Zootaxa 4766 (1): 139–156 ISSN 1175-5326 (print edition)

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Copyright © 2020 Magnolia Press
Article ZOOTAXA
ISSN 1175-5334 (online edition)
https://doi.org/10.11646/zootaxa.4766.1.7
http://zoobank.org/urn:lsid:zoobank.org:pub:A8B9071B-1DA9-436C-BD3D-131E526298E3

Description of Pallisentis thapari n. sp. and a re-description of Acanthosentis

seenghalae (Acanthocephala, Quadrigyridae, Pallisentinae) using morphological
and molecular data, with analysis on the validity of the sub-genera of Pallisentis
NEELAM KUMARI GAUTAM1, PAWAN KUMAR MISRA2, ANAND MURARI SAXENA1 & SCOTT MONKS3
1
Department of Zoology, University of Lucknow, Lucknow 226007, India. � neelamkumari.gautam54@gmail.com
https://orcid.org/0000-0001-7133-9157, � anandmsaxena@rediffmail.com https://orcid.org/0000-0001-5596-8738
2
Department of Zoology, Babasaheb Bhimrao Ambedkar University, Lucknow 226025, India. � pawanmshr17@gmail.com
https://orcid.org/0000-0002-8711-8006
3
Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo 42184, Mexico.
� monks.scott@gmail.com https://orcid.org/0000-0002-5041-8582

Abstract

One new species of Pallisentis Van Cleave, 1928 is described from Channa punctatus (Bloch) from Gomti River (tributary
of the Ganga River), in Uttar Pradesh, Lucknow district, India. Pallisentis thapari n. sp. is characterized morphologically
by individuals having a globular proboscis armed with rooted hooks in 4 circles of 8–10 hooks each, the first circle more
than 100 long and hooks gradually declining in size posteriorly. The trunk is cylindrical, with collar spines comprised of
15–17 complete circles of spines, each ring with 12–22 spines. As common in members of the genus, a narrow spine-free
zone lies in between the rings of collar and trunk spines. Field of spines extends posteriorly to half of the body length,
ending above the level of testes in males and slightly past mid-body in females; trunk spines have an optically-dense Y-
shaped core. The trunk is only slightly wider at the anterior end. The syncytial cement gland of males contained 23–30
nuclei. Individuals of Channa striatus Bloch from the same locality also were infected with the new species. A second
species of Acanthocephala, Acanthosentis seenghalae Chowhan, Gupta, Khera, 1988, was found as a parasite of Puntius
sophore (Hamilton) from the same locality. The proboscis is short, globular, with 3 circles of hooks each circle bearing 6
hooks. The trunk is broad in the middle and tapered at both ends, with the posterior end narrower than the anterior end.
Twelve to 16 circles of spines, each with 21–40 spines, extend from anterior end to just past mid-body in males and only to
mid-body in females. The syncytial cement gland of males contains 6–10 nuclei. The analysis of 18s rDNA identified two
clades of a monophyletic Pallisentis and placed the isolate of P. thapari n. sp. within that clade; previously established
subgenera were not supported by the results of the analysis.

Key words: Acanthosentis, Freshwater, Morphological, Pallisentis, SEM, 18s

Introduction

India is one of the 17 megadiverse countries of the world, with about 2.4% of the world’s landmass but over 8%
of all recorded species of the world (IUCN 2019; Raghunathan et al. 2012). There are genera of Acanthocephala
absent, or nearly so, from the Indian subcontinent (Abdel-Ghaffar et al. 2014; Bhattacharya 2007; Braicovich et
al. 2014), but members of two genera, Pallisentis Van Cleave, 1928 and Acanthosentis Verma and Datta, 1929,
are more abundant in India than the rest of the world (Bhattacharya 2007). Of all known species of Pallisentis and
Acanthosentis, 76% and 51%, respectively, are reported only from India. Despite the importance of these helminths
as parasites of commercially important species of fish, many authors described new species only on the basis of a
single specimen and for most of the species neither voucher specimens nor a holotype specimen are available for
further study (Gautam et al. 2019). Amin et al. (2000) reviewed Pallisentis and recognized 26 species as valid, a
number that has grown to 33 valid species. Amin et al. (2017b) reviewed Acanthosentis and recognized 23 species
as valid, all on the Indian subcontinent, a number that has grown to 45 valid species worldwide.
Earlier descriptions of taxa and the classification of Acanthocephala focused almost exclusively on morpho-

Accepted by N. Dronen: 6 Mar. 2020; published: 16 Apr. 2020 139

logical criteria (Yamaguti 1963; Bullock 1969; Petrochenko, 1971; Amin 1985) with very little consideration of
phylogenetic methods, as is true for all major taxonomic groups. One of the first descriptions of a species of Acan-
thocephala to combine molecular techniques (in this case, electrophoretic techniques) with classical morphology
was that of de Buron et al. (1986), Acanthocephaloides geneticus de Buron, Renaud, and Euzet, 1986. Since then,
many studies combine molecular and morphological techniques—pertinent recent studies of members of Pallisentis
have included both types of data (Gautam et al. 2017; Chaudhary et al. 2019).
As part of a continuing study of the helminth parasites of fish of India, two species of acanthocephalans, one
new and one known, were collected from the Gomti River, tributary of the Ganga, in Uttar Pradesh. In this work
we describe the morphology of a new species, P. thapari n. sp., and re-describe a known species, A. seenghalae
(Chowhan et. al. 1988), providing new information not given in the original description (Chowhan et al. 1988). A
phylogenetic hypothesis from the species of Pallisentinae is presented and the previously established of sub-genera
of the genus Pallisentis is analyzed.

Materials and methods

Specimen collection
Live fish were purchased from the fish market of Malihabad, Lucknow, Uttar Pradesh, India, and transported to the
laboratory; fishermen reported that they captured the fish from Nabi Panah Pond (26.5830° N, 80.4322° E). Fish
were identified according to Srivastava (2002) and the validity information of scientific name, common name and
the authority for the scientific name agrees with Fishbase (Froese & Pauly 2018). Each fish was dissected and the
digestive tract was removed and placed in normal saline (0.9 %). Acanthocephalans recovered from the intestine
and intestine were washed thoroughly and maintained at 4° C for 12 hours to relax the worms. Some worms were
preserved in 100% ethanol, and stored at 4 °C for molecular study.
Once the proboscis was everted, worms were fixed in Alcohol-Formalin-Acetic acid (AFA), stained with ace-
toalum carmine, dehydrated in ascending grades of ethanol, cleared in xylene and mounted in DPX mounting
medium. Drawings were made with the aid of an Olympus CX-41 microscope with a drawing attachment. Measure-
ments were given in micrometers (µm) as the range followed by the mean in parentheses. To provide standardized
shape names, nomenclature for plane shapes follows Clopton (2004). The holotype was deposited at the Gangetic
Plane Regional Centre, Zoological Survey of India (ZSI), Patna, India and registered in Zoobank. Paratypes were
deposited at the Helminthology Laboratory, Department of Zoology, University of Lucknow, U.P., India.

Scanning Electron Microscopy

Specimens for scanning electron microscopy (SEM) were fixed in 2.5% glutaraldehyde for 2–4 hours at 4˚C, washed
with phosphate buffer (0.1M, pH 7.2) three times at the interval of 15 min. Specimens were post-fixed with 1%
osmium tetroxide for 2 hours at 4 ˚C and again washed in 0.1 M phosphate buffer three times each of 15 min at
4˚C. Subsequently the specimens were dehydrated using an ethanol (100%) for at least 10 min and placed in critical
point drying baskets. All specimens were coated with palladium and examined using a JEOL (6490 LV) scanning
electron microscope.

Molecular Analysis
Three specimens that had been stored in 100% ethanol were processed for genomic DNA isolation, with slight
modifications in the protocol of Qiagen’s DNeasy Blood and Tissue Kit (QIAGEN N.V., Hulsterweg 82, 5912 PL
Venlo, The Netherlands). The region of nuclear rDNA using primers corresponding to conserved regions at the
extreme ends of the 18S rRNA gene (5’-AGATTAAGCCATGCATGCGTAAG-3’ and 5’-TGATCCTTCTGCAG-
GTTCACCTAC-3’) (Garey et al. 1996) were amplified. Each reaction mixture contained 100 ng of genomic DNA,
a 10 mM concentration of dNTP, 10 pmol of each primer, 5µl 10X PCR buffer (100mM Tris, pH 9.0), 50mM KCl
and 15mM MgCl2), and 1 U of Taq polymerase (final volume 50 µl). Thermal cycling conditioned were 94°C for
4 min initial denaturing followed by 30 cycles: 94°C for 30 s, 60°C for 30 s, 72°C for 90 s and a final extension at
72°C for 10 min. The PCR products were evaluated on 1.5% agarose gels. Sequencing was performed by Amnion
Biosciences, Bangalore, on an automated sequencer (Model Name 3130 × 1/3130x/GA-1203-019) by the method
of chain termination by dideoxynucleotides (Sanger & Nicklen 1977).

140 · Zootaxa 4766 (1) © 2020 Magnolia Press Gautam et al.

Phylogenetic analysis
The out-group for the analysis was chosen based on the results of the analyses of Verweyen et al. (2011); Poly-
acanthorhynchus caballeroi Diaz-Ungria and Garcia-Rodrigo, 1960 (GenBank: AF388660; see Table 1). Because
phylogenetic relationships within Eoacanthocephala are not well known, the three eoacanthocephalans included in
that analysis also were included in this one: Floridosentis mugilis (Machado, 1951) Bullock, 1962 (AF064811);
Neoechinorhynchus crassus Van Cleave, 1919 (AF001842); and, N. saginata Van Cleave and Bangham, 1949
(AY830150) (Van Cleave 1919; Van Cleave & Bangham 1949; Machado-Filho 1951; Díaz-Ungria & Gracia-Ro-
drigo 1960; Bullock 1962;). Sequence data for the 18s rDNA gene from additional putative species of Pallisentis
and Acanthosentis were acquired from GenBank (Appendix I). The sequences obtained above and those of the
additional taxa downloaded from GenBank were aligned using the online software Multiple Alignment using Fast
Fourier Transformation (MAFFT) (Katoh et al. 2002, 2009, 2017; Pais et al. 2014). Two data sets were prepared:
1. A matrix with all sequences left as downloaded from GenBank (i.e., lengths not trimmed, gaps not removed, and
all gaps treated as missing data); and 2. A matrix with all sequences trimmed to approximately the same lengths and
gaps were removed by eye. Identical analyses were run on both matrices and they both produced the same single
tree.
Based on the study and conclusions of Brooks et al. (2007) and others (Grandcolas et al. 2001; Peña 2011; Yang
& Rannala 2012), a homology-based parsimony method was chosen to elucidate phylogenetic lineage relation-
ships. Analyses were conducted with parsimony in TNT 1.5 (Goloboff et al. 2003) using new technology analyses
(Goloboff 1999; Nixon 1999; Goloboff et al. 2008). Initial test runs were conducted in order to establish whether
the resulting cladogram was different with or without trimming and gap removal: no differences were found. The
cladogram was prepared with TNT and edited with Adobe Illustrator CS5.1.

Results

Pallisentis thapari n. sp.

(Figs. 1–8 and 15–18)
urn:lsid:zoobank.org:act:294DD60F-1233-47D0-A85E-CDC3B24FABC1

Type host: Spotted snakehead Channa punctatus (Bloch, 1793) (Perciformes, Channidae).
Type locality: Nabi Panah Pond, Malihabad, Lucknow, Uttar Pradesh, India (26.5830° N, 80.4322° E).
Additional host: Channa striatus (Bloch, 1793) (Perciformes, Channidae).
Site of infection: Intestine.
Specimens deposited: Holotype deposited in Gangetic Plane Regional Centre, Zoological Survey of India
(ZSI), Patna, India- male ZSI/GPRC, IV–4356a and allotype female ZSI/GPRC, IV–4356b. Paratypes deposited at
the Helminthology Laboratory, Department of Zoology, University of Lucknow, U.P., India- males LU/Z/2019/1–
LU/Z/2019/07 and females LU/Z/2019/08–LU/Z/2019/014.
Zoobank Registration: The Life Science Identifier (LSID) urn:lsid:zoobank.org:pub:53DFA57F-4D6A-49DE-
8664-0C8B080C58E8
Etymology: The new species is named in honor of the late Dr. Gobind Singh Thapar, Department of Zoology,
University of Lucknow, Lucknow, India, for his outstanding contributions in Helminthology.
Diagnosis: Quadrigyridae, Pallisentinae, with characters of the genus Pallisentis as diagnosed by Amin et al.
(2000). Worms cylindrical, relatively large sized, range of length of males and females overlapping but mean length
of males slightly longer than females. Trunk with Y-shaped spines in rings, with anterior set of 14–17 closely spaced
rings of collar spines and posterior set of 14–41 more widely spaced rings of trunk spines; posterior set reaching
posteriorly only to mid body. Anterior and posterior sets of trunk spines separated by a narrow region lacking spines.
Trunk spines conical with an optically-dense Y-shaped core. Proboscis short, broadly ovoid, wider anteriorly, with
4 circles of 8–10 hooks each, hooks decreasing in size posteriorly. Proboscis receptacle single-walled, with cerebral
ganglion located at base. Lemnisci cylindrical, much longer than the receptacle, posterior ends hanging loose in
body cavity, about equal in length. In males, testes dollioform-depressed in shape (width to length ratio 1.0:3.3),
contiguous and slightly overlapping. Cement gland oblong-very depressed in shape, width to length ratio 1:6, syn-
cytial, about as long as testicular field, containing 23–30 giant nuclei. Cement reservoir and Saefftigen’s pouch
present. Parasites of freshwater fishes in India.

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 141
 Description: Material examined: seven male and seven female specimens, and two specimens for SEM.
Male. Worms relatively large in size (Fig. 1). Trunk 4.9–8.0 (6.6) mm long, 420–560 (484) µm a widest point.
Anterior part of trunk (about 1/2) (Fig. 1) covered with 29–58 rings of tegmental spines, pointing posteriorly (Figs.
1 and 16), number of spines per ring declining in numbers from anterior to posterior. Circles of spines separated into
two fields, anterior set composed of 15–17 closely spaced rings, each ring consisting of 12–22 spines, called collar
spines, and posterior set of 21–41 more widely spaced rings of trunk spines, each anteriormost ring composed of
12–16 spines (Figs. 16 and 17). Area covered by collar spines 340–800 (457) long. Anterior and posterior groups
of spines separated by narrow spine-free zone 100–180 (139) in length. Circles of trunk spines reaching posteriorly
only to mid-body, ending anterior to testicular field. Individual spines 40–50 (47) long, 20–40 (30) wide at the
base. Proboscis globular, broadly ovoid in shape, wider anteriorly and narrowing posteriorly, 230–280 (243) long,
220–260 (241.4) wide anteriorly (Figs. 1, 3 and 15). Proboscis with rooted hooks in 4 circles of 8–10 hooks each
(Fig. 3). Hooks longest in first circle, decreasing in size posteriorly in succeeding rings (Figs. 3, 4 and 15). Blades
of hooks in anterior circles relatively straight and oriented laterally because of position of insertion point or root;
blades increasingly curved posteriorly. Length of hooks from anterior to posterior, 100–130 (112), 80–110 (96),
40–70 (52), 30–50 (34); size ratios anterior to posterior 1.0:0.9:0.5:0.3. Neck short, robust, 150–290 (199) long,
160–290 (213) wide posteriorly. Proboscis receptacle 340–890 (547) long by 130–220 (161) wide. Lemnisci paired,
cylindroid, much longer than the receptacle and almost equal sized; right lemniscus 730–2450 (1560) long, 40–60
(52) wide posteriorly, left lemniscus 1200–2450 (1625) long, 40–60 (52) wide. Reproductive system in posterior
half of trunk. Testes dollioform-depressed in shape, contiguous with slight overlap. Anterior testis 440–720 (521)
long, 140–210 (161) wide; posterior testis 440–730 (53) long, 140–220 (167) wide. Cement gland contiguous with
testes, about size of testicular field, 680–1550 (1061) long by 130–240 (180) wide, containing 23–30 nuclei. Ce-
ment reservoir contiguous with cement gland, branching posteriorly into two ducts, 550–1070 (824) long, 140–230
(184) wide. Saefftigen’s pouch obdeltoid/very narrowly spatulate in shape, 160–340 (272) long, 130–200 (17) wide
anteriorly. Seminal vesicle 400–800 (596) long, 70–140 (104) wide. Gonopore terminal (Figs. 1 and 2). Bursa, when
extended (Figs. 1, 2 and 18), 200–300 (183) long and 100–240 (164) wide.
Female. Worms slightly smaller in size than males (Fig. 6). Trunk 4.0–7.7 (6.3) mm long, 490–590 µm (503)
µm at widest point. Anterior part of trunk (approximately anterior half) (Fig. 6) covered with 30–41 rings of tegmen-
tal spines, pointing posteriorly (Fig. 5), number of spines per ring declining in number from anterior to posterior.
Circles of spines separated into two fields, anterior set composed of 14–15 closely spaced rings, called collar spines,
each ring composed of 16–26 spines and posterior set of 14–40 more widely spaced rings of trunk spines, each ring
made up of 12–24 spines. Length of trunk covered by collar spines 430–570 (530) long. Spine-free zone narrow
100–190 (147) in length. Circles of trunk spines reaching posteriorly only to mid-body, ending anterior to testicular
field. Individual spines 40–51 (48) long, 20–40 (31) wide at the base. Proboscis globular, broadly ovoid in shape, wider
anteriorly and narrowing posteriorly, 180–260 (213) long, 190–250 (220) wide anteriorly (Fig. 6). Proboscis with
rooted hooks in 4 circles of 8–10 hooks each (Fig. 6). Hooks longest in first circle, hooks decreasing in size posteriorly
in succeeding rings (Figs. 6). Blades of hooks in anterior circles relatively straight and oriented laterally because of
position of insertion point or root; blades increasingly curved posteriorly. Length of hooks of proboscis of paratype
female, from anterior to posterior, 140, 120, 70, 40; size ratios anterior to posterior 1.0:0.9:0.5:0.3. Neck short, robust,
170–460 (296) long, 160–240 (197) wide posteriorly. Proboscis receptacle 440–840 (643) long, 80–250 (173) wide.
Lemnisci paired, cylindroid, much longer than the receptacle and equal in size, 1,600–2,600 (2,110 ± 500.0) long,
40–60 (53) wide posteriorly. Reproductive system in posterior end of trunk, uterine bell cylindrical shaped, 240–330
(280) long and 50–110 (78) wide (Fig. 7). Uterus 50–260 (110) long, 30–110 (63) wide. Vagina 30–90 (53.3 ± 20.7)
long, 30–50 (38.3 ± 9.8) wide. Ovarian balls present in some females, 40–80 (59) long, 20–60 (41) wide. Eggs 15–20
(18) long, 10–10 wide (Fig. 8) (width to length ratio about 2:3). Gonopore ventro-terminal (Figs. 6 and 7).
Remarks: Amin et al. (2000) re-described Pallisentis and provided a more detailed diagnosis for the genus. The
new species is placed in Pallisentis because it has an anterior set of closely arranged rings of collar spines (15–17
rings) and a posterior set of more widely spaced rings of trunk spines (14–41) separated from the collar spines by a
region lacking spines. Giant hypodermal nuclei are present in the trunk wall, the proboscis is broadly ovoid in shape
with rooted hooks in 4 circles of 8–10 hooks in each, and the proboscis receptacle is single-walled with the cerebral
ganglion located near the base. The lemnisci are much longer than the receptacle, and the syncytial cement gland is
long (about as long as the testicular field) with many giant nuclei. The new species is placed in this genus because
it shares these characters.

142 · Zootaxa 4766 (1) © 2020 Magnolia Press Gautam et al.

FIGURES 1–8. Drawing of specimens of Pallisentis thapari n. sp.: 1. Male worm; 2. Posterior part of male; 3. Proboscis; 4.
Hooks of the proboscis; 5. Trunk spines with optically dense Y-shaped core; 6. Female worm containing ovarian balls; 7.
Posterior end and gonopore with ovarian balls shown in uterine bell; 8. Eggs. Scale bars: 1, 2 and 6 = 1.0 mm; 3, 4, 5 and 7 =
100 µm; 8 = 10 µm.

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 143
 If the subgenera of Pallisentis established by Amin et al. (2000) is accepted, P. thapari n. sp. is similar to spe-
cies previously assigned to the subgenus P. (Pallisentis) Amin, Heckmann, Ha, Luc, and Doanh, 2000 because the
longest hooks are in first circle and the hooks of succeeding circles decrease in size posteriorly (Amin et al. 2000).
The new species can be distinguished from all known species of Pallisentis because the trunk spines do not extend
to the posterior end of either sex, the proboscis hooks in first circles are more than 100 long, and the average length
of the proboscis receptacle is more than 500 long.
Pallisentis thapari n. sp. is most similar in form to P. clupei Gupta and Gupta, 1979); the length of the first hook
in both is about 100, but P. clupei is reported to have conical trunk spines and the new species has spines with a Y-
shaped core (Gupta & Gupta 1979). In P. clupei, the number of circles of collar spines (12–13 circles in males and
13–14 in females) is similar bit less than that of the new species (15–17 in males and 14–15 in females), although
the number of spines per circle in males of P. clupei overlaps with that of the new species (14–20 spines vs. 12–22,
respectively). The number of circles of trunk spines in males in both species overlaps (P. clupei 28–30; P. thapari
n. sp. 21–41), but the number of circles of trunk spines in females of P. clupei (61) is greater than for females of
the new species (14–40). The number of trunk spines in P. clupei and that of the new species also overlaps (males
8–16; females 10–16 vs. males 12–16; females 12–24, respectively). Additionally, the new species can be distin-
guished from P. clupei in that the trunk spines of the new species extend only to mid-body but they extend to the
posterior end in P. clupei. The number of spines in each circle of trunk spines in P. clupei (8–16 in males and 10–16
in females) is less than in P. thapari n. sp. (12–16 in males and 12–24) in females, although there is some overlap
in range. Finally, in males, the size and position of testes and the size of the cement gland are similar in P. clupei
(1390–1550 long) and the new species (680–1550 long), but in P. clupei there are 9–16 giant nuclei in the cement
gland and that of the new species has 23–30 nuclei.
Seven species of Pallisentis have been reported to have spines that are Y-shaped (Amin et al. 2000; Gautam et
al. 2019): P. basiri Farooqi, 1958; P. cavasii Gupta and Verma, 1980; P. fasciata Gupta and Verma, 1980; P. guptai
Gupta and Fatma, 1986; P. indica Mithal and Lai, 1981; P. mehrai Gupta and Fatma, 1986; P. panadei Rai, 1967;
and P. unnaoensis Gautam, Misra, and Saxena, 2019 (Farooqi 1958; Rai 1967; Gupta & Verma 1980; Mithal & Lal
1981; Gupta & Fatma 1986; Gautam et al. 2019). In some species the spines are more flattened and evidence of the
Y-shape core can be seen externally (Gautam et al. 2019). In the new species the spines are conical but with the Y-
shaped core (Figs. 5 and 17).
Comparisons among species of Pallisentis are difficult because many of the type specimens are not available
and one must rely on descriptions in the literature. However, the new species can be distinguished from the above-
mentioned species by various features. Pallisentis thapari n. sp. is larger than P. cavasii, P. fasciata, and P. unnaoen-
sis (4.9–8.0 mm vs. 2.1–3.0, 2.9–4.0, and 3.4–4.4, respectively) and the new species has more nuclei in the cement
gland (23–30 vs. 6–8, 8–10, and 7–8, respectively), and the proboscis hooks of the first row of the new species are
longer (100–130) than those of P. cavasii (50–51), P. fasciata (60–70), and P. panadei (70–50) and they are shorter
than those of P. unnaoensis (220). The hooks in the first row of the new species and those of P. basiri are about the
same size (approximately 100), but the roots of the hooks of the new species are elongate and those of P. basiri are
described as being knob-like (Amin et al. 2000). The cement gland of males of the new species is longer than that of
P. guptai (680–1550 vs. 500–580, respectively) and the number of nuclei is greater (28–30 vs. 10–12, respectively).
Males of the new species have more nuclei in the cement gland than those of P. indica (23–30 vs. 9–18) (Amin et al.
2017a). The trunk spines of the new species reach posteriorly only to about mid-body but those of P. mehrai reach to
the posterior end in both males and females. Finally, in results of the molecular analysis, P. unnaoensis is the sister
taxa to the clade comprised of the three specimens of the new species. The new species is similar to P. unnaoensis in
the number of rings and spines per ring of collar and trunk spines, but the cement gland is longer in the new species
(680–1550 vs. 380–520) and the number of nuclei in the cement gland is greater (23–30 vs. 7–8).

Acanthosentis seenghalae Chowhan, Gupta, and Khera, 1988

(Figs. 9–14 and 19–20)

Type host: Gangetic mystus, Mystus seenghala (Hamilton) (Siluriformes, Bagridae).

Type locality: Nabi Panah Pond, Malihabad, Lucknow, Uttar Pradesh, India (26.5830° N, 80.4322° E).
Additional host: Pool barb, Puntius sophore (Hamilton) (Cypriniformes, Cyprinidae).
Site of infection: Intestine.

144 · Zootaxa 4766 (1) © 2020 Magnolia Press Gautam et al.

 Specimens deposited: Voucher male ZSI/GPRC, IV–4362a and female ZSI/GPRC, IV–4362b. Paratype males
LU/Z/2019/15–LU/Z/2019/18 and females LU/Z/2019/19–LU/Z/2019/21.
Diagnosis: Quadrigyridae, Pallisentinae, with characters of the genus Acanthosentis Verma and Datta, 1929
(Verma & Datta 1929; Bhattacharya 2007). Shared structures smaller in females than in males. Body small, broader
at about 1/3 distance from anterior end, tapering to posterior end, slightly curved ventrally. Trunk with rings of
spines closely set anteriorly but gradually more widely apart as they proceed backward to just past mid-body, spines
pointing posteriorly. Proboscis short, orbicular (length to width ratio 1:1), with 3 circles of 6 hooks each; hooks
declining in size from first circles to third circles, anterior to posterior. Proboscis receptacle ovoid (length to width
ratio 1:0.5), with single layered wall, and with cerebral ganglion at base. Hypodermic nuclei few, not branched, in
posterior half of trunk. Lacunar system reticular, with prominent transverse vessels. Lemnisci longer than proboscis
receptacle, cylindrical in males and narrowly deltoid in females, longer in males than in females. Testes eliptoid,
tandem, contiguous in posterior region of body. Cement gland syncytial with 6–10 nuclei, about 1/4 as long as tes-
tes. Genital pore terminal in males, slightly ventro-terminal in females. Eggs narrowly rhomboid (length to width
ration 1:0.3).
Description: Material Examined: Four males, three females, and two specimens for SEM.
Male. Worm small in size, broadest at about 1/3 distance from anterior end, tapering to posterior end, anterior
2/3 of trunk straight, posterior 1/3 slightly curved ventrally. Trunk 2.43–2.16 (2.4) mm long, 320–370 (343) µm
wide at widest point (measurements of type material from original description given in Table 2). Five pairs of gi-
ant hypodermal nuclei present. Trunk with rings of spines closely set anteriorly but gradually more widely apart
as they proceed backward to just past mid-body to level of posterior testis, 12–16 circles of 21–40 spines, almost
equal in size (Figs. 9 and 19). Proboscis short, orbicular in shape (length to width ratio 1:1), 40–60 (45 ± 10) long,
40–60 (45) wide (Figs. 9, 10 and 20). Proboscis with rooted hooks in 3 circles of 6 hooks each (Figs. 10 and 20).
Hooks longest in first circle, decreasing in size posteriorly in succeeding rings (Figs. 10, 11 and 20). Blades of hooks
curved to point posteriorly. Length of hooks from anterior to posterior, 500 250, 200; size ratios anterior to posterior
1.0:0.6:0.5. Neck short, 16–23 (20). Proboscis receptacle very deeply ovoid, 120–160 (143) long, 50–80 (63) wide.
Lemnisci cylindrical, much longer than proboscis receptacle, approximately equal in size, 690–700 (693) long,
40–50 (43) wide. Reproductive system in posterior 3/4 of body (Fig. 9). Testes eliptoid, tandem, contiguous. Ante-
rior testis 420–490 (465) long, 300–300 wide; posterior testis 370–540 (470) long, 270–300 (290) wide. Anterior
testis 1130–1700 (1367) from anterior end of body, posterior testes 1600–2060 (1767) from anterior end. Cement
gland much smaller than testes, contiguous with posterior testis, 150–240 (198) long, 110–240 (193) wide, contain-
ing 6–10 giant nuclei. Cement reservoir larger than cement gland, contiguous, 280–390 (335) long, 130–150 (140)
wide. Saefftigen’s pouch pyriform; 70–130 (100) long, 80–90 (88) wide, duct wide anteriorly tapering to posterior
connection. Gonopore terminal (Fig. 9). Bursa, when everted, 80–140 (113) long, 90–160 (123) wide.
Female. Females smaller than males, broadest at about 1/3 distance from anterior end, tapering to posterior end,
anterior 1/1 of trunk straight, posterior 1/1 slightly curved ventrally (Fig. 12). Trunk 1.7–1.8 (1.7) mm long, 158–237
(198) µm wide at widest point. Five pairs of giant hypodermal nuclei present, posteriormost three nuclei clumped
together at posterior part of trunk. Trunk with rings of spines closely set anteriorly but gradually more widely apart
as they proceed backward to just past mid-body, 18 circles of 34 spines, fifth ring from last, third from last, and
second from last rings double (12 single rings and 3 double rings) (Fig. 12). Spines almost equal in size. Proboscis
short, orbicular in shape (length to width ratio 1:1), 29–31 (30) long, 29–31 (30) wide (Fig. 12). Neck short, 16–20
(18). Proboscis receptacle very deeply ovoid, 142–150 (146) long, 67–70 (68) wide. Lemnisci cylindrical, much
longer than proboscis receptacle, compressed by ligament sac, approximately equal in size, 196–200 (198) long,
56–60 (58) wide. Reproductive system in posterior 1/5 of trunk, ligament sac persistent, attached to anterior opening
of uterine bell. Uterine bell 79–83 (81) long, 54–58 (56) wide (Fig. 13). Uterus 73–75 (74) long, 40–43 (41) wide.
Vagina 32–35 (33) long, 38 wide. Gonopore approximately terminal, protruding slightly from body wall (Fig. 13).
Ovarian balls somewhat elliptical in shape. Eggs narrowly fusiform in shape, with thick outer shell, 35–44 (39) long,
14–17 (15) wide (Fig. 14).
Remarks: The original authors (Chowhan et al. 1988) placed A. seenghalae in the Acanthosentis because the
proboscis hooks are in three circles of six hooks in each; the present form is consistent with those specimens. Using
the key provided by Amin (2005), the specimens collected for this study also were identified as A. seenghalae. The
present form possesses only one sperm duct and Saftigen’s pouch is present. The rings of trunks spines extend over
the anterior half of the body to of the posterior end of the testicular field. In the original description of A. seenghalae,
12–14 circles of 20–24 spines in each ring were reported, but in present form males had 12–16 rings of 21–40 spines

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 145
in each and in females had 12 circles of 34 in each with the fifth from the last ring, third from last, and second from
last ring are double (not reported in the original description), increasing the total number of rings to 18 unless you
count the double rings as a single ring. The difference between the spination of the present material and that of the
type material of the species could not be evaluated.

FIGURES 9–14. Drawing of specimens of Acanthosentis seenghalae Chowhan, Gupta, Khera, 1988: 9. Male worm; 10. Pro-
boscis; 11. Hooks of the proboscis; 12. Female worm; 13. Posterior end and gonopore; 14. Eggs. Scale bars: 9 = 1 mm; 10 and
12 = 500 µm; 11 = 20 µm; 13 = 200 µm; 14 = 100 µm.

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FIGURES 15–20. Scanning electron micrographs: 15. Proboscis of Pallisentis thapari n. sp. showing hooks that de-
crease in size from anterior to posterior rings; 16. Trunk spines of P. thapari n. sp. with optically dense Y-shaped
core and conical exterior; 17. Hooked point of a collar spine of P. thapari n. sp; 18. Bursa of a male worm of P.
thapari n. sp.; 19. Trunk spines of a specimen of Acanthosentis seenghalae; 20. Proboscis of a male worm of A. seeng-
halae. Scale bars: 15 = 50 µm; 16, 18 and 19 = 100 µm; 17 = 10 µm; 20 = 20 µm.

The proboscis of the holotype of A. seenghalae is as long as wide (60–80 long, 70–80 wide); the proboscis of
the present form also is as long as wide (40–60 long, 40–60 wide) but it is a little smaller. The uterine bell, vagina
and eggs were not mentioned by Chowhan et al. (1988) and they were obscured by eggs in their figures, and in the
present material the females also were filled with ovarian balls and mature eggs. The para-receptacle organ was not
seen. A comparison of the measurements of the type specimens of A. seenghalae with those of the present material
is given in Table 2.

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 147
TABLE 1. Sequences of Acanthocephala included in the analysis with some specific names updated as verified in this
study and the accession numbers.
Species of Acanthocephala GenBank accession No.
Acanthosentis sp. 2 NKG-2016 KY305530
A. seenghalae (as Acanthosentis sp. 1 NKG-2016 in GenBank) KY305529
Floridosentis mugilis AF064811
Neoechinorhynchus crassus AF001842
N. saginata AY830150
Pallisentis thapari n. sp. (as Pallisentis sp. 1 NKG-2016 in GenBank) KU715091
Pallisentis sp. 3 NKG-2016 KU715089
Pallisentis sp. 4 NKG-2016 KY305516
Pallisentis sp. 5 NKG-2016 KY305517
Pallisentis sp. 6 NKG-2016 KY305518
Pallisentis sp. 8 NKG-2016 KY305520
Pallisentis sp. 9 NKG-2016 KY305521
Pallisentis sp. 10 NKG-2016 KY305522
Pallisentis sp. 11 NKG-2016 P. amini KY305523
Pallisentis sp. 1 NKG-2017 KY491740
Pallisentis sp. BR-2017 isolate Mani1 MF437351
P. indica (as Pallisentis sp. HS/CCSU/2017/17-ISL1 in GenBank) MG582597
P. indica (as Pallisentis sp. HS/CCSU/2017/17-ISL2 in GenBank) MG582598
P. lucknowensi (as Pallisentis sp. 14 NKG-2016 in GenBank) i KY305526
P. meyeri (as Pallisentis sp. 13 NKG-2016 in GenBank) KY305525
P. thapari n. sp. (as Pallisentis sp. 12 NKG-2016 in GenBank) KY305524
P. thapari n. sp. (as Pallisentis sp. 15 NKG-2016 in GenBank) KY305527
P. unnaowensis (as Pallisentis sp. 16 NKG-2016 in GenBank) KY305528
Polyacanthorhynchus caballeroi AF388660

Molecular characterization

The amplification sizes of the rRNA of P. thapari (KY305524, KY305527 and KU715091) were 927, 909, 782 base
pairs long and A. seenghale (KY305529) was 913 base pairs long. The G+C contents were estimated as 45–46.1%.
Sequences for the remaining taxa included in the analysis were downloaded from GenBank by referencing the ac-
cession numbers (Table 1).
In the resultant cladogram (Fig. 21), three clades were identified: Pallisentis Clade 1, Pallisentis Clade 2, and a
clade comprised of the two species of Acanthosentis as sister group to a clade of the other three species of Eoacan-
thocephala included in the analysis. Members of the genus Pallisentis were separated into the two clades mentioned
above, one (Clade 1) comprised of sequences in GenBank that have not yet been identified to species and one clade
(Clade 2) composed of nine identified species (including three sequences of P. thapari n. sp.) and two sequences
from different putative species that have not yet been identified (Fig. 21).

Discussion

This study has reported a new species of Pallisentis and provided new information on a known species of Acantho-
sentis. Seven sequences from GenBank were identified as belong to P. thapari n. sp., P. lucknowensi Gautam, Misra,
and Saxena, 2019, P. meyeri Gautam, Misra, and Saxena, 2019, P. unnaowensis, and Acanthosentis seenghalae; all
sequences had been reported previously as unidentified members of their respective genus.

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TABLE 2. Comparative measurements of Acanthosentis seenghalae Chowhan, Gupta and Khera 1988 form the original
study with the data from the present study. Measurements in millimeters, given as Length X width; Abbreviation: NM =
not measured.
Morphological characters Chowhan et al. 1988 Present study
Host Mystus seenghla (Sykes) Puntius sophore (Hamilton)
Body Length- Male 2.87–3.18 X 0.72–1.00 1.80–2.47 X 0.28–0.43
Proboscis 0.06–0.08 X 0.07–0.08 0.03–0.06 X 0.03–0.07
Proboscis receptacles NM 0.12–0.15 X 0.06–0.07
Proboscis Hooks 3 rows / 6 hooks in each row 3 rows / 6 hooks in each row
Proboscis Hooks 1 row
st
0.029–0.030 0.50
Proboscis Hooks 2nd row 0.020–0.027 0.25
Proboscis Hooks 3 row
rd
0.016–0.024 0.20
Hypodermal nuclei NM 5 pair
Right lemnisci 0.59–0.91 X 0.30–0.41 0.20–0.69 X 0.05–0.06
Left lemnicsi 0.59–0.91 X 0.30–0.41 0.20–0.55 X 0.05–0.06
Trunk Spine 12- 14 /20 -24 16 / 40 hooks each
Anterior testis NM 0.48 X 0.30
Posterior testis NM 0.53 X 0.30
Cement gland NM 0.24 X 0.24
Cement gland nuclei 6–11 nuclei 6–10 nuclei
Saefftigen’s Pouch NM 0.13 X 0.09
Bursa NM 0.12–0.15 X 0.06–0.07
Body length- Female 2.87–4.35 X 0.70–0.98 1.60 X 0.26
Uterine bell NM 0.083 X 0.058 (Funnel shaped)
Uterus NM 0.12 X 0.12
Vagina NM 0.070–035 X 0.038–0.043
Eggs NM 0.035–0.044 X 0.015–0.017

The results of the molecular analysis supported the monophyly of Pallisentis, comprised of two clades. The
majority of the sequences deposited in GenBank for this genus still are identified only as Pallisentis sp. The genus
Acanthosentis was not supported as a member of Pallisentinae, suggesting that Pallisentinae may be paraphyletic.
Some ambiguity exists as to the complete description of the character of the trunk spines that have been de-
scribed as being Y-shaped (see Amin et al. 2000 and Gautam et al. 2019 for use of the term previously in descrip-
tions). Close examination of the material reported herein and comparison with the form of the spines in photographs
taken using SEM, indicated that the spines are not Y-shaped, as reported previously. There is an optically-dense core
of the spines that is Y-shaped and not the entire spine. Further studies, such as that of Heckmann et al. (2012) are
necessary to elucidate the micro-structure of the trunk spines.
In the specimens of Acanthosentis the para-receptacle organ was not observed. The presence of this structure,
also called the para-receptacle structure, was first reported by Amin et al. (2002) and was further described by Amin
et al. (2007). Amin et al. (2007) considered the structure to be evidence that Neoechinorhynchidae and Quadrigy-
ridae are the most basal families of Acanthocephala. Those authors consider the organ to be a remanent of the hy-
drostatic structure of ancient acanthocephalans. Furthermore, they proposed that all acanthocephalans with a single-
walled proboscis receptacle have para-receptacle organs, but no explanation was made for the lack of identification
of it in many taxa. Our failure to observe this structure cannot be taken as conclusive evidence that individuals of
A. seenghalae do not have a para-receptacle organ, and the significance of the presence or lack of it in a particular
species is unclear.

Selection of the most appropriate out-group

The out-group in a phylogenetic analysis functions to polarize the character states within the in-group (Bryant
2001). Thus, it should be closely related to the members of the in-group; ideally, the sister-group to the members

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 149
of the in-group. Following this reasoning, species that have been identified as potential sister-taxa to Pallisentinae
were selected for the present study based on several previous analyses (García-Varela et al. 2002; Verweyen et al.
2011; García-Varela & Pinacho-Pinacho 2018), three potential sister-group taxa were selected in the present study to
polarize the sequence data for members of Pallisentinae because relationships within Eoacanthocephala are not well
studied. Polyacanthorhynchus caballeroi was chosen as the distant out-group, sister-group to Eoacanthocephala in
order to test the monophyly of Pallisentinae.

Paraphyly and redefinition of Pallisentinae

Van Cleave (1928) established Pallisentis for P. umbellatus Van Cleave, 1928. Since then, the genus has grown to
include at least 33 valid species (34 with the present description). In the present classification, for which there is
not complete agreement (for two examples, see Golvan, 1994 and Amin, 2013), Family Quadrigyridae (Eoacantho-
cephala, Gyracanthocephala) consists of two subfamilies, Quadrigyrinae and Pallisentinae. Pertinent to this study,
Pallisentinae is comprised of four genera: Pallisentis, Acanthogyrus Thapar, 1927, Raosentis Datta, 1947, and Ac-
anthosentis (Verma & Datta 1929). The numbers of recognized species became so great in certain genera that some
authors felt compelled to form subgenera (Golvan 1959; Amin 1985; Golvan 1994; Amin 2013), although none of
these efforts were based on the results of an objective phylogenetic analysis. For this reason, not all authors have
agreed with the division of Pallisentis into subgenera (Bhattacharya 2007).
The two most recent phylogenetic analyses of Pallisentinae, by Chaudhary et al. (2019) and this study, included
sequences of some identified and some unidentified species (Table 1). Although only part of the 34 recognized spe-
cies are included, the results of the present study (Fig 21) do not support the division of Pallisentis into the three
subgenera proposed previously, most recently by Amin (2013). In the results of the study by Chaudhary et al. (2019)
(Fig. 22), the species of Pallisentis are divided into two clades; one clade comprised of P. indica Mithal and Lal,
1981 and the other is a polytomy containing 15 then unnamed putative species.
Pallisentis indica previously was P. (Brevitritospinus) Amin, Heckmann, Ha, Luc and Doanh, 2000 (Mithal
& Lal 1981; Amin et al. 2000). In the cladogram presented in that study (Chaudhary et al. 2019), the larger clade
contains a taxa now identified as P. lucknowensis Gautam, Misra, and Saxena, 2019, also formerly assigned to P.
(Brevitritospinus), and three species, P. meyeri Gautam, Misra, and Saxena, 2019, P. amini Gautam, Misra, and Sax-
ena, 2019, and P. unnaoensis, formerly assigned to P. (Pallisenti). Pallisentis thapari n. sp., also is part of that clade,
and, according to Amin et al. (2000) would be considered as part of subgenus P. (Pallisenti) because the proboscis
hooks gradually declining in size posteriorly and the cement gland is relatively long with many giant nuclei (Amin
et al. 2000). It also should be mentioned that the two putative species of Acanthosentis has been suggested as a
sister-group to Pallisentis, but this was not supported in the results of the present analysis. The results of the present
study also indicate that at least two of the three subgenera are paraphyletic. Affinities of the putative species in Clade
1 (Fig. 21) cannot be determined from the sequences alone because they all are identified only to genus. However,
Clade 2 is a mix of the same two subgenera indicated as paraphyletic in the study by Chaudhary et al. (2019); this
was not mentioned by those authors. The number of putative species included in this study are still a fraction of
those currently assigned to the genus, but these two studies each separately show that the current subgenera are not
monophyletic, and thus, they should not be used in future studies and classification.
We recognize the risk in making decisions about classification that is intended to reflect phylogenetic rela-
tionships using only a single type of data. Despite its great importance, the peril of using data from a single gene
also must be recognized—gene trees and species trees are not the same thing (Pamilo & Nei 1988; Nichols 2001;
Szöllősi et al. 2014). It should be added that one must be careful and not draw sweeping conclusions from a limited
data set, particularly when only a partial group of pertinent taxa are included (Giribet et al. 2016). Delimiting taxa
on the basis of partial molecular data also can be risky (Doyle 1995; DeSalle et al. 2005; de Queiroz 2007; Wiens
2007; Haszprunar 2011; Camargo & Sites, Jr. 2013). However, because studies of acanthocephalans sometimes base
taxonomic decisions on inadequate methodology, or on no objective methodology at all, these problems must be
addressed here.
First, the designations of subgenera in Pallisentis, as presently conceived (Amin et al. 2000) is without objective
justification. Neither the previous analysis (Chaudhary et al. 2019) nor the present results support the recognition of
monophyletic subgenera in Pallisentis. Thus, the recognition of the subgenera is not acceptable and pertinent taxa
should be recognized only as members of the genus Pallisentis sensu Amin et al. (2000). Secondly, the monophyly
of Pallisentinae is in doubt because Acanthosentis was not identified as closely related to Pallisentis. In the results of
the analyses of Chaudhary et al. (2019) (Fig. 22), it is the sister-group to Pallisentis, but in the results of the present

150 · Zootaxa 4766 (1) © 2020 Magnolia Press Gautam et al.

analysis, the clade comprised of A. seenghalae and Acanthosentis sp. 2 NKG-2016 is basal in a clade comprised of
that genus and species of Floridosentis and Neoechinorhynchus (Fig. 21).

FIGURE 21. Single phylogenetic tree produced by the parsimony analysis of 18s rDNA sequences including the sequence of
Pallisentis thapari n. sp., Acanthosentis seenghalae Chowhan, Gupta, Khera, 1988, and the sequences of the taxa downloaded
from GenBank: names of species associated with sequences in the present work are included (see Table 1 for the taxon names
given in GenBank). Putative sub-genera (sensu Amin et al. 2000) indicated in color.

FIGURE 22. Phylogenetic tree generated by Chaudhary et al. (2019) using maximum likelihood (ML) analysis of 18s rDNA
sequence data of Pallisentis indica and related species. Tree modified from Chaudhary et al. (2019) to remove distance values
and different branch lengths but retaining hypothesized phylogenetic relationships and to include the names of species associ-
ated with the sequences identified in the present work. Putative sub-genera (sensu Amin et al. 2000) indicated in color.

New species of Pallisentis from India Zootaxa 4766 (1) © 2020 Magnolia Press · 151
 While attempting to modernize the classification of Acanthocephala, it is important to remember that premature
conclusions without data are inappropriate. The gene sequence used in this study is from a non-coding region of
DNA. Thus, drawing conclusions about the evolution of morphological structures or life cycles can only be tenta-
tive (Near 2002 as an example). However, even taking this advice to heart, it still is interesting to hypothesize about
one notable morphological feature from this study; the presence/absence of trunk spines in Eoacanthocephala. The
results of a phylogenetic analysis of a limited number of genera of Acanthocephala by Monks (2001) suggested that
the presence of trunk spines within the Phylum was quite homoplastic, appearing and disappearing often in distantly
related groups. Nevertheless, if we accept the results of this and the previous studies cited herein, that Polyacan-
thorhynchus is the sister-group to Eoacanthocephala, or even consider it to be a basal member of that clade, then an
interesting hypotheses emerge. Species of Polyacanthorhynchus have rings of spines in the anterior portion of the
trunk. For example, Polyacanthorhynchus kenyensis Schmidt and Canaris, 1967 has about 9–12 circles of spines
with 18–32 spines in each ring (Amin & Dezfuli 1995). Rings of spines on the trunk in various patterns are present
in members of both Acanthosentis and Pallisentis. The results of this study suggest that the presence of trunk spines
in rings is a synapomorphy for the clade Polyacanthorhynchus + Eoacanthocephala, and the secondary lose of trunk
spines in the portion of Eoacanthocephala represented by Floridosentis Ward, 1953 and Neoechinorhynchus Stiles
and Hassall, 1905 may be a secondarily derived state. However, of course, the evolution of trunk spines in eoac-
anthocephalans and other taxa must be studied further. Nevertheless, it demonstrates how the selection of a proper
out-group provides a springboard to the study of the evolution of acanthocephalans.
Finally, this study has reported a new species of Pallisentis and provided new information on a known species
of Acanthosentis, but it has not even touched the surface of the many questions about the interesting members of
Pallisentinae. Members of the subfamily are attractive candidates as model organisms for studying ecology and co-
evolutionary history of host-parasitic interactions, among other topics. However, even with the advances presented
herein, the lack of more comprehensive phylogenetic studies prevents detailed comparison of this group to other
acanthocephalans. It is hoped that this study will suggest future research on the species composition, zoogeography,
and evolution of the Pallisentinae, allowing comparisons to be made on the ecology of this taxa and other groups
that have diversified under similar conditions.

Acknowledgement

We thank the University Grants Commission, New Delhi, India, for the financial assistance through a Rajeev Gan-
dhi National Fellowship (F1-17.1/2013-14/RGNF-2013-14-SC-UTT-43684/(SAIII/website) to Neelam Kumari
Gautam. Francisco Zaragoza-Tapia edited the images.

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