Parasitology Research (2022) 121:933–944

https://doi.org/10.1007/s00436-022-07447-1

HELMINTHOLOGY - ORIGINAL PAPER

The life cycle of Philophthalmus aylacostoma n. sp. (Trematoda:

Philophthalmidae), a new eye fluke species transmitted
by Aylacostoma spp. (Gastropoda: Thiaridae) in Brazil
Eduardo A. Pulido‑Murillo1 · Vasyl V. Tkach2 · Hudson A. Pinto1

Received: 9 November 2021 / Accepted: 24 January 2022 / Published online: 2 February 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Philophthalmus is a cosmopolitan genus of digeneans that includes ocular parasites of birds and mammals. Despite broad
distribution and veterinary importance of these digeneans, there are still gaps in knowledge about their diversity and biol-
ogy, especially in South America. Herein, we conducted morphological, life cycle, and molecular studies of megalurous
cercariae found in aquatic gastropod molluscs Aylacostoma chloroticum and A. tuberculatum collected in the São Francisco
River, Brazil. Adult parasites reared experimentally in the eyes of chicks are described here as Philophthalmus aylacostoma
n. sp. The new species differs from its congeners known in the Americas by a combination of traits, including the sucker
width ratio, the oral sucker to pharynx width ratio, egg size, and the type of vitellarium in adult forms. The new species is
morphologically closest to Philophthalmus megalurus, from which it differs by the smaller body and larger eggs, as well as
by the measurements of cercariae and the family of snails that act as the intermediate host. Molecular phylogenetic analysis
based on 28S rDNA and comparison of cox1 sequences confirm that P. aylacostoma n. sp. is distinct from four previously
sequenced named species of the genus. Moreover, cox1 sequences revealed conspecificity of our specimens with an isolate
of Philophthalmus sp. previously reported, also in thiarid snails, in Paraná River, Brazil. The interspecific divergence in cox1
between the new species and other species with sequences available for comparison varied between 12 and 15%.

Keywords Philophthalmidae · Thiaridae · Life cycle · São Francisco River · Molecular phylogenetic analysis

Introduction Philophthalmus were described worldwide, however, after

Members of the cosmopolitan genus Philophthalmus Looss,
1899, are ocular parasites of birds and mammals, includ-
ing humans (Kanev et al. 2005; Chalkowski et al. 2021).
The taxonomy of the genus has always been problematic,
especially because the morphological variability may have
resulted in an inflated number of formally described spe-
cies of these parasites. More than 50 nominal species of
Section Editor: Konstans Wells
* Hudson A. Pinto
hudsonalves13@icb.ufmg.br
1
Laboratório de Biologia de Trematoda, Department
of Parasitology, Institute of Biological Sciences,
Universidade Federal de Minas Gerais, Av. Pres. Antônio
Carlos, 6627, Pampulha, Belo Horizonte 31270‑901, Brazil
2
Department of Biology, University of North Dakota,
Grand Forks, ND 58202, USA
several subsequent revisions, it was presumed that the num-
ber of valid species should be reduced to less than 10 (Kanev
et al. 1993; Nollen and Kanev 1995). The differentiation
between these species is based on morphometric charac-
teristics, although it is known that they can be affected by
several factors, such as the parasite age (Dronen and Fried
2008), worm burden (Nollen 1983) and host-induced varia-
tion (Ching 1961). In this sense, integrative approaches com-
bining adult morphology, information on larval stages, and
molecular data can contribute to a more robust taxonomic
identification of Philophthalmus spp.
The life cycle pattern typical of species of Philophthalmus
was elucidated through experimental infection studies, which
resulted in obtaining adult stages in birds exposed to cercar-
iae and metacercariae by oral or ocular routes (Alicata 1962;
Nollen and Kanev 1995). This approach allowed not only to
reveal the role of freshwater and marine caenogastropods as
intermediate hosts of these eye flukes, but also to describe
new species, e.g., Philophthalmus distomatosa (Looss,

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1896), Philophthalmus hegeneri Penner and Fried, 1963, and
Philophthalmus megalurus (Cort, 1914) (Penner and Fried
1963; McMillan 1971; McMillan and Macy 1972; Radev et al.
2000). The natural infections of birds with the latter two spe-
cies were reported (West 1961; Penner and Fried 1963), while
the definitive host for P. distomatosa remains unknown.
In the last few years, molecular markers have been used in
the studies of Philopthalmus spp., contributing to taxonomic
identification, life cycle elucidation, and a preliminary phylog-
eny (Church et al. 2013; Literák et al. 2013; Heneberg et al.
2018; Bennett and Presswell 2019). A study of a zoo infec-
tion of captive rheas with Philophthalmus gralli Mathis and
Leger, 1910 (Church et al. 2013) and the recent description
of Philophthalmus attenuatus Bennett and Preswell, 2019
are examples of studies where larval and adult stages were
matched using DNA sequences. Nevertheless, the molecular
data on philophthalmids remain scarce, with sequences avail-
able only for some isolates of P. attenuatus, P. gralli, Philoph-
thalmus lachrymosus Braun, 1902, and Philophthalmus luc-
ipetus (Rudolphi, 1819). The lack of sequence data for the
majority of species hampers the identification of larval stages,
which nowadays is usually done using the molecular approach.
As a result, multiple publications only provide the identifica-
tion to the genus level (Keeney et al. 2009; Leung et al. 2009;
Born-Torrijos et al. 2014; Onaca et al. 2020; Preston et al.
2021). In some cases, the specific identification of cercariae
occurs retrospectively years later, when a corresponding adult
form is being sequenced (Bennett and Presswell 2019).
Thus far, 8 species of Philophthalmus spp. have been
reported from the Americas, namely Philophthalmus ander-
soni Dronen and Penner, 1975, Philophthalmus larsoni Pen-
ner and Trimble, 1970, Philophthalmus hegeneri Penner and
Fried, 1963, and Philophthalmus zalophi Dailey, Ellin, and
Parás, 2005, found in the marine environment; P. gralli,
and Philophthalmus megalurus (Cort, 1914) transmitted by
freshwater snails; and P. lachrymosus, and Philophthalmus
semipalmatus Nasir and Díaz, 1972, with no knowledge on
the type of life cycle and intermediate hosts (Chalkowski
et al. 2021). In this study, we describe different stages of
the experimentally completed life cycle of a new species of
the genus Philophthalmus transmitted by aquatic gastropods
Aylacostoma spp. in the São Francisco River, Brazil. We
also use partial sequences of the mitochondrial cox1 gene
and nuclear ribosomal 28S gene for comparison of the new
species with its congeners and phylogenetic inference.
Material and methods
Malacological study and trematode infection
Malacological surveys were conducted along the banks
of the São Francisco River, municipality of Januária,
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northern Minas Gerais State, Brazil (15°29′43.53′′S;
44°21′45.41′′W), from July 2017 to August 2019 aiming
to study the diversity of larval trematodes transmitted by
molluscs in this lotic environment. The snails were col-
lected using a D-shaped nylon hand net and transported
to the laboratory. Two species of thiarids were identified
morphologically as Aylacostoma chloroticum Hylton Scott,
1954, and Aylacostoma tuberculatum Spix, 1827 (Fig. S1).
Voucher specimens of each morphotype were deposited
in the Malacological Collection of the Instituto Oswaldo
Cruz, Rio de Janeiro, Brazil (CMIOC).
To detect the infection by larval trematodes, the thi-
arids were placed in 6-well polystyrene plates containing
dechlorinated water and exposed to a period in darkness
and under light to stimulate emergence of cercariae with
both positive and negative phototaxis. After that, the wells
in the plates were examined under a stereomicroscope for
the presence of cercariae. A repeated examination was car-
ried out on the following day. Live philophthalmid cer-
cariae (megalurous type) were studied under a light micro-
scope with vital stains (0.05% neutral red and 0.05% Nile
blue). Metacercariae found on plate walls and snail shells
were excysted in warm water (42°C) according to Cheng
and Thakur (1967). Samples of cercariae were killed in
hot water (70°C) and fixed in 10% formalin. Additionally,
cercariae or thermally excysted metacercariae were fixed
in 95% ethanol and kept at −20°C for molecular analysis.
Intramolluscan stages (rediae) were obtained from infected
gastropods crushed between glass plates and examined
under a stereomicroscope; rediae were also fixed in 10%
formalin.
Permission to collect molluscs was obtained from the
Brazilian Institute of Environment and Renewable Natural
Resources (IBAMA/SISBIO) under registration SISBIO
52870-1.
Experimental infection
Forty-five excysted metacercariae suspended in water were
deposited on the outer surface of each eye of two young
chicks, Gallus gallus domesticus (L.) with the aid of a 50-μL
micropipette. Each chicken was necropsied at 36 and 45 days
post-infection (dpi). Worms were removed from the nicti-
tating membrane and conjunctival sacs, lightly compressed
between glass slides, relaxed with hot water, and fixed in
10% formalin. Eggs freshly laid by adults and miracidia
were also detected during the necropsy and collected for
live observation and fixation, as previously described for
cercariae.
The use of vertebrate animals in experiment followed the
protocol approved by the Local Ethics Committee in Animal
Experimentation (CEUA-UFMG, protocol 68/2017).
Parasitology Research (2022) 121:933–944
Morphological data
Adult parasites were stained with alum acetocarmine, dehy-
drated in an ethanol series, cleared in beechwood creosote,
and mounted in Canada balsam. Morphological and mor-
phometric studies were based on permanent (adults) and
temporary wet mounts (formalin-fixed cercariae, eggs, mira-
cidia, and rediae) analyzed under an Olympus BH2 optical
microscope (Olympus, Tokyo, Japan). Measurements were
obtained using a calibrated ocular micrometer. Photographs
were taken using Leica DM500 and DM750 microscopes
(Leica Microsystems, Wetzlar, Germany) equipped with a
Leica ICC50 HD digital camera, using Leica Application
Suite version 2.0 software. All measurements are in microm-
eters. For larval stages, mean values are followed by the
range in parentheses. In the description of adults, measure-
ments of the holotype are followed by ranges for the type
series in parentheses.
Drawing of an adult specimen was made with the aid
of a drawing tube and digitally inked using a Wacom tab-
let (Wacom Co., Vancouver, USA) in Adobe Photoshop
v.22.4.3 (Adobe Inc., California, USA). Type and voucher
material were deposited in the Helminthological Collection
of the Oswaldo Cruz Institute (CHIOC) and the Collection
of Trematodes of the Universidade Federal de Minas Gerais
(UFMG-TRE).
Molecular data and analysis
DNA extraction from larval stages (cercariae from A. tuber-
culatum and excysted metacercariae from A. chloroticum)
was performed using the QIAamp DNA Micro Kit (Qiagen,
Stanford, California, USA) according to the manufacturer’s
instructions. The concentration of the extracted DNA was
estimated using a microvolume spectrophotometer Nan-
oDrop® ND-1000 (Thermo Fisher Scientific, Wilmington,
DE). Molecular characterization was based on partial frag-
ments of 28S rDNA and cox1 mtDNA genes. PCR reactions
were performed using Platinum Hot Start PCR Master Mix
(Thermo Fisher Scientific Inc., Massachusetts, São Paulo,
Brazil). For 28S, primers dig12 and 1500R (Tkach et al.
2003) were used with annealing temperature of 56°C; for
cox1, primers JB3 and CO1 R-trema (Bowles et al. 1993;
Miura et al. 2005) were used with annealing temperature
of 45°C. All products were visualized on a 1% agarose gel,
stained with UniSafe Dye 20,000 × (Uniscience, São Paulo,
Brazil). Amplicons were purified using 20% polyethylene
glycol (PEG 8000, Promega, Wisconsin, USA), resuspended
in 20 μL of ultrapure water and dosed as described for
extracted DNA. PCR products were sequenced in both direc-
tions utilizing the PCR primers in an ABI 3730 sequencer
using the BigDye Terminator Cycle Sequencing kit 3.1
(Applied Biosystems, California, USA).
935
Consensus sequences were assembled using ChromasPro
version 2.0.1 (Technelysium Pty Ltd, Tewantin, Australia)
and submitted to Basic Alignment Tool (BLAST) for homol-
ogy search. Newly generated sequences and those retrieved
from GenBank (Supplementary Table 1) were aligned using
Clustal W implemented in MEGA7 (Kumar et al. 2016).
Two members of the family Cyclocoelidae, Morishitium
polonicum malayense Urabe, Nor Hashim & Uni, 2020, and
Typhlocoelum cucumerinum (Rudolphi, 1809), were chosen
as outgroups according to previously published broader phy-
logeny (Tkach et al. 2016). The alignments were trimmed
to the length of the shortest sequence, which resulted in
alignments 903-bp long for 28S and 774-bp long for cox1.
Phylogenetic analyses were run using Bayesian inference
(BI) and maximum likelihood (ML) algorithms. The best
nucleotide substitution models were estimated based on
the Bayesian information criterion in MEGA7. The general
time-reversible model with gamma distribution (GTR +
G) was selected for 28S and the Hasegawa-Kishino-Yano
incorporating gamma-distributed among-site variation
(HKY + G) for cox1. The BI analysis was conducted using
Mr. Bayes v.3.2.6 (Ronquist et al. 2012), running two inde-
pendent Markov Chain Monte Carlo runs of four chains
settings for 1,000,000 generations with every 100th tree
saved. The first 25% of the trees sampled were discarded
as “burn-in,” and consensus topology and posterior prob-
ability values were calculated from the remaining trees and
visualized in FigTree v.1.4.3 (http://​tree.​bio.​ed.​ac.​uk/​softw​
are/fi
​ gtre​ e/). ML analysis was performed using MEGA7 and
nodal supports were inferred based on 1000 bootstrap repli-
cates. Genetic distances (uncorrected p-distance) were esti-
mated with MEGA7 from the alignment. Newly generated
sequences were deposited in GenBank (28S: OM273471;
cox1: OM273172,- OM273173).
Results
From 3879 specimens of Aylacostoma spp. collected, 1/1157
(0.09%) of Aylacostoma chloroticum and 1/2722 (0.04%) of
A. tuberculatum were infected with megalurous cercariae,
characteristic of the family Philophthalmidae Looss, 1899.
In the experimental study, 20 (left eye = 14; right eye = 6)
and 5 (left eye = 2; right eye =3) adult parasites were recov-
ered from each chick at 36 and 45 days post-infection (dpi),
respectively. Measurements and morphological description
of the adult stage were based on fully mature (ovigerous)
worms.
Philophthalmus aylacostoma n. sp. (Figs. 1, 2, 3)
Description of developmental stages
Rediae (Fig. 1a)
Based on fixed specimens from A. chloroticum (n=20):
body elongated, 1385 (1220–1602) × 302 (255–355);
13
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pharynx small, 59 (54–71) × 59 (52–71). Caecum long,
47–57% of body length. Lateral pair of locomotory append-
ages present in posterior region.
Cercariae from A. tuberculatum (Fig. 1b, c)
Based on fixed larvae (n=8): body elongated, 374
(317–415) × 118 (107–125), with slight constriction at level
of ventral sucker; tegument armed with minute spines. Tail
simple, 376 (353–406) × 39 (36–43), with adhesive glands at
terminal portion. Oral sucker subterminal, 61 (53–67) × 59
(56–63), followed by short prepharynx. Pharynx muscular,
32 (28–34) × 20 (17–23). Esophagus long with bifurcation
anterior to ventral sucker. Intestinal caeca long, terminating
near excretory vesicle. Ventral sucker slightly post-equato-
rial, 70 (66–73) × 75 (67–80).
Cercariae from A. chloroticum
Based on fixed larvae (n=4): larvae with the same char-
acteristics as mentioned above but with slightly more elon-
gated body, 459 (440–475) × 96 (88–100). Oral sucker 69
(66–71) × 54 (52–55). Pharynx 36 (32–38) × 20 (32–38
× 18–21).; Vventral sucker 71 (68–75) × 75. Tail, 372
(340–404) × 37 (34–41).
Metacercariae (Fig. 1c)
Based on live encysted (n=4) and formalin-fixed excysted
(n=20) metacercariae, obtained from cercariae emerged
from A. chloroticum: cyst pyriform, with thin wall, 336
(310–366) × 196 (188–205). Morphology of metacercar-
iae is identical to that of cercarial body without tail. Body
covered with spines, 462 (408–528) × 133 (113–148). Oral
Fig. 1  Philophthalmus aylacostoma n. sp. found in Aylacostoma spp. from Brazil: a redia; b whole cercaria; c detail of the cercarial body; d
encysted metacercaria
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sucker 64 (54–69) × 56 (52–67). Pharynx 34 (29–40) × 23
(19–28). Ventral sucker 70 (52–78) × 74 (69–81).
Adult (Figs. 2, 3)
Based on mature ovigerous worms obtained at 45 dpi
(n=5) (Fig. 2): body elongated, 4405 (3823–4405) ×
1453 (1312–1453) with a constriction at level of ventral
sucker, attenuated anteriorly, with rounded anterior and
posterior ends. Maximum width always posterior to ven-
tral sucker. Tegument smooth. Oral sucker subterminal,
298 (284–312) ×312 (312–390), followed by short pre-
pharynx 17 (12–22). Pharynx muscular, 262 (248–262) ×
277 (262–291). Oral sucker to pharynx width ratio 1:0.9
(1:0.7 –0.9). Esophagus very short, 36 (29–100), bifurcat-
ing just posteriorly to pharynx. Caeca extending to near
posterior margin of posterior testis. Ventral sucker larger
than oral sucker, pre-equatorial, 610 (567–610) × 581
(560–588). Sucker width ratio 1:1.9 (1:1.4–1.9). Testes
in tandem, oval, entire or slightly lobate. Anterior tes-
tis 496 (425–553) × 553 (532–695); posterior testis 553
(489–610) × 531 (532–638). Cirrus sac elongated, 879
(759–1064) × 150 (118–164) (Fig. 3c), corresponding to
20% (18–26%) of body length, in most cases not surpass-
ing posterior margin of ventral sucker (exception for one
specimen). Cirrus with minute spines. Seminal vesicle
internal to cirrus sac, elongated, sausage-shaped. Genital
pore median, at level of caecal bifurcation. Ovary oval,
213 (191–255) × 284 (255–291), pre-testicular. Uterus
long, coiled, occupying the space between ventral sucker
Parasitology Research (2022) 121:933–944
Fig. 2  Line drawing of Philophthalmus aylacostoma n. sp., obtained
experimentally at day 45 pi. (holotype), ventral view
and level of anterior margin of posterior testis. Metraterm
developed, alongside the cirrus sac. Seminal receptacle
oval to elongated. Vitellarium tubular (Fig. 3d), bilaterally
extracecal, occupying 73% (72 to 89%) of length between
ventral sucker and anterior testis. Excretory pore terminal.
937
Excretory vesicle Y-shaped. The main excretory ducts
extend to level of pharynx.
Based on mature ovigerous worms obtained at 36 dpi
(n=10) (Fig. 3a, b): morphologically similar to the described
above, except for the smaller size of the body and most
organs (Table 1). Likewise, the sucker ratio and the size of
mature eggs among these samples overlap.
Eggs (Fig. 3e)
Based on eggs fixed after being shed by live worms
(n=10): oval-shaped, thin-walled, 108 (93–125) × 47
(43–55), embryonated, non-operculated.
Miracidia (Fig. 3f)
Based on formalin-fixed specimens (n=23): body oval,
158 (148–168) × 64 (57–75), ciliated, with a pigmented
eyespot, 20 (16–23) × 15 (11–21) and a developed mother
redia inside.
Taxonomic summary
Experimental definitive host: Gallus gallus domesticus
Site of infection: nictitating membrane and conjunctival
sacs
Etymology: the species is named after the genus of its
first intermediate hosts.
First intermediate host: Aylacostoma chloroticum
(CHIOC 11749) and Aylacostoma tuberculatum (CHIOC
11750) (Thiaridae)
Type locality: São Francisco River, Januária City, Minas
Gerais, Brazil.
Other locality: Garças lagoon, upper Paraná River, Batay-
porã City, state of Mato Grosso do Sul, Brazil (Onaca et al.
2020).
Z o o B a n k r e g i s t r a t i o n : u r n : l s i d : z o o b a ​ n k .​
org:pub:FBDD68DA-BD9C-400A-91D5-BADA9506CF56
Deposited specimens: Holotype: CHIOC 39720; Para-
types: CHIOC 39721-39722, UFMG-TRE 122-123.
Remarks The morphological characteristics and site of
infection of P. aylacostoma n. sp. conform with the diag-
nosis of genus Philophthalmus Looss, 1899 (Kanev et al.
2005; Dronen and Fried 2008). Morphometric comparison
of the species reported herein with other freshwater species
(P. gralli, P. megalurus) or species with yet unknown life
cycle (P. lachrymosus, P. semipalmatus) reported from the
Americas is provided in Table 1. The new species described
herein shares the type of vitellarium (tubular) with P. gralli
but differs from the latter species by a slightly higher aver-
age sucker ratio and larger eggs (Muniz-Pereira and Amato
1993; Pinto and Melo 2010). Philophthalmus lachrymosus
further differs from the new species in having follicular
more compact vitellarium, and the pharynx width equal or
slightly larger than that of the oral sucker (Freitas 1955;
Nasir and Díaz 1972). Philophtalmus aylacostoma n. sp. can
be differentiated from another species described from South
America, P. semipalmatus (Nasir and Díaz 1972), by having
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Fig. 3  Philophthalmus aylacostoma n. sp.: living worms recov-
ered from experimentally infected chicken 36 days post-infection, a
in vivo, b stained; c detail of the posterior part of the cirrus sac and
a smaller ventral sucker and larger eggs. Finally, the spe-
cies described here can be distinguished from P. megalurus
by the smaller body and larger eggs (West 1961; McMillan
1971).
Morphometric data of the cercarial stage of the new spe-
cies along with other Philophthalmus spp. reported from
the Americas are provided in Table 2. The larvae of P. ayla-
costoma n. sp. differ from Philophthalmus sp. reported in
the Paraná River (Onaca et al. 2020), by having a narrower
body and longer tail. The cercariae of the invasive species,
P. gralli, in turn, differ from the larvae described herein in
having a larger body and longer tail (Díaz et al. 2002; Pinto
and Melo 2010). The cercariae of P. megalurus can be read-
ily distinguished from the cercariae of the new species by the
larger body and tail (West 1961; McMillan 1971). Admit-
tedly, the comparison of cercarial measurements is usually
greatly affected by the mode of fixation and/or use of fixed
vs live cercariae for measurements. Moreover, P. megalurus
is transmitted by snails of the families Pleuroceridae Fischer,
1885 (Goniobasis virginica (Gmelin, 1791) and Pleurocera
acuta Rafinesque, 1831) and Semisulcospiridae (Juga plic-
ifera (Lea, 1838)) (West 1961; McMillan 1971; McMillan
and Macy 1972; Huffman and Fried 1983).
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seminal vesicle (arrow); d detail of the tubular vitellaria (arrow); e
freshly laid egg; f hatched miracidium containing a developed redia
inside.
Molecular study
Partial sequences of 28S (1143 bp) were successfully gen-
erated for cercariae of P. aylacostoma n. sp. emerged from
A. tuberculatum. For this molecular marker, phylogenetic
analyses inferred by BI and ML yielded trees with iden-
tical topologies, showing P. aylacostoma n. sp. nested
in a strongly supported clade with other members of the
genus Philophthalmus with available sequences (Fig. 4a).
Philophthalmus aylacostoma n. sp. appears as a sister taxon
to a clade P. lucipetus + (Philophthalmus sp. + P. lach-
rymosus). The interspecific divergence in 28S gene among
species of Philophthalmus was 0.7−1.7%, while the inter-
generic distances between Philophthalmus and species of
other philophthalmid genera, Parorchis Nicoll, 1907, and
Cloacitrema Yamaguti, 1935, range from 5.9 to 6.9% and
3.9 to 5%, respectively.
Sequences of cox1 obtained for cercariae isolated from A.
chloroticum (784 bp) and A. tuberculatum (793 bp) showed
a divergence of 2.1%, thus supporting the conspecificity
between cercariae found in the two species of thiarid mol-
luscs. Compared with data available for Philophthalmus
spp. in the GenBank, our sample turned out to be very close
 Table 1  Morphometric comparison of Philophthalmus aylacostoma n. sp. with other species of the genus reported in the Americas. The means obtained for the type series, in the respective time
of infection (36 and 45 days post-infection), are followed by the standard deviation and range in parenthesis. d, diameter
Philophthalmus aylacostoma n. sp. P. gralli P. lachrymosus P. megalurus P. semipalmatus

Reference Present study Muniz-Pereira Pinto and Melo Freitas 1955 Nasir and Díaz West 1961 McMillan 1971 Nasir and Díaz
and Amato 2010 1972 1972
1993
Locality Brazil Brazil Brazil Brazil Venezuela USA USA Venezuela
Parasitology Research (2022) 121:933–944

Specimens n 5 (45 dpi) 10 (36 dpi) 9 13 4 1 − 4 −

Body L 4114 (3823– 2860 2710 3610 4190−4620 4000 3420−8360 4800−6000 2624−4475
4405) (2257−3404) (2120−3710) (3100−4070)
W 1392 759 (631−950) 834 (604−1280) 1020 (860−1210) 1380−1640 1500 670−2070 1530−2000 960−1794
(1312−1453)
Oral sucker (OS) L 301 (284−312) 244 (206−277) 260 (204−329) 330 (316−339) 300−310 416d 200−370 280−370 216−363
W 366 (312−390) 277 (234−319) 332 (277−421) 401 (374−421) 360−430 − 210−470 300−470 253−485
Pharynx (PHX) L 255 (248−262) 170 (142−191) 226 (183−293) 299 (279−316) 310−350 543d 190−350 290−330 225−333d
W 274 (262−291) 194 (163−213) 277 (183−403) 339 (326−358) 380−460 − 170−310 270−370 −
Ventral sucker L 598 (567−610) 486 (425−546) 418 (343−549) 514 (479−542) 610−690d 782d 430−700d 480−670d 889
(VS) W 576 (560−588) 448 (404−496) 391 (343−494) 514 (479−542) − − − − 944
Ovary L 217 (191−255) 130 (99−156) 153 (88−219) 237 (211−258) 200−210 400d 120−370d 220−320 188−394d
W 274 (255−291) 167 (121−213) 181 (88−256) 266 (245−300) 180−220 − − 280−300 −
Anterior testis L 492 (425−553) 291 (184−347) 258 (161−416) 222 (179−263) 360−480 347d 270−1100 370−600 188−297
W 594 (532−695) 387 (305−432) 329 (241−504) 403 (279−453) 460−550 − 250−870 630-740 206−563
Posterior testis L 545 (489−610) 320 (262−440) 248 (161−438) 216 (174−247) 380−480 347d 250−1010 380−590 188−454
W 580 (532−638) 372 (305−468) 319 (241−504) 351 (268−421) 450−530 − 210−890 480−830 216−669
Eggs L 108 (104−114) 106 (100−114) 70 (64−90) 73 (60−87) 94−97 − 80 − 60−69
W 37 (36−38) 38 (36−40) Collapsed 32 (27−36) 38−42 − 40 − 18−30
OS/VS 1:1.6 (1.4−1.9) 1:1.6 (1.5−1.8) 1:1.1−1.3 1:1.2 (1−1.5) 1:1.5−1.7* 1:1.9 1:1.4* 1:1.6−1.8 1:2.2*
OS/PHX 1:0.8 (0.7−0.9) 1:0.7 1:0.6−1 1:0.9 (0.5−1.1) 1:1−1.1* 1:1.3* 1:0.7* 1:1.1* 1:0.7*
Vitellaria % 82 (72−89) 74 (61−84) 79−89 87 (73−87) 45 (37−54)* 41 & 54* 92 & 84* 85%* 63 and 74*
Type of vitellaria Tubular Tubular Tubular Tubular Follicular Follicular Tubular to fol- Tubular to fol- −
licular licular

*Values calculated from illustrations of the respective studies

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940 Parasitology Research (2022) 121:933–944

Table 2  Measurements of cercariae of Philophthalmus aylacostoma n. sp. and other species reported in freshwater snails in the Americas
Philophthalmus aylacostoma Philophthalmus P. gralli P. megalurus
n. sp. sp.

Study Present study Onaca et al. Díaz et al. Pinto and Melo West (1961) McMillan (1971)
(2020) (2002) (2010)
Locality Brazil Brazil Venezuela Brazil USA USA
Intermediate hosts Aylacostoma A. tubercula- A. chloroticum Melanoides M. tuberculata Goniobasis sp., Juga plicifera
chloroticum tum tuberculata Pleurocera
acuta
Specimens n 4 8 7 − − − −
Body L 459 (440−475) 374 (317−415) 487 (420−553) 456−598 535 (420−580) 540−670 550 (470−790)
W 96 (88−100) 118 (107−125) 157 (145−171) 101−162 128 (110−140) 120−130 120 (110−120)
Oral sucker L 69 (66−71) 61 (53−67) − 55−64 58 (50-69) 60 50 (50−60)
W 54 (52−55) 59 (56−63) − 51−61 55 (49-65) 40−50 40 (30−60)
Pharynx L 36 (32−38) 32 (28−34) − 26−40 − 30−40 30
W 20 (18−21) 20 (17−23) − 18−28 − 10−20 20
Ventral sucker L 71 (68−75) 70 (66−73) − − 68 (65−78) 60−70d 60 (50−70)d
W 75 75 (67−80) − − 75 (60−80) − −
Tail L 372 (340−404) 376 (353−406) 259 (206−318) 294−446 434 (302−485) 930−1140 630 (470−890)
W 37 (34−41) 39 (36−43) 46 (43−53) 30−50 54 (36−62) 40−50 20 (10−20)

genetically to the larvae of Philophthalmus sp. found in A.
chloroticum from Paraná River (Onaca et al. 2020), with the
divergence of 0.16−2.27%. Moreover, those isolates formed
a highly supported clade with P. aylacostoma n. sp. in the
phylogenetic trees generated by both BI and ML (Fig. 4b).
Thus, we consider the form found in the upper Paraná River,
Mato Grosso do Sul, to be conspecific with P. aylacostoma
n. sp. The new species from Brazil appeared on the tree as
a sister taxon to P. gralli; the genetic divergence in cox1
between sequences of these two species was 11.97−12.29%.
The interspecific divergence in sequences of the same mito-
chondrial gene among all Philophthalmus spp. varied from
11.97 to 14.72%. The genetic divergence in cox1 between the
new species and Parorchis spp. was 16.50–16.99%.
Discussion
Philophthalmus aylacostoma n. sp. is described here based
on morphological characteristics of larval stages found in
two species of Aylacostoma Spix, 1827, from Brazil and
adult parasites obtained experimentally. The adult stage was
distinguished from closely related species described in the
Americas by a few morphometric characters (sucker ratio,
width ratio of oral sucker to pharynx, and size of eggs).
The new species is morphologically closest to P. megalurus;
however, the cercarial stages of these eye flukes are clearly
different, especially by the larger tail of the North Ameri-
can species (West 1961; McMillan 1971). Regarding the
size of the egg, Dronen and Fried (2008) concluded through
an experimental study that the length of eggs is a helpful
characteristic to differentiate older specimens of P. ander-
soni and P. larsoni. Our findings reveal that the eggs shed
by adult worms (fixed in formalin) and those from mounted
specimens had, on average, the same length but with slight
variation in width (43–55 vs. 36–38). This may have been
due to eggs from mounted parasites being shrunk as the
result of the dehydration inherent to the staining process.
Despite the difference in size between specimens of the new
species collected at 36 dpi (smaller) vs. 45 dpi (larger), the
eggs did not show significant difference in size, or level of
development, between the two samples. Our observations
concur with the results of Nollen (1983), who showed that
crowding did not affect the larval development in intrauter-
ine eggs of P. gralli. In the new species described herein, the
sucker ratio and the oral sucker width to pharynx width ratio
were consistent in specimens of different sizes collected at
two different times post-infection, which suggests that these
characteristics can be used for species differentiation despite
the existing age variability. Interestingly, the width of the
pharynx of the new species never exceeded the width of oral
sucker, while in P. lachrymosus, the pharynx width is equal
or slightly greater than that of oral sucker (Freitas 1955;
Lamothe-Argumedo et al. 2003).
The description of a new species of Philophthalmus
based on experimentally obtained specimens, as in the pre-
sent study, is not uncommon and previously occurred with
other species of Philophthalmus, e.g., North American P.
andersoni, P. hegeneri., P. larsoni, and P. megalurus. Natu-
ral avian definitive hosts were also reported for all these spe-
cies, either at the time of the original descriptions or later, as
in the case of P. megalurus (Penner and Fried 1963; Krygier

13
Parasitology Research (2022) 121:933–944 941

Fig. 4  Consensus trees from

phylogenetic analyses for
members of the family Philoph-
thalmidae based on 28S (a) and
cox1 (b). Numbers in the nodes
represent posterior probabilities
and bootstrap values; values
lower than 0.90 (Bayesian infer-
ence) and 70 (maximum likeli-
hood) are not shown. Differ-
ences of the topology obtained
by ML analysis are indicated
with an asterisk instead of a
bootstrap value. The scale bar
indicates the expected number
of substitutions per site. Taxa in
bold were newly sequenced in
this study

and Macy 1969; Dronen and Fried 2008). There is always
a question whether the possible host-induced morphologi-
cal variation may potentially result in differences between
naturally and experimentally obtained parasites. Although
this possibility cannot be completely ruled out for P. ayla-
costoma n. sp., a host-induced variation, resulting in signifi-
cant changes in organ size and ratios, or the type of vitel-
larium, was not previously reported for Philophthalmus spp.
In fact, consistency in morphology of specimens obtained
from natural and experimental infections has been reported
for some species of Philophthalmus (Penner and Fried 1963;
Radev et al. 1999; Dronen and Fried 2008; Pinto and Melo
2010). Therefore, we are confident that the differentiating
characters observed in the type series of our new species are
valid and will be confirmed once a natural definitive host of
this species is found.
Based on cox1 DNA, two genotypes of P. aylacostoma n.
sp. were detected infecting A. chloroticum and A. tubercu-
latum. By comparison with data available in GenBank, the
isolate from A. tuberculatum almost exactly matched the
sequences obtained by Onaca et al. (2020) from cercariae
collected from A. chloroticum in Paraná River, located about
1500 km from the type locality of P. aylacostoma n. sp. The
28S rDNA was also sequenced in that study, but the frag-
ments were smaller (524 bp), which prevented their inclu-
sion in our phylogenetic analyses. Nevertheless, the 28S
sequences published by Onaca et al. (2020) were identical
to the one obtained in the present study. Morphologically,
the cercariae of that isolate had a slightly larger body and a
shorter tail when compared with those of the new species.
This could be attributed to the fact that Onaca et al. (2020)
measured larvae collected from the digestive gland of the

13
942
snail, where they are found at different stages of develop-
ment, while in the present study, measures were taken from
fully developed, heat-killed, formalin-fixed specimens.
Additionally, the molecular data were useful to distin-
guish the new species from P. gralli and P. lachrymosus,
the only species previously reported in Brazil (Freitas 1955;
Muniz-Pereira and Amato 1993; Pinto and Melo 2010). The
first species also has a freshwater life cycle and is broadly
distributed in the Americas, being transmitted by the inva-
sive thiarid M. tuberculata (Chalkowski et al. 2021). This
snail is widely distributed in Brazil, including the type local-
ity of P. aylacostoma n. sp. (Coelho et al. 2018), and the
possibility of occurrence of these eye flukes in sympatry
cannot be ruled out. In relation to P. lachrymosus, currently
available molecular data were generated from specimens
obtained from gulls in Portugal (Heneberg et al. 2018).
Sequence data from specimens collected in Brazil is nec-
essary to confirm the broad, transcontinental distribution
of P. lachrymosus. Unfortunately, only cox1 sequences are
currently available for a single marine species, P. attenuatus
(Bennett and Presswell 2019).
This study reports the fifth digenean species transmitted
by A. chloroticum that had its life cycle elucidated experi-
mentally in South America (Ostrowski de Núñez and Quin-
tana 2008; Quintana and Ostrowski de Núñez 2014, 2016;
Ostrowski de Núñez et al. 2020). This mollusc species is
listed as vulnerable in Argentina due to the dramatic changes
in its natural environment (Vogler 2012). Thus, the effects of
the changes in the populations of these thiarids on parasite
ecology, and even parasite coextinction, cannot be ruled out.
In Brazil, a molecular approach was used in parasitological
examinations of A. chloroticum, resulting in reporting of
three cercarial types, including the new species presented
herein. Noteworthy, according to Simone (2006), 32 spe-
cies of Aylacostoma are known in Brazil. Considering this
diversity combined with our data confirming P. aylacostoma
n. sp. in different species of Aylacostoma from different
states, we anticipate that other members of this large thiarid
genus may participate in life cycles of other philophthalmid
digeneans.
A rich avifauna has been documented along the São
Francisco River (Schunck et al. 2012). Therefore, future
helminthological studies of birds in the region are required
to reveal the natural definitive host of the new species and
fill the gap in our knowledge of its biology. Although the
prevalence of infected snails in our study was very low, we
nevertheless consider the potential for zoonotic human infec-
tions, especially because the local population heavily uses
the studied stretch of the São Francisco River for recreation
such as swimming and fishing.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00436-0​ 22-0​ 7447-1.
13
Parasitology Research (2022) 121:933–944
Acknowledgements We thank the members of the Center of Advanced
Treatment and Research on Tegumentary Leishmaniasis, Januária City,
especially Jailton Xavier and Prof. Stefan Geiger, for the laboratory
infrastructure facilities used during the malacological surveys. We are
indebted to Dr. Monica Fernandez and Dr. Silvana Thiengo (Instituto
Oswaldo Cruz, Rio de Janeiro), and Dr Luiz Simone (Universidade de
São Paulo) for their help in the taxonomic identification of molluscs.
This work was also supported by National Council for Scientific and
Technological Development (CNPq), Brazil (research scholarship to
HAP and doctoral scholarship to EAPM), and U. S. National Science
Foundation grants DEB-1120734 to VVT.
Declarations
Ethics approval Applicable national and/or institutional guidelines for
the care and use of animals were followed in all procedures performed.
Conflict of interest The authors declare no competing interests.
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