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Description and phylogenetic characterization of common hydra from India

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CURRENT SCIENCE, VOL. 101, NO. 6, 25 SEPTEMBER 2011
736
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ACKNOWLEDGEMENTS. We thank the
Director, National Institute of Ocean Tech-
nology (NIOT), Chennai and authorities of the
Ministry of Earth Sciences, Government of
India for providing necessary facility to con-
duct this study. We also thank Dilip Kumar
Jha, NIOT, Port Blair for help in the prepara-
tion of the map of the study site.
Received 21 April 2011; revised accepted 22
August 2011
N. MARIMUTHU1,*
G. DHARANI2
N. V. VINITHKUMAR1
M. VIJAYAKUMARAN2
R. KIRUBAGARAN2
1Andaman and Nicobar Centre for
Ocean Science and Technology,
National Institute of Ocean Technology
(Ministry of Earth Sciences,
Government of India),
Dollygunj,
Port Blair 744 103, India
2Marine Biotechnology,
National Institute of Ocean Technology,
Ministry of Earth Sciences,
Government of India, Pallikaranai,
Chennai 600 100, India
*For correspondence.
e-mail: marinemari@hotmail.com
Description and phylogenetic characterization of common hydra from
India
Hydra, a freshwater polyp belonging to
phylum Cnidaria and class Hydrozoa, is
globally distributed except in the Antarc-
tic region and Oceanic islands1. Although
this organism has been extensively used
as a model system in biology, there has
been considerable uncertainty over its
taxonomy, primarily due to lack of taxo-
nomically distinct features. This created
a doubt whether to put different hydra
species under the genus Hydra, which
was first reported by Carl Linné2, or to
follow Schulze’s three genera classifica-
tion3. Campbell1 has classified the genus
Hydra into four different groups: ‘oligac-
tis group’ (stalked hydra), ‘vulgaris
group’ (common hydra), ‘braueri group’
(gracile hydra) and ‘viridissima group’
(green hydra), based on morphological
differences. A recent study based on mo-
lecular phylogenetic analysis has shown
the reliability of Campbell’s system of
grouping different hydra species4. Hydra
types were first studied in India by An-
nandale5,6. A detailed study based on
morphological and physiological charac-
ters of Indian hydra types was conducted
by Prasad and Mookerjee7. However, they
have refrained from naming any species,
except Chlorohydra (green hydra) col-
lected from Hyderabad7. In India, a local
species referred to as Pelmatohydra oli-
gactis, is being used as a model system
for studying regeneration, pattern forma-
tion and development8–12, but has not
been taxonomically described till date. It
has so far been referred to as P. oligactis
based on a personal communication
between late L. H. Hyman and late Leela
Mulherkar. However, a detailed taxo-
nomic study of Pune hydra ecotype,
especially in view of the prevailing prin-
ciples of hydra taxonomy, has not been
carried out. With increasing use of this
organism as a model system, it is neces-
sary to describe the taxonomic position
and phylogenetic relationship of Indian
hydra with other species of hydra.
Polyps collected from a local pond were
cultured by standard method13. Live
polyps were collected randomly from the
culture and their body length was mea-
sured by placing a graph paper under
the glass beaker containing the animals.
Hydra at various stages of budding were
randomly selected from a mass culture,
relaxed by exposure to 2% urethane for
2 min and fixed in 4% paraformaldehyde
overnight at 4°C (ref. 14). The pattern of
emergence of tentacles was studied with
an Olympus SZX16 stereomicroscope.
Nematocysts were prepared for observa-
tion as described by David15 and photo-
graphed with a Zeiss Axio ImagerZ1.
Total DNA was isolated from 50 polyps
by the phenol/chloroform method16. Prim-
ers reported earlier4 were used to amplify
regions of mitochondrial 16S rRNA
gene. PCR product was sequenced and a
379 bp sequence was submitted to Gen-
Bank (accession no. GU591886). Mito-
chondrial 16S rRNA sequences from
other hydra species reported recently17
were used for comparison with the
sequence from Indian hydra. Sequence
alignments were carried out using
ClustalW18 and cured manually.
Phylogenetic tree was constructed by
neighbour joining (NJ) method based on
p-distance using MEGA 4.0 software19.
A separate analysis by maximum parsi-
mony (MP) and maximum likelihood
(ML) methods was carried out with
PAUP* 4.0b10 (ref. 20). The MP analy-
sis was performed with heuristic searches
of 100 random additions with characters
weighed equally and tree bisection and
reconnection (TBR) branch swapping
algorithm. The ML analysis was per-
formed with general time reversible (GTR)
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CURRENT SCIENCE, VOL. 101, NO. 6, 25 SEPTEMBER 2011 737
Figure 1. ac, Hydra vulgaris Ind-Pune. a, Polyp with bud; b, Polyp with testes () and c, polyp with female gonads (). dg,
Asynchronous emergence of tentacles on bud: d, No tentacles; e, Two tentacle rudiments (); f, Both pairs of tentacle rudiments
() and g, Two big and three small tentacle rudiments (). hk, Nematocysts: h, Stenotele; i, Holotrichous isorhiza; j, Atrichous
isorhiza and k, Desmoneme. Scale bar: ac = 2.5 mm, dg = 200 μm and hk = 5 μm.
model, selected by Akaike information
criterion (AIC) implemented in
DAMBE21. A discrete gamma distribu-
tion with four rate categories across the
site heterogeneity was estimated in PAUP*
4.0b10 and used in the ML method. In
both the methods gaps were treated as
missing, initial tree was obtained by
stepwise addition and TBR algorithm
was used for branch-swapping. L5 strain
of Hydra oligactis was used as outgroup.
In all methods bootstrap test with 1000
replicates was conducted.
Taxonomy
Phylum: Cnidaria
Class: Hydrozoa
Family: Hydridae
Hydra vulgaris Ind-Pune (Figure 1 ac)
Etymology
Ind-Pune: Ind refers to India and Pune is
the type locality of the described strain.
Collection site
Samples were collected from a pond in
University of Pune Campus, Pune,
18°31N lat. and 73°51E long., Mahara-
shtra, India.
Description
General morphology: Polyps are without
a distinct stalk and the number of tenta-
cles per polyp is 5–6 (n = 30, where n is
the sample size); rarely animals with
seven tentacles have been observed. Ten-
tacles are shorter than body column and
stretched tentacles can extend a little
longer than the body column. In all the
48 animals studied, tentacles emerged
asynchronously on buds. In 56% of the
buds, two tentacles emerged first, oppo-
site each other, followed by two more
perpendicular to the first pair, whereas
the fifth one appeared randomly (n = 48;
Figure 1 dg).
Colour: Polyps are light brown in col-
our after feeding and appear pale on
starvation (48 h). On prolonged starva-
tion (> 72 h), they become white.
Measurements: Adult polyps (with
bud) measure about 4–8 mm (n = 25) in
the relaxed state and 0.6–4 mm (n = 27)
when fixed. Polyps without buds can ex-
tend up to 12 mm (n = 102) in length.
Sexuality: Usual mode of reproduction
is asexual (by budding). Sexual repro-
duction is rarely observed and the condi-
tions under which gonads are induced are
not clear. Polyps are dioecious in nature
and many male gonads are found on the
body column starting from just below the
sub-hypostomal region to the budding
zone. The testes are broadly triangular in
shape with a slightly constricted apex,
without a distinct nipple. They are alter-
nately and spirally arranged on the body
axis (Figure 1 b). More than one female
gonad occur along the axis starting from
the sub-hypostomal region to budding
zone (Figure 1 c).
Nematocysts: Stenoteles are pyriform,
9.72 ± 1.37 μm × 7.73 ± 1.13 μm (n = 24;
Figure 1 h), holotrichous isorhizae are
paramecium-like and some cylindrical,
10 ± 0.43 μm × 4.18 ± 0.28 μm (n = 15;
Figure 1 i), atrichous isorhizae are cylin-
drical, 8 ± 0.69 μm × 3.53 ± 0.29 μm (n =
20; Figure 1 j) and desmonemes are
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CURRENT SCIENCE, VOL. 101, NO. 6, 25 SEPTEMBER 2011
738
small, pyriform, 4.28 ± 0.28 μm × 3.16 ±
0.28 μm (n = 12; Figure 1 k). The tubule
is transversely coiled in holotrichous
isorhiza (Figure 1 i).
Phylogenetic affinities: H. vulgaris
Ind-Pune has characteristic morphologi-
cal features of the H. vulgaris species,
such as absence of a distinct stalk, the
length of stretched tentacles equal to or
slightly longer than the body axis and the
transversely coiled pattern of tubule
within the holotrichous isorhiza. The
asynchronous emergence of tentacles on
buds is the only feature it shares with the
H. oligactis (P. oligactis). Molecular
phylogenetic analysis based on mito-
chondrial 16S rRNA clearly shows its
close affinity with members of common
hydra, H. vulgaris (Figure 2). Pairwise
distance (p-distance) of the Indian strain
from Hydra magnipapillata 105 (105
strain of H. vulgaris) is 0.005 and from
other members of Eurasia analysed here
is 0.003.
A recent report based on molecular
phylogeny shows the species relation-
ships within the genus Hydra22 found
across the globe. Another report has
shown further clear clades within the
vulgaris group based on their geographi-
cal distribution (North America, South
America, Oceania, South Africa and
Eurasia)17. The hydra described here
Figure 2. Phylogenetic tree based on 16S
rRNA datasets using neighbour joining (NJ)
method. Bootstrap values of NJ, maximum
parsimony and maximum likelihood analysis
are indicated above/below each node. Branch
lengths are proportional to the scale bars
given in substitutions per site. C5, HUN01C,
B11, C7, J7, 105, ARG37a, ARG38a, 849a
and AEP are strains of H. vulgaris17; L5 is a
strain of H. oligactis17 and Ind-Pune is the
strain used in the present study.
clusters with Eurasian members of vul-
garis species (Figure 2). The genetic
divergence among the Eurasian members
of vulgaris species is low17, but morpho-
logical features like total polyp size,
colour, emergence pattern of tentacles on
buds and consistency in gonad produc-
tion vary. The Indian strain shows very
little genetic divergence from other
members of the clade. Morphological
and cytological evidence also strongly
suggests close affinity of this species
with the vulgaris group and not with
P. oligactis, as erroneously called earlier
in non-taxonomic literature. Though it
has peculiar characters like alternatively
arranged male gonads and asynchronous
emergence of tentacles, we cannot attrib-
ute the novel species status as these
characters are variable within the
vulgaris group1. The ecotype from Pune
previously studied7 has similar morpho-
logical features as H. vulgaris Ind-Pune,
and appears to be the same species. This
strain differs by > 40% (in terms of mor-
phology and physiology described by
Prasad and Mookerjee7) from the Cal-
cutta ecotype, which was presumed to be
H. vulgaris phase orientales, by Annan-
dale (cited by Prasad and Mookerjee7).
On the basis of evidence presented
here, this hydra can be classified only as
a different strain of the vulgaris species,
particular to the type locality mentioned,
and for further referencing we call it
as H. vulgaris Ind-Pune. Our data show
that H. vulgaris Ind-Pune is closely re-
lated to H. magnipapillata 105, whose
genome has been sequenced.
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London, 1987, 91, 253–263.
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miae, laurentii Salvii, 1767, 12th edn.
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Monog No. 13, Zoological Society of
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ACKNOWLEDGEMENTS. We thank Prof.
Richard Campbell, UCI, USA and Dr Hemant
Ghate, Modern College, Pune, for valuable
suggestions and comments on an earlier draft
of this manuscript. P.C.R. and A.B. were
recipients of SRF–NET from UGC and CSIR
respectively. This work was funded by
Department of Biotechnology, Government of
India (Centre of Excellence in Epigenetics).
Received 20 December 2010; revised accep-
ted 27 July 2011
P. CHANDRAMOULI REDDY
APURVA BARVE
SURENDRA GHASKADBI*
Division of Animal Sciences,
Agharkar Research Institute,
Pune 411 004, India
*For correspondence.
e-mail: ghaskadbi@gmail.com
... Clonal culture of Hydra vulgaris Ind-Pune (Reddy et al., 2011) was maintained in Hydra medium by following standard methods at 18 ± 1°C (Horibata et al., 2004). Polyps were fed daily with freshly hatched Artemia nauplii larvae and washed 6-8 h after feeding. ...
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... A clonal culture of Hydra vulgaris Ind-Pune (Reddy et al. 2011) was maintained in hydra medium (1 mM CaCl 2 , 0.1 mM MgSO 4 , 0.1 mM KCl, 1 mM NaCl and 1 mM Tris Cl, pH 8) at 18°C with 12:12 h light and dark cycle. The polyps were fed with freshly hatched Artemia nauplii on alternate days. ...
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... Clonal culture of Hydra vulgaris Ind-Pune (Reddy et al., 2011) was maintained in Hydra medium by 121 following standard methods at 18±1°C (Horibata et al., 2004). Polyps were fed daily with freshly 122 hatched Artemia nauplii larvae and washed 6-8 h after feeding. ...
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The Hippo signaling pathway has been shown to be involved in the regulation of cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and hence abrogates its function in transcription of target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report for the first time the existence of a complete set of Hippo pathway core components in Hydra. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested conservation of YAP-TEAD interaction in Hydra. We also characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. The YAP expressing cells are recruited early during head regeneration and budding implicating the Hippo pathway in early response to injury or establishment of oral fate. These cells exhibit a non-clustered existence at the site of regeneration and budding, indicating the involvement of a new population of YAP expressing cells during oral fate specification. Collectively, we posit that the Hippo pathway is an important signaling system in Hydra, its components are ubiquitously expressed in the Hydra body column, and may play crucial role in Hydra oral fate specification.
... A clonal culture of Hydra vulgaris Ind-Pune was maintained at 18 °C using standard protocols described previously [83,84]. Hydra polyps were fed with freshly hatched Artemia nauplii larvae daily and cleaned 6-8 h post feeding. ...
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... A clonal culture of Hydra vulgaris Ind-Pune was maintained at 18 °C using standard protocols described previously [83,84]. Hydra polyps were fed with freshly hatched Artemia nauplii larvae daily and cleaned 6-8 h post feeding. ...
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