previously described  and hybridization signals were detected
using an anti-digoxigenin alkaline phosphatase-conjugated anti-
body and chemiluminescence (CDP-STAR, Roche, Mannheim,
Germany). Chemiluminescent signals were detected using a
FluorChem Q (Alpha Innotech, San Leandro, CA).
Sequences of EST clones corresponding to pc2 [43,98] were
assembled with one another and the S. mediterranea genome
(Sequencher 4.7, Gene Codes, Ann Arbor, MI) to determine the
full-length sequence and genomic structure of the pc2 gene.
For RNAi analysis of pc2, EST clone PL05006A1C09 ,
which corresponds to pc2, was shuttled to plasmid pPR244 using a
Gateway reaction (Invitrogen, Carlsbad, CA) . For npy-8
RNAi, a 39 RACE product specific to npy-8 was cloned in
pJC53.2. RNAi feedings were performed essentially as described
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org17October 2010 | Volume 8 | Issue 10 | e1000509
previously , with some modifications. In pc2 RNAi
experiments, ,6.25 mL of IPTG-induced culture was pelleted,
frozen at 280uC, and resupended in 30 mL of a mixture of
homogenized beef liver and water. ,5 mature sexual animals
(.1 cm in length) received 1–2 feedings over the course of ,48 h.
npy-8 RNAi experiments were performed similarly to pc2 RNAi
except feedings included 50% less bacteria and animals were fed
every 5–7 d over the indicated time course; for some feedings,
bacteria were omitted. On occasion, because of either refusal to
feed or improper nutrition, some animals (both controls and
treatment groups) decreased in size over the long time courses of
the npy-8 RNAi experiments. Therefore, only animals .1 cm in
length at the time of fixation were included in our analyses at time
points greater than 4 wk. For all RNAi experiments with
bacterially expressed dsRNA, control feedings were performed
with bacteria containing empty plasmid pPR242.
For RNAi experiments conducted with juvenile planarians,
dsRNA was generated by in vitro transcription [113,114]. To
generate dsRNA, templates cloned in pJC53.2 were amplified with
a modified T7 oligonucleotide (GGATCCTAATACGACTCAC-
TATAGGG), cleaned up using the DNA Clean & Concentrator kit
(Zymo Research, Orange, CA, D4003), and eluted in 10 mL of
water. 4 mL of each PCR product was used as template for in vitro
transcription in a reaction containing 5.5 mL DEPC-treated water,
5 mL 100 mM mix of rNTPs (Promega, E6000), 2 mL high-yield
transcription buffer (0.4 M Tris pH 8.0, 0.1 M MgCl2, 20 mM
spermidine, 0.1 M DTT), 1 mL thermostable inorganic pyrophos-
phatase (New England Biolabs, Madison, WI, M0296S), 0.5 mL
Optizyme recombinant ribonuclease inhibitor (Fisher Scientific,
Pittsburg, PA, BP3222-5), and 2 mL HIS-Tagged T7 RNA
polymerase . Samples were incubated at 37uC for 4–5 h and
then treated with RNase-free DNase (Fisher Scientific, Pittsburg,
PA, FP2231). Synthesized RNA was then melted by heating at
75uC, 50uC, and 37uC each for 3 min. 2.5–10 mg of each dsRNA
solution was mixed with 45 mL of 3:1 liver to water mix and used to
feed up to 8 worms. For these experiments, animals without visible
gonopores (juveniles) were fed every 4–5 d for the indicated time
period and starved 1 wk before fixation. Unless otherwise specified,
as a negative control, animals were fed dsRNA synthesized from the
ccdB and camR-containing insert of pJC53.2.
To analyze the structure of the testes, animals were killed in 2%
HCl for 3 min, fixed in either Methacarn (6 MeOH:3 Chloro-
form: 1 Glacial Acetic Acid) or 4% formaldehyde for 1–2 h,
dehydrated in MeOH, bleached in 6% H2O2in MeOH, and
stained with 49,6-diamidino-2-phenylindole (DAPI) (Sigma-Al-
drich, St. Louis, MO). Alternatively, samples were processed for in
situ hybridization, as described above. Following staining, animals
were mounted in Vectashield, flattened, and imaged on either a
Zeiss SteREO Lumar (Carl Zeiss, Germany) or a Zeiss LSM 710
confocal microscope (DAPI was excited with a 405 nm laser).
Animal Size Measurements
To examine if npy-8(RNAi) affected overall growth, animals were
immobilized on ice and imaged on a Leica M205A stereomicro-
scope. The area of each animal was determined using ImageJ.
To examine transcript levels in npy-8 knockdowns, juvenile
animals were fed either liver homogenate or 45 mL of liver
homogenate mixed with 2.5 mg of in vitro synthesized npy-8
dsRNA. 7 d later RNA was extracted from individual planarians
using Trizol Reagent (Invitrogen, Carlsbad, CA). Following
DNase treatment (DNA-free RNA Kit, Zymo Research, Orange,
CA), reverse transcription was performed (iScript cDNA Synthesis
Kit, Bio-Rad, Hercules, CA) and quantitative PCR was conducted
using Power SYBR Green PCR Master Mix (Applied Biosystems,
Warrington, UK) and a 7900HT real-time PCR system (Applied
Biosystems). Standard curves were generated from serial dilutions
of either plasmid DNA containing the gene of interest (npy-8 and
npy-1) or from genomic DNA (b-tubulin GB: DN305397). All
samples were measured in triplicate to account for pipetting error.
Absolute quantities of each transcript were determined from the
standard curves and the levels of npy-8 or npy-1 were normalized to
the level of b-tubulin in each sample. The mean value (i.e. npy-8/b-
tubulin or npy-1/b-tubulin) for each treatment (i.e. control or
npy-8(RNAi)) was then compared using a Student’s t test. The
primers used for these studies were npy-8 Forward AATCA-
GAAAGGCATCAG; npy-1 Forward GTCGACCAAGATTCGG-
TAAACG, Reverse CATTCTTTTATGAAAATCCCCTGT; b-tubulin
Analysis of Prohormone Processing Following pc2 RNAi
To investigate the effect of pc2 RNAi on the proteolytic
processing of prohormones, peptide profiles were measured by
MALDI-TOF MS and compared by principal component analysis
followed by a t test in tissue extracts prepared from 7 individual
control and 7 individual RNAi-treated animals. Extracts were
prepared by homogenizing each specimen in 100 mL of acidified
acetone (see above). Following centrifugation at 14,0006 g for
15 min, supernatant was collected, dried in SpeedVac concentra-
tor (Thermo Scientific, San Jose, CA), and reconstituted in 30 mL
of 0.01% TFA. For MALDI-TOF MS analysis, 0.7 mL of each
extract was spotted on a stainless steel sample holder and co-
crystallized with 0.7 mL of freshly prepared concentrated DHB
matrix (DHB: 2,5-dihydroxybenzoic acid, 50 mg/mL 50%
acetone). Three technical replicates were sampled for each
biological sample, 42 spots total. Positive ion mass spectra were
acquired manually in 600–6,000 m/z region using a Bruker
Ultraflex II mass spectrometer in linear mode with external
calibration. For each spot 700 laser shots in 7 acquisitions were
accumulated into a sum spectrum representative of a replicate.
For comparison of peptide profiles in control and pc2(RNAi)
animals, raw MALDI-TOF MS data were loaded into an
evaluation version of ClinProTools software (Bruker Daltonics,
Bremen, Germany) using the following processing parameters:
convex hull baseline subtraction, baseline flatness 0.2, mass range
1,000–6,000 m/z, Savizky-Golay smoothing over 1 m/z width
with 11 cycles, data reduction factor of 10, null spectra exclusion
enabled, recalibration with maximum peak shift of 200 ppm. All
spectra were normalized to the total ion count (TIC) prior to PCA
calculations. Sum spectra from technical replicates were grouped
into a representative sample spectrum in ClinProTools, thus
representing a biological replicate for statistical calculations. From
representative sample spectra a mean spectrum was generated by
ClinProTools to reveal general peptide features for control and
pc2(RNAi) groups. Standard deviation of signal intensities among
biological replicates was derived for each peak in the group profile.
Unlimited peak picking on the base of maximal peak intensity and
minimal signal-to-noise ratio of 6 was done on the mean spectrum
representative of each sample group in order to take advantage of
noise reduction effect due to spectra addition. Peptide profiles of
mean spectra representative of biological replicates were com-
pared by principal component analysis followed by Anderson-
Darling (AD) normality test and paired Student’s t test for peaks
showing normal distribution. Peaks not showing a normal
distribution (pAD#0.05) were evaluated by the Wilcoxon or
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org18October 2010 | Volume 8 | Issue 10 | e1000509
Kruskal-Wallis tests, respectively [116–118]. To decrease the
number of false positives while computing individual peak statistics
on highly complex spectra, the Benjamini-Hochberg procedure
incorporated into ClinProTool was automatically applied for p
value adjustment during analysis .
2435 bp Smed-pc2 transcript: 59 untranslated region (UTR)
(Purple, nucleotides 1–295), Coding region (Red, nucleotides
296–2243), and 39 UTR (Yellow, nucleotides 2244–2435). An
additional putative transcriptional start site was also detected 12
nucleotides upstream of the initiator methionine (unpublished
data). The Smed-pc2 locus occupies ,20 kB on supercontig 98 of
the S. mediterranea genome. (B) The Smed-pc2 gene encodes a
predicted 649 amino acid (AA) protein that shares significant
identity with Proprotein Convertase Substilisin/Kexin Type 2
NP_002585.2), D. melanogaster (60% identities, 72% positives;
NP_477318.1), and S. mansoni (69% identities, 81% positives;
CAY17138.1). SMED-PC2 domains and functional regions are
color coded as follows: secretory signal sequence (Purple; AA 1–
16), autocatalytic cleavage site (Gray; AA 98–101), Peptidase-S8
domain (Pink; AA 158–465; PFAM domain PF00082, E-value
4.26102108), and Proprotein Convertase P-Domain (Yellow; AA
525–613; PFAM domain PF01483, e-value 1.9610231). Asterisks
shown above bolded residues indicate amino acids comprising the
putative catalytic core of SMED-PC2.
Found at: doi:10.1371/journal.pbio.1000509.s001 (0.30 MB TIF)
The Smed-pc2 gene. (A) Predicted structure of the
encode multiple prohormone convertase proteins. Shown
is a ClustalW alignment of a region of the Peptidase-S8 domain
from PC2 with three related proteins predicted from the S.
mediterranea genome . Although these are the only predicted
proteins with similarity to this region of the Peptidase-S8 domain,
additional sequences in the S. mediterranea genome show similarity
to other regions of PC2.
Found at: doi:10.1371/journal.pbio.1000509.s002 (4.34 MB TIF)
The S. mediterranea genome is predicted to
ClustalW alignment of prohormones SMED-SPP-6, -7, -8, -9, and
-17. Matching residues are highlighted in yellow, basic cleavage
sites are highlighted in green, and the signal sequence is
highlighted in magenta. (B) The genomic organization of
prohormone genes Smed-spp-6, 7. These genes are located in close
proximity to one another and are transcribed in opposite
orientations. Given their sequence similarity and genomic
organization, it is likely that the Planarin family of genes was
expanded by a series of recent gene duplication events.
Found at: doi:10.1371/journal.pbio.1000509.s003 (0.20 MB TIF)
The Planarin family of prohormones. (A) A
of prohormone gene expression in the planarian cephal-
ic ganglia. Cartoon depicting the distribution of some prohor-
mone genes expressed in distinct regions of the cephalic ganglia
(gray) and photoreceptors. Although npp-12, eye53-1, mpl-2, and
eye53-2 are all expressed in the cephalic ganglia, their expression is
only depicted in the photoreceptors. Abbreviations: LB, Lateral
branches; PR, photoreceptors; PC, pigment cups; OC, optic
chiasma; and VNC, ventral nerve cords.
Schematic representation of the distribution
Found at: doi:10.1371/journal.pbio.1000509.s004 (1.45 MB TIF)
the activity of other npy genes. (A) Area measurements of
animals after 1 mo of being fed either control or npy-8 dsRNA. p
value from Student’s t test is given above and error bars represent
95% confidence intervals. n=19 for controls and npy-8(RNAi). (B)
Levels of either npy-8 (left) or npy-1 (right) transcripts normalized to
b-tubulin mRNAs. n=3 animals for controls and n=5 animals for
npy-8(RNAi). p value from Student’s t test is given above and error
bars represent 95% confidence intervals. (C) DAPI staining
showing distribution of testes in npy-8(RNAi), npy-1(RNAi), and
npy-2(RNAi) animals ,2 mo after the first RNAi treatment. n=4
animals for each treatment. Scale bars: 1 mm.
Found at: doi:10.1371/journal.pbio.1000509.s005 (9.06 MB TIF)
npy-8(RNAi) does not affect animal growth or
asexual S. mediterranea.
Found at: doi:10.1371/journal.pbio.1000509.s006 (0.08 MB PDF)
Summary of MS analysis from sexual and
Found at: doi:10.1371/journal.pbio.1000509.s007 (0.12 MB PDF)
Peptides characterized by MS from sexual S.
Found at: doi:10.1371/journal.pbio.1000509.s008 (0.13 MB PDF)
Peptides characterized by MS from asexual S.
nea prohormone genes.
Found at: doi:10.1371/journal.pbio.1000509.s009 (0.07 MB PDF)
Peptide families encoded from S. mediterra-
Found at: doi:10.1371/journal.pbio.1000509.s010 (0.25 MB PDF)
Sequence information for S. mediterranea
ized peptides following pc2(RNAi) treatment.
Found at: doi:10.1371/journal.pbio.1000509.s011 (0.09 MB PDF)
Changes in characterized and uncharacter-
encode peptides related to peptides from S. mediterra-
Found at: doi:10.1371/journal.pbio.1000509.s012 (0.10 MB PDF)
Prohormone genes from Schistosoma that
We would like to thank Gene Robinson, Alejandro Sa ´nchez Alvarado,
David Forsthoefel, Ryan King, Labib Rouhana, and James Sikes for
providing valuable feedback on the manuscript; David Forsthoefel for
helpful discussions on vector design; Bret Pearson, George Eisenhoffer,
Kyle Gurley, Jochen Rink, Otto Guedelhoefer, and Alejandro Sa ´nchez
Alvarado for sharing in situ hybridization methods prior to publication;
Kiyokazu Agata for providing the VC-1 antibody; and Edwin Hadley for
assistance with artwork. Planarian genomic sequence data were generated
by the Washington University Genome Sequencing Center in St. Louis.
The sequences reported in this paper have been deposited in GenBank
(accession nos. GU295175-GU295180 and BK007007-BK007043).
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: JJC EVR JVS
PAN. Performed the experiments: JJC XH EVR BGL CMM AS.
Analyzed the data: JJC XH EVR BGL CMM AS JVS PAN. Wrote the
paper: JJC PAN.
1. Littlewood DTJ, Bray RA (2001) Interrelationships of the platyhelminthes
Warren A, ed. London: Taylor & Francis. 356 p.
2. Hyman L (1951) The invertebrates: platyhelminthes and rhynchocoela the
acoelomate bilateria. New York: McGraw-Hill Book Company, Inc. 550 p.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org 19October 2010 | Volume 8 | Issue 10 | e1000509
3. Curtis WC (1902) The life history, the normal fission and the reproductive
organs of Planaria maculata. Proceedings of the Boston Society of Natural
History 30: 515–559.
4. Severinghaus AE (1928) Memoirs: sex studies on Schistosoma japonicum.
Quarterly Journal of Microscopical Science 71: 653–702.
5. Erasmus DA (1973) A comparative study of the reproductive system of mature,
immature and ‘‘unisexual’’ female Schistosoma mansoni. Parasitology 67:
6. Popiel I (1986) The reproductive biology of schistosomes. Parasitol Today 2:
7. Loverde PT, Chen L (1991) Schistosome female reproductive development.
Parasitol Today 7: 303–308.
8. LoVerde PT, Andrade LF, Oliveira G (2009) Signal transduction regulates
schistosome reproductive biology. Curr Opin Microbiol 12: 422–428.
9. Clough ER (1981) Morphology and reproductive organs and oogenesis in
bisexual and unisexual transplants of mature Schistosoma mansoni females.
J Parasitol 67: 535–539.
10. Strand F (1999) Neuropeptides: regulators of physiological processes Stevens C,
ed. Cambridge: The MIT Press. 658 p.
11. Steinberger E (1971) Hormonal control of mammalian spermatogenesis.
Physiol Rev 51: 1–22.
12. Gnessi L, Fabbri A, Spera G (1997) Gonadal peptides as mediators of
development and functional control of the testis: an integrated system with
hormones and local environment. Endocr Rev 18: 541–609.
13. Schwartz NB, McCormack CE (1972) Reproduction: gonadal function and its
regulation. Annu Rev Physiol 34: 425–472.
14. Ghirardelli E (1965) Differentiation of the germ cells and generation of the
gonads in planarians. In: Kiortsis V, Trampusch H, eds. Regeneration in
animals and related problems. Amsterdam: North-Holland Publishing
15. Fedecka-Bruner B (1967) Etudes sur la regeneration des organes genitaux chez
la planaire Dugesia lugubris. I. Regeneration des testicules apres destruction. Bull
Biol Fr Belg 101: 255–319.
16. Wang Y, Zayas RM, Guo T, Newmark PA (2007) nanos function is essential for
17. LaFever L, Drummond-Barbosa D (2005) Direct control of germline stem cell
division and cyst growth by neural insulin in Drosophila. Science 309:
18. Ueishi S, Shimizu H, Y HI (2009) Male germline stem cell division and
spermatocyte growth require insulin signaling in Drosophila. Cell Struct Funct
19. Michaelson D, Korta DZ, Capua Y, Hubbard EJ (2010) Insulin signaling
promotes germline proliferation in C. elegans. Development 137: 671–680.
20. Mita M, Yoshikuni M, Ohno K, Shibata Y, Paul-Prasanth B, et al. (2009) A
relaxin-like peptide purified from radial nerves induces oocyte maturation and
ovulation in the starfish, Asterina pectinifera. Proc Natl Acad Sci U S A 106:
21. Brown MR, Clark KD, Gulia M, Zhao Z, Garczynski SF, et al. (2008) An
insulin-like peptide regulates egg maturation and metabolism in the mosquito
Aedes aegypti. Proc Natl Acad Sci U S A 105: 5716–5721.
22. Girardie J, Girardie A (1996) Lom OMP, a putative ecdysiotropic factor for the
ovary in Locusta migratoria. Journal of Insect Physiology 42: 215–221.
23. Cerstiaens A, Benfekih L, Zouiten H, Verhaert P, De Loof A, et al. (1999) Led-
NPF-1 stimulates ovarian development in locusts. Peptides 20: 39–44.
24. Schoofs L, Clynen E, Cerstiaens A, Baggerman G, Wei Z, et al. (2001) Newly
discovered functions for some myotropic neuropeptides in locusts. Peptides 22:
25. Hook V, Funkelstein L, Lu D, Bark S, Wegrzyn J, et al. (2008) Proteases for
processing proneuropeptides into peptide neurotransmitters and hormones.
Annu Rev Pharmacol Toxicol 48: 393–423.
26. Fricker LD, Lim J, Pan H, Che FY (2006) Peptidomics: identification and
quantification of endogenous peptides in neuroendocrine tissues. Mass
Spectrom Rev 25: 327–344.
27. Hummon AB, Amare A, Sweedler JV (2006) Discovering new invertebrate
neuropeptides using mass spectrometry. Mass Spectrom Rev 25: 77–98.
28. Boonen K, Landuyt B, Baggerman G, Husson SJ, Huybrechts J, et al. (2008)
Peptidomics: the integrated approach of MS, hyphenated techniques and
bioinformatics for neuropeptide analysis. J Sep Sci 31: 427–445.
29. Husson SJ, Landuyt B, Nys T, Baggerman G, Boonen K, et al. (2009)
Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-
MALDI-TOF MS. Peptides 30: 449–457.
30. Husson SJ, Mertens I, Janssen T, Lindemans M, Schoofs L (2007)
Neuropeptidergic signaling in the nematode Caenorhabditis elegans. Prog
Neurobiol 82: 33–55.
31. Husson SJ, Clynen E, Baggerman G, De Loof A, Schoofs L (2005) Discovering
neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatogra-
phy and mass spectrometry. Biochem Biophys Res Commun 335: 76–86.
32. Hummon AB, Richmond TA, Verleyen P, Baggerman G, Huybrechts J, et al.
(2006) From the genome to the proteome: uncovering peptides in the Apis
brain. Science 314: 647–649.
33. Baggerman G, Boonen K, Verleyen P, De Loof A, Schoofs L (2005)
Peptidomic analysis of the larval Drosophila melanogaster central nervous system
by two-dimensional capillary liquid chromatography quadrupole time-of-flight
mass spectrometry. J Mass Spectrom 40: 250–260.
34. Yew JY, Wang Y, Barteneva N, Dikler S, Kutz-Naber KK, et al. (2009)
Analysis of neuropeptide expression and localization in adult Drosophila
melanogaster central nervous system by affinity cell-capture mass spectrometry.
J Proteome Res 8: 1271–1284.
35. Wegener C, Reinl T, Jansch L, Predel R (2006) Direct mass spectrometric
peptide profiling and fragmentation of larval peptide hormone release sites in
Drosophila melanogaster reveals tagma-specific peptide expression and differential
processing. J Neurochem 96: 1362–1374.
36. Li B, Predel R, Neupert S, Hauser F, Tanaka Y, et al. (2008) Genomics,
transcriptomics, and peptidomics of neuropeptides and protein hormones in
the red flour beetle Tribolium castaneum. Genome Res 18: 113–122.
37. Li L, Sweedler JV (2008) Peptides in the brain: mass spectrometry–based
measurement approaches and challenges. Annual Review of Analytical
Chemistry 1: 451–483.
38. Moroz LL, Edwards JR, Puthanveettil SV, Kohn AB, Ha T, et al. (2006)
Neuronal transcriptome of Aplysia: neuronal compartments and circuitry. Cell
39. McVeigh P, Mair GR, Atkinson L, Ladurner P, Zamanian M, et al. (2009)
Discovery of multiple neuropeptide families in the phylum Platyhelminthes.
Int J Parasitol 39: 1243–1252.
40. Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, et al. (2009) The
genome of the blood fluke Schistosoma mansoni. Nature 460: 352–358.
41. McVeigh P, Kimber MJ, Novozhilova E, Day TA (2005) Neuropeptide
signalling systems in flatworms. Parasitology 131 Suppl: S41–S55.
42. Newmark PA, Sa ´nchez Alvarado A (2002) Not your father’s planarian: a classic
model enters the era of functional genomics. Nat Rev Genet 3: 210–219.
43. Robb SM, Ross E, Sa ´nchez Alvarado A (2008) SmedGD: the Schmidtea
mediterranea genome database. Nucleic Acids Res 36: D599–D606.
44. Newmark PA, Wang Y, Chong T (2008) Germ cell specification and
regeneration in planarians. Cold Spring Harb Symp Quant Biol 73: 573–581.
45. Miller R, Toneff T, Vishnuvardhan D, Beinfeld M, Hook VY (2003) Selective
roles for the PC2 processing enzyme in the regulation of peptide
neurotransmitter levels in brain and peripheral neuroendocrine tissues of
PC2 deficient mice. Neuropeptides 37: 140–148.
46. Scamuffa N, Calvo F, Chretien M, Seidah NG, Khatib AM (2006) Proprotein
convertases: lessons from knockouts. FASEB J 20: 1954–1963.
47. Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sa ´nchez Alvarado A
(2005) Identification of genes needed for regeneration, stem cell function, and
tissue homeostasis by systematic gene perturbation in planaria. Dev Cell 8:
48. Agata K, Soejima Y, Kato K, Kobayashi C, Umesono Y, et al. (1998) Structure
of the planarian central nervous system (CNS) revealed by neuronal cell
markers. Zoolog Sci 15: 433–440.
49. Franquinet R, Lender T (1973) Etude ultrastructurale des tesicules de Polycelis
tenuis et Polycelis nigra (Planaires). Evolution des cellules germinales males avant
la spermiogenese. Z Mikrosk Anat Forsch 87: 4–22.
50. Sato K, Shibata N, Orii H, Amikura R, Sakurai T, et al. (2006) Identification
and origin of the germline stem cells as revealed by the expression of nanos-
related gene in planarians. Dev Growth Differ 48: 615–628.
51. Handberg-Thorsager M, Salo ´ E (2007) The planarian nanos-like gene Smednos is
expressed in germline and eye precursor cells during development and
regeneration. Dev Genes Evol 217: 403–411.
52. Southey BR, Amare A, Zimmerman TA, Rodriguez-Zas SL, Sweedler JV
(2006) NeuroPred: a tool to predict cleavage sites in neuropeptide precursors
and provide the masses of the resulting peptides. Nucleic Acids Res 34:
53. Maule AG, Shaw C, Halton DW, Curry WJ, Thim L (1994) RYIRFamide: a
turbellarian FMRFamide-related peptide (FaRP). Regul Pept 50: 37–43.
54. Maule A, Shaw C, Halton D, Thim L (1993) GNFFRFamide: a novel
FMRFamide-immunoreactive peptide isolated from the sheep tapeworm,
Moniezia expansa. Biochem Biophys Res Commun 193: 1054–1060.
55. Maule AG, Shaw C, Halton DW, Thim CF, Johnson CF, et al. (1991)
Neuropeptide F: a novel parasitic flatworm regulatory peptide from Moniezia
expansa (Cestoda: Cyclophyllidea). Parasitology 102: 309–316.
56. Wu Q, Wen T, Lee G, Park JH, Cai HN, et al. (2003) Developmental control
of foraging and social behavior by the Drosophila neuropeptide Y-like system.
Neuron 39: 147–161.
57. Cerda-Reverter JM, Larhammar D (2000) Neuropeptide Y family of peptides:
structure, anatomical expression, function, and molecular evolution. Biochem
Cell Biol 78: 371–392.
58. Brown MR, Crim JW, Arata RC, Cai HN, Chun C, et al. (1999) Identification
of a Drosophila brain-gut peptide related to the neuropeptide Y family. Peptides
59. Leung PS, Shaw C, Maule AG, Thim L, Johnston CF, et al. (1992) The
primary structure of neuropeptide F (NPF) from the garden snail, Helix aspersa.
Regul Pept 41: 71–81.
60. Cebria ` F (2008) Organization of the nervous system in the model planarian
Schmidtea mediterranea: an immunocytochemical study. Neurosci Res 61:
61. Mair GR, Halton DW, Shaw C, Maule AG (2000) The neuropeptide F (NPF)
encoding gene from the cestode, Moniezia expansa. Parasitology 120 (Pt 1):
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org20 October 2010 | Volume 8 | Issue 10 | e1000509
62. Larhammar D, Ericsson A, Persson H (1987) Structure and expression of the Download full-text
rat neuropeptide Y gene. Proc Natl Acad Sci U S A 84: 2068–2072.
63. Inoue T, Kumamoto H, Okamoto K, Umesono Y, Sakai M, et al. (2004)
Morphological and functional recovery of the planarian photosensing system
during head regeneration. Zoolog Sci 21: 275–283.
64. Umesono Y, Agata K (2009) Evolution and regeneration of the planarian
central nervous system. Dev Growth Differ 51: 185–195.
65. Carpenter KS, Morita M, Best JB (1974) Ultrastructure of the photoreceptor of
the planarian Dugesia dorotocephala. I. Normal eye. Cell Tissue Res 148: 143–158.
66. Sakai F, Agata K, Orii H, Watanabe K (2000) Organization and regeneration
ability of spontaneous supernumerary eyes in planarians- eye regeneration field
and pathway selection by optic nerves. Zoolog Sci 17: 375–381.
67. Okamoto K, Takeuchi K, Agata K (2005) Neural projections in planarian
brain revealed by fluorescent dye tracing. Zoolog Sci 22: 535–546.
68. Schoofs L, Holman GM, Hayes TK, Nachman RJ, De Loof A (1991) Isolation,
primary structure, and synthesis of locustapyrokinin: a myotropic peptide of
Locusta migratoria. Gen Comp Endocrinol 81: 97–104.
69. Holman GM, Cook BJ, Nachman RJ (1986) Primary structure and synthesis of
a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea
maderae. Comp Biochem Physiol C 85: 219–224.
70. Chitsulo L, Engels D, Montresor A, Savioli L (2000) The global status of
schistosomiasis and its control. Acta Trop 77: 41–51.
71. Liu F, Zhou Y, Wang ZQ, Lu G, Zheng H, et al. (2009) The Schistosoma
japonicum genome reveals features of host-parasite interplay. Nature 460:
72. Guillemin R (1978) Peptides in the brain: the new endocrinology of the neuron.
Science 202: 390–402.
73. Nathoo AN, Moeller RA, Westlund BA, Hart AC (2001) Identification of
neuropeptide-like protein gene families in Caenorhabditis elegans and other
species. Proc Natl Acad Sci U S A 98: 14000–14005.
74. Li C, Kim K (2008) Neuropeptides. WormBook. pp 1–36.
75. Park D, Veenstra JA, Park JH, Taghert PH (2008) Mapping peptidergic cells in
Drosophila: where DIMM fits in. PLoS One 3: e1896. doi:10.1371/journal.
76. Santos JG, Vomel M, Struck R, Homberg U, Nassel DR, et al. (2007)
Neuroarchitecture of peptidergic systems in the larval ventral ganglion of
Drosophila melanogaster. PLoS One 2: e695. doi:10.1371/journal.pone.0001848.
77. Reuter M, Halton DW (2001) Comparative neurobiology of Platyhelminthes.
In: Littlewood DT, Bray RA, eds. Interrelationships of the Platyhelminthes.
London: Taylor & Francis. pp 239–249.
78. Umesono Y, Watanabe K, Agata K (1999) Distinct structural domains in the
planarian brain defined by the expression of evolutionarily conserved
homeobox genes. Dev Genes Evol 209: 31–39.
79. Shinn GL (1993) Formation of egg capules by flatworms (phylum platyhel-
minthes). Transactions of the American Microscopical Society 112: 18–34.
80. Skuce PJ, Johnston CF, Fairweather I, Halton DW, Shaw C, et al. (1990)
Immunoreactivity to the pancreatic polypeptide family in the nervous system of
the adult human blood fluke, Schistosoma mansoni. Cell Tissue Res 261: 573–581.
81. Kuroki Y, Kanda T, Kubota I, Fujisawa Y, Ikeda T, et al. (1990) A molluscan
neuropeptide related to the crustacean hormone, RPCH. Biochem Biophys Res
Commun 167: 273–279.
82. de Lange RP, van Minnen J (1998) Localization of the neuropeptide
APGWamide in gastropod molluscs by in situ hybridization and immunocy-
tochemistry. Gen Comp Endocrinol 109: 166–174.
83. Di Cristo C, Van Minnen J, Di Cosmo A (2005) The presence of APGWamide
in Octopus vulgaris: a possible role in the reproductive behavior. Peptides 26:
84. Trent C, Tsuing N, Horvitz HR (1983) Egg-laying defective mutants of the
nematode Caenorhabditis elegans. Genetics 104: 619–647.
85. Kass J, Jacob TC, Kim P, Kaplan JM (2001) The EGL-3 proprotein convertase
regulates mechanosensory responses of Caenorhabditis elegans. J Neurosci 21:
86. Rayburn LY, Gooding HC, Choksi SP, Maloney D, Kidd AR, 3rd, et al. (2003)
amontillado, the Drosophila homolog of the prohormone processing protease PC2,
is required during embryogenesis and early larval development. Genetics 163:
87. Rayburn LY, Rhea J, Jocoy SR, Bender M (2009) The proprotein convertase
amontillado (amon) is required during Drosophila pupal development. Dev Biol 333:
88. Rajpara SM, Garcia PD, Roberts R, Eliassen JC, Owens DF, et al. (1992)
Identification and molecular cloning of a neuropeptide Y homolog that
produces prolonged inhibition in Aplysia neurons. Neuron 9: 505–513.
89. Blomqvist AG, Soderberg C, Lundell I, Milner RJ, Larhammar D (1992)
Strong evolutionary conservation of neuropeptide Y: sequences of chicken,
goldfish, and Torpedo marmorata DNA clones. Proc Natl Acad Sci U S A 89:
90. Kalra SP, Fuentes M, Fournier A, Parker SL, Crowley WR (1992) Involvement
of the Y-1 receptor subtype in the regulation of luteinizing hormone secretion
by neuropeptide Y in rats. Endocrinology 130: 3323–3330.
91. Contijoch AM, Malamed S, McDonald JK, Advis JP (1993) Neuropeptide Y
regulation of LHRH release in the median eminence: immunocytochemical
and physiological evidence in hens. Neuroendocrinology 57: 135–145.
92. Advis JP, Klein J, Kuljis RO, Sarkar DK, McDonald JM, et al. (2003)
Regulation of gonadotropin releasing hormone release by neuropeptide Y at
the median eminence during the preovulatory period in ewes. Neuroendocri-
nology 77: 246–257.
93. Terasawa E, Fernandez DL (2001) Neurobiological mechanisms of the onset of
puberty in primates. Endocr Rev 22: 111–151.
94. Kalra SP, Kalra PS (1996) Nutritional infertility: the role of the interconnected
hypothalamic neuropeptide Y-galanin-opioid network. Front Neuroendocrinol
95. Jing J, Vilim FS, Horn CC, Alexeeva V, Hatcher NG, et al. (2007) From
hunger to satiety: reconfiguration of a feeding network by Aplysia neuropeptide
Y. J Neurosci 27: 3490–3502.
96. Xu M, Hill JW, Levine JE (2000) Attenuation of luteinizing hormone surges in
neuropeptide Y knockout mice. Neuroendocrinology 72: 263–271.
97. Bagun ˜a ` J, Carranza S, Pala M, Ribera C, Giribet G, et al. (1999) From
morphology and karyology to molecules. New methods for taxonomical
identification of asexual populations of freshwater planarians. A tribute to
Professor Mario Benazzi. Italian Journal of Zoology 66: 207–214.
98. Zayas RM, Hernandez A, Habermann B, Wang Y, Stary JM, et al. (2005) The
planarian Schmidtea mediterranea as a model for epigenetic germ cell specification:
analysis of ESTs from the hermaphroditic strain. Proc Natl Acad Sci U S A
99. van der Werf MJ, de Vlas SJ, Brooker S, Looman CW, Nagelkerke NJ, et al.
(2003) Quantification of clinical morbidity associated with schistosome
infection in sub-Saharan Africa. Acta Trop 86: 125–139.
100. Ross AG, Bartley PB, Sleigh AC, Olds GR, Li Y, et al. (2002) Schistosomiasis.
N Engl J Med 346: 1212–1220.
101. Humphries JE, Kimber MJ, Barton YW, Hsu W, Marks NJ, et al. (2004)
Structure and bioactivity of neuropeptide F from the human parasites
Schistosoma mansoni and Schistosoma japonicum. J Biol Chem 279: 39880–39885.
102. Cebria ` F, Newmark PA (2005) Planarian homologs of netrin and netrin receptor are
required for proper regeneration of the central nervous system and the
maintenance of nervous system architecture. Development 132: 3691–3703.
103. Liu F, Baggerman G, Schoofs L, Wets G (2008) The construction of a bioactive
peptide database in Metazoa. J Proteome Res 7: 4119–4131.
104. Sa ´nchez Alvarado A, Newmark PA, Robb SM, Juste R (2002) The Schmidtea
mediterranea database as a molecular resource for studying platyhelminthes, stem
cells and regeneration. Development 129: 5659–5665.
105. Reddien PW, Newmark PA, Sa ´nchez Alvarado A (2008) Gene nomenclature
guidelines for the planarian Schmidtea mediterranea. Dev Dyn 237: 3099–3101.
106. Ichihara Y, Kurosawa Y (1993) Construction of new T vectors for direct
cloning of PCR products. Gene 130: 153–154.
107. Bernard P, Gabant P, Bahassi EM, Couturier M (1994) Positive-selection
vectors using the F plasmid ccdB killer gene. Gene 148: 71–74.
108. Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed
dsRNAs can produce specific and potent genetic interference in Caenorhabditis
elegans. Gene 263: 103–112.
109. Pearson BJ, Eisenhoffer GT, Gurley KA, Rink JC, Miller DE, et al. (2009)
Formaldehyde-based whole-mount in situ hybridization method for planarians.
Dev Dyn 238: 443–450.
110. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image Processing with ImageJ.
Biophotonics International 11: 36–42.
111. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual
Irwin N, Janssen KA, eds. Cold Spring Harbor: Cold Spring Harbor
112. Gurley KA, Rink JC, Sa ´nchez Alvarado A (2008) Beta-catenin defines head
versus tail identity during planarian regeneration and homeostasis. Science 319:
113. Rouhana L, Shibata N, Nishimura O, Agata K (2010) Different requirements
for conserved post-transcriptional regulators in planarian regeneration and
stem cell maintenance. Dev Biol 341: 429–443.
114. Pellettieri J, Fitzgerald P, Watanabe S, Mancuso J, Green DR, et al. (2010) Cell
death and tissue remodeling in planarian regeneration. Dev Biol 338: 76–85.
115. He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, et al. (1997) Rapid
mutagenesis and purification of phage RNA polymerases. Protein Expr Purif 9:
116. Wilcoxon F (1945) Individual comparisons by ranking methods. Biometrics 1:
117. Kruskal W, Wallis W (1952) Use of ranks in one-criterion variance analysis.
Journal of American Statistical Association 47: 588–621.
118. Stephens M (1974) EDF for goodness of fit and some comparisons. Journal of
American Statistical Association 69: 730–737.
119. Dudoit S, Shaffer J (2003) Multiple hypothesis testing in microarray
experiments. Statistical Science 18: 71–103.
Global Analysis of Planarian Neuropeptides
PLoS Biology | www.plosbiology.org21 October 2010 | Volume 8 | Issue 10 | e1000509