ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043
ORIGINAL ARTICLE / ARTÍCULO ORIGINAL
MITOCHONDRIAL DNA AND MORPHOLOGY DATA OF HELMINTHOXYS FREITASI QUENTIN,
1969 REVEALS ITS PHYLOGENETIC RELATIONSHIPS IN THE TRIBE PROTOZOOPHAGINI
LOS DATOS DEL ADN MITOCONDRIAL Y DE LA MORFOLOGÍA DE HELMINTHOXYS FREITASI
QUENTIN, 1969 REVELAN SUS RELACIONES FILOGENÉTICAS EN LA TRIBU
PROTOZOOPHAGINI
1Programa de Pós-graduação em Biologia Parasitária, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz.
2Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz, Fundação Oswaldo
Cruz, Av. Brasil 4365, Rio de Janeiro, RJ, 21040-360, Brasil.
3Instituto Federal do Acre, Rio Branco, Acre, Brazil.
4Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz.
*Corresponding author: maldonad@ioc.ocruz.br
1,2 2* 3
Beatriz Elise Andrade-Silva ; Arnaldo Maldonado Junior ; ; Charle Crisóstomo
4 2
Alena Mayo Iñiguez & Roberto Val Vilela
ABSTRACT
Keywords: Cytochrome c subunit I – Integrative Taxonomy – Mesomys hispidus – Scanning Electron Microscopy – Syphaciinae
Nematodes from the genus Helminthoxys Freitas, Lent & Almeida, 1937 are intestinal parasites of
caviomorph rodents with a wide Neotropical distribution. This study detailed the morphology of
Helminthoxys freitasi Quentin, 1969 using light microscopy and scanning electron microscopy (SEM),
and inferred a phylogeny for the tribe Protozoophagini using partial mitochondrial cytochrome c oxidase
subunit I gene sequences (MT-CO1). Rodents Mesomys hispidus (Desmarest, 1817) were collect in three
distinct areas in the state of Acre, Brazil. The helminths were recovered and morphology of their surfaces,
such as lateral alae reaching the level of the anus, the posterior region of the body in the male having three
pair of sessile papillae and one pair papillae pedunculated were detailed. Genetic sequence of H. freitasi
suggested a close relationship with the genus Wellcomia Sambon, 1907, corroborating a previous
morphological phylogeny. A new host species, M. hispidus, and a new locality in the Amazon rainforest is
recorded.
Neotropical Helminthology
135
Neotropical Helminthology, 2019, 13(2), jul-dic:135-149.
ÓrganooficialdelaAsociaciónPeruanadeHelmintologíaeInvertebradosAfines(APHIA)
Lima-Perú
VersiónImpresa:ISSN2218-6425VersiónElectrónica:ISSN1995-1043
Volume13,Number2(jul-dec2019)
INTRODUCTION
136
RESUMEN
Palabras clave: Subunidad I del citocromo C – Taxonomía Integrativa – Mesomys hispidus – Microscopía Electrónica de Barrido – Syphaciinae
Los nematodos del género Helminthoxys Freitas, Lent & Almeida, 1937 son parásitos intestinales de
roedores caviomorfos con una amplia distribución neotropical. Este estudio detalló la morfología de
Helminthoxys freitasi Quentin, 1969 por microscopía óptica y microscopía electrónica de barrido (MEB),
e infirió una filogenia para la tribu Protozoophagini con las secuencias del gene parcial Citocromo c
Oxidasa subunidade 1 (MT-CO1). Los roedores Mesomys hispidus (Desmarest, 1817) fueron colectados
en tres áreas distintas en el estado de Acre, Brasil. Los helmintos encuentrados observaron alas laterales
que alcanzan el nivel del ano, la región posterior del cuerpo en el hombre con tres pares de papilas sésiles y
un par de papilas pedunculadas. La secuencia COI de H. freitasi revelan una estrecha relación con el
género Wellcomia Sambon, 1907, corroborando con la filogenia morfológica anterior. Además,
reportamos una nueva especie huésped, M. hispidus, con una nueva localidad en la selva amazónica.
composed of three genera which include the
following: Helminthoxys Freitas, Lent & Almeida,
1937; Wel lc o m i a Sambon, 19 07; and
Protozoophaga Travassos, 1923 (Hugot, 1988).
Later studies based on molecular phylogenetic
inference have confirmed the evolutionary
relationship between Wellcomia Sambon, 1907 and
Protozoophaga Travassos, 1923 (Nadler et al.,
2007), but not included Helminthoxys Freitas,
Almeida & Lent, 1937. Nevertheless, so far, no
molecular phylogeny has included molecular
sequence Helminthoxys and representatives of the
sister Syphaciini tribe.
This study detailed the morphology of
Helminthoxys freitasi Quentin, 1969 by light
microscopy and scanning electron microscopy
(SEM), adding further taxonomic characteristics
for species and inferred a phylogeny for the tribe
Protozoophagini using partial mitochondrial
cytochrome c oxidase sub unit I gene (MT-CO1). In
addition, both a new host species and new
geographic locality were recorded.
Collection sites
This study was developed in three distinct areas
within the Amazon rainforest in the state of Acre, in
t h e m u n i c i p a l i t i e s o f P o r t o A c r e
(9°54'17.70"S;67°17'8.01"W), Senador Guiomard
(10°09'39.0"S;67°44'17.6"W), and Xapuri
(10°49'40.79"S; 68°21'38.89"W). Rodents were
The genus Helminthoxys Freitas, Almeida and
Lent, 1937 currently comprises eight species. The
type species Helminthoxys caudatus (syn. H. pujoli
Quentin, 1973) was first described infecting the
rodent Microcavia australis in Argentina (Freitas-
Texeira et al., 1937). Subsequently other species
were described: H. tiflophila Vigueras, 1943 in
Mysateles prehensilis (Vigueras, 1943); H.
effilatus Schuurmans-Stekhoven, 1951 (syn. H.
velizi Parra Ormeño, 1953) in Lagidium viscacia
(Schuurmans-Stekhoven, 1951); H. urichi
Cameron & Reesal, 1951 in Dasyprocta leporina
(Cameron & Reesal, 1951); H. quentini Barus,
1972 in Capromys pillorides (Barus, 1972); H.
gigantea Quentin, Courtin & Fontecilla 1975 in
Octodon degus (Quentin, Courtin & Fontecilla
1975); H. freitasi Quentin, 1969 in Thrichomys
laurentius (syn. Thrichomys apereoides) (Quentin,
1969); and H. abrocomae Hugot & Gardner, 2000
in Abrocoma cinerea (Hugot & Gardner, 2000).
These nematodes inhabit the large intestine of
caviomorph rodents of seven different families,
which include the following: Caviidea,
Capromyidae, Chinchilidae, Dasyproctidae,
Echimyidae, Octodontidae, and Abrocomidae
(Hugot, 1988).
Studies on the morphologic phylogeny of the order
Oxyurida based on morphologic characteres of the
reproductive structures of the male and cephalic
plate, proposed that the tribe Protozoophagini is
MATERIALS AND METHODS
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
137
trapped using Tomahawk (model 201, Hazelhurst,
Wisconsin) and Sherman (model XLK, H.B.
Sherman Traps, Tallahassee, Florida) live traps. To
capture arboreal mammals, traps were tied to tree
branches placed in the forest understory. Captures
occurred during five consecutive nights in 2014,
2015, and 2016. Euthanasia followed the
guidelines of the American Society of
Mammologists for the use of wild mammals in
research and the Brazilian Guide to Good Practices
for Euthanasia in Animals (Sikes, 2016). Permits
for rodent capture and handling were issued by the
Instituto Chico Mendes de Conservação da
Biodiversidade (ICMBio), and experimental
procedures on animals were approved by the Ethics
Committee on Animal Use (CEUA) of the Instituto
Oswaldo Cruz.
Helminths collection
After collection, worms were washed in saline,
sodium chloride solution (NaCl 0.9%) and
maintained in 70% ethanol solution. For
examination under light microscopy, nematodes
were clarified in lactophenol 90% and drawings
were produced with aid of a Camera Lucida,
attached to a Zeiss Scope Z1 light microscope
(Zeiss, Göttingen, Germany). All measurements
were in micrometers. The structures were
measured through digital images captured by a
Zeiss Axio Cam HRC (Zeiss, Germany) using the
accessory software Axio Vision Rel. 4.7 (2009).
For SEM analyses, six specimens (three males and
three females) of post-fixed helminths were
dehydrated in increasing ethanolic series (70%,
80%, 90%, and absolute ethanol), for 20 min at
each stage, and dried by the critical point method
with CO (Souza et al., 2017). The samples were
2
then submitted to gold metallization with layer
thickness of approximately 20nm. The specimens
were then analyzed in a SEM JEOL JSM6390LV at
the Plataforma de Microscopia Eletrônica Rudolf
Barth, Instituto Oswaldo Cruz, Fiocruz (Electron
Microscopy Platform of the Oswaldo Cruz
Institute).
A paratype of H. freitasi from the Coleção
Helmintológica do Instituto Oswaldo Cruz –
CHIOC (Nº 30936) was used to compare
morphological characteristics. Voucher specimens
were deposited in CHIOC under the following
number: CHIOC 38502.
Molecular and phylogenetic analyses
Genomic DNA was isolated from three individual
pinworms from the Senador Guiomard and Porto
Acre localities using the QIAamp DNA Mini Kit,
applying the manufacturer's protocol (QIAGEN,
Hilden, Germany).
DNA amplification by polymerase chain reaction
(PCR) was conducted using the following primers:
SyphaCO1_F (5'-ACCCGCTGAATTTAA-
GCAT-3') and SyphaCO1_R (5'-AACC-
ACCCAACGTAAACATAAA-3') to produce
amplicons of the mitochondrial cytochrome c
oxidase subunit I gene (MT-CO1) fragments
(Okamoto et al., 2007).
Each PCR contained 1x PCR buffer, 4 mM MgCl ,
2
0.2 µM of each primer, 0.2 mM of each
deoxynucleotide triphosphate solution (dNTPs),
1U of Platinum TaqDNA polymerase (Invitrogen,
São Paulo, Brazil), 2.0 µL of genomic DNA, and
ultrapure water, in a total reaction volume of 25 µL.
PCR-cycling parameters followed Okamoto et al.,
(2007). Resulting amplicons were visualized on
1.5% agarose gels after electrophoresis, using Gel
Red™ nucleic acid gel stains (Biotium, Hayward,
California, USA), and UV transilluminator.
Successfully amplified amplicons were purified
using the Illustra™ GFX™ PCR DNA and Gel
Band Purification Kit following the manufacturer's
protocol (GE Healthcare, Little Chalfont, UK).
Amplicons were cycle-sequenced using the Big
Dye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, USA) on both strands using
the PCR primers mentioned, resulting in
bidirectional sequencing for better data accuracy.
Sequencing was performed using the ABI3730
DNA Analyzer. Both procedures and cycle-
sequenced products precipitation were conducted
at the Plataforma de Sequenciamento de DNA do
Instituto Oswaldo Cruz, PDTIS/Fiocruz (DNA
Sequencing Platform of the Oswaldo Cruz
Institute). Fragments were assembled into contigs
and edited for ambiguities using the software
Geneious 9.1.8 (Kearse et al., 2012), resulting in
consensus sequences.
Our dataset included sequences from the closest
relatives of Helmintoxy genus, oxyurids of the
subfamily Syphaciinae. These oxyurids belong to
four genera each representing a different tribe as
foll o w s : P a s s a l u r u s Dujard i n , 1845;
Neotropical Helminthology, 2019, 13(2), jul-dic Mitochondrial DNA and morphology data of Helminthoxys
138
RESULTS
Rauschtineria Hugot, 1980; Syphacia Seurat,
1916, and Wellcomia Sambon, 1907. We also
included sequences of genera Enterobius Leach,
1853 and Lemuricola Chabaud et Petter, 1959 as a
representative of the oxyurid subfamily
Enterobiinae. The oxyuroid Aspiculuris tetraptera
Schulz, 1924, from the family Heteroxynematidae,
was included as outgroup. The subfamilies, tribes
of Syphaciinae, species, GenBank accession
numbers, and references of specimens used in this
study are listed in Table 1.
We aligned the MT-CO1 sequences using the
Translator X online software (Abascal et al., 2011).
Resulting alignments were trimmed of poorly
aligned regions using the Mesquite package
software (Maddison & Maddison, 2011).
Substitution saturation in the dataset was assessed
via the Test by Xia (Xia et al., 2003; Xia & Lemey,
2009) using the DAMBE program, Version 6.4.79
(Xia & Xie, 2001).
Phylogenetic reconstructions using maximum
likelihood (ML) were carried out using PhyML 3.0
software (Guindon et al., 2010). Nucleotide
evolutionary model selection was executed with
SMS (Smart Model Selection) (Lefort et al., 2017)
in PhyML, using the Bayesian information
criterion (BIC). Node support in ML trees was
assessed by the Approximate Likelihood-Ratio
Test for Branches (aLRT) (Anisimova & Gascuel,
2006) and by nonparametric bootstrap percentages
(ML-BP) after 1000 pseudoreplications. Bayesian
phylogenetic inference (BI) was carried out using
the MrBayes program, version 3.2.6 (Ronquist et
al., 2012) on XSEDE using the CIPRES Science
Gateway (Miller et al., 2010). To account for
different evolutionary processes at each of the three
codon positions, BI analyses were performed using
the GTR+G model for each codon position, with
unlinked base frequencies and parameters. Markov
chain Monte Carlo samplings were performed for
10,000,000 generations with four simultaneous
chains in two runs. The robustness of nodes was
assessed by Bayesian posterior probabilities (Bpp)
calculated from tree samples every 100
generations, after removal of a “burn-in” fraction
of 25%. To assess the adequacy of our sampling, we
used the Tracer v1.6 program (Rambaut et al.,
2014) to calculate the Effective Sample Sizes
(ESS) of parameters. Values above 1000
effectively independent samples were considered
sufficient. To assess the level of variation in the
(COI) among the selected samples of different
taxa, uncorrected (p) pairwise genetic distances
were calculated using PAUP* 4.0b10 software
(Swofford, 2002).
Ethical standards
License for animal capture was provided by the
Instituto Chico Mendes de Conservação da
Biodiversidade- ICMBio (permanent license
13373-1). All protocols followed the guidelines for
capture, hand ling and care of the Ethics
Committee on Animal Use of the Oswaldo Cruz
Institute (according to license L-049/08) (protocol
P-70/13-2; license LW-39/14).
Conflict of interest
The authors declare no conflict of interest.
Morphology by scanning electron and light
microscopy
Adult helminths exhibited sexual dimorphism. In
both sexes (Figures 1A and 3A) the anterior
extremity had three prominent pseudolabia, one
ventral and two dorsolateral, interspersed with
three strong conical esophageal teeth (Figure 3B),
which were intercalated with cuticularized
thickenings of the inner part of the pseudolabia and
the vestibule (Figure 1B). In the external part,
surrounded by a rough cuticular area located on
each dorsolateral pseudolabia, two labial papillae
were closely grouped laterally with corresponding
amphids (Figure 1C). Morphological analysis by
scanning electron microscopy showed the cuticular
expansions which formed the cervical alae extend
in lateral alae reaching the level of the anus, in the
light microscope only the cervical wing was clearly
seen (Figure 3A).
Males (Figures 1A and 2A) had two cuticular
mamelons protruding as cuticular expansions with
longitudinal ridges located in the posterior part of
the body (Figures 2C and 3F). There was 18 ventral
trimmings after the second mamelon in the form of
small longitudinal cuticular ridges (Figures 2B and
3E), one long spicule, gubernaculum, accessory
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
Figure 1. Helminthoxys freitasi A) Complete male view; B) Head, left lateral view; C) En face view of head; D) Ventral view of
caudal bursa showing one pair of sessile papillae and one pair of pedunculate papillae; E) Cross-section of the body at the level of
the cervical alae; F) Cross-section of the body at the level of the rst mamelon; G) Cross-section of the body at the level of the area
rugosa posterior to the second mamelon; H) Area rugosa posterior to the second mamelon, ventral view; I) Uterus didelphic and a
pair of spermatheca (arrow). Scale: A, E, F, G, H, I, 50µm; B, C, D, 10µm.
Neotropical Helminthology, 2019, 13(2), jul-dic Mitochondrial DNA and morphology data of Helminthoxys
139
Figure 2. Helminthoxys freitasi A) Adult male, general view; B) Bursal caudal, detail of the area rugosa posterior to second
mamelon (asterisk); C) First and second mamelon (arrow), lateral view; D) Detail, spicule (S) and gubernaculum (arrow), lateral
view; E) Detail, vulva (large arrow), lateral view; F-G) Eggs. Scale: A, 100µm; B, C, D, E, 50µm; F, 10µm.
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
140
hooks at the base of the cloacal opening, three pair
of sessile ad-cloacal papillae, and one pair of
pedunculate posterior papillae (Figures 1D, 1H,
2D, 3C, and 3D). Phasmids were located anteriorly
to the pedunculate pair of papillae.
In the females, the vulva was in the posterior part of
the body (Figure 2E). The uterus folded on itself,
opening in two oviducts with a pair of spermatheca
(Figure 1I). Eggs were asymmetrical and not
operculated (Figure 2F).
Morphometric data including all the species of the
genus Helminthoxys, from their original
descriptions were compared, emphasizing
distinctions of our specimen and added new data
with the measurements of eggs not previously
described (listed in Table 2).
Taxonomic Summary
Host: Mesomys hispidus (Desmarest, 1817)
(Rodentia: Echimyidae).
Site infection: large intestine
Locality: Municipalities of Porto Acre
(9°54'17.70"S; 67°17'8.01"W), Senador Guimard
(10º09'39.0''S;67º44'17.6''W), and Xapuri
(10°49'40.79"S; 68°21'38.89"W), State of Acre,
Brazil.
Mean intensity: 6.2 (31 specimens out of 5 host
infected)
Prevalence: 45.4 (5 host infected out of 11 host
examined)
Abundance: 2.81 (31 specimens out of 11 host
examined).
Specimens deposited: CHIOC Nº 38502
Molecular and phylogenetic analyses
We obtained consensus MT-CO1 sequences from
three adult Helminthoxys freitasi recovered from
two hosts from different localities. Two consensus
sequences were obtained from Porto Acre (A) and
one was obtained from Senador Guiomard (B). The
sequence from A were identical 975 bp whereas the
sequence from B had 954 bp and differed from A by
a single transition, representing two distinct MT-
CO1 haplotypes. Both sequences were deposited in
the GenBank database under accession numbers:
MH212135 and MH212136.
The resulting aligned matrix with GenBank
sequences comprised of 18 taxa (shown in Table 1)
and 819 characters, of which 482 characters were
constant, 118 variable characters were parsimony-
uninformative, and 201 were parsimony
informative. The test by Xia (Xia et al., 2003; Xia
& Lemey, 2009) provided evidence for substantial
saturation only at the third codon positions,
whereas at the first and second positions, and
overall there was little saturation in the matrix.
As the best-fit model, PhyML-SMS selected the
GTR+G model nucleotide substitution, with ML
optimized frequencies, estimated Gamma-shape
parameter (α=0.280), and four rate categories. The
best log-likelihood ML tree score was -
4469.546140.
For the BI, the mean estimated marginal likelihood
was -4077.7366 and the median was -4077.421.
ESSs for all parameters were above 1000
effectively independent samples and for most
parameters, indicating the robustness of our
sampling.
The pairwise uncorrected p-distances for
representatives of tribes of the subfamily
Syphaciinae and subfamily Enterobiinae are
summarized in Supplementary Table S1. Overall,
our matrix had pairwise genetic intraspecific p-
distances from 0.1% to Helminthoxys and to
Passarulus genera and 17.9% interspecific
distances between W. siamensis Nadler, 2007 and
E. vermicularis (Linneu, 1758) Leach, 1853 (mean
= 13.1%). The genetic distance between
Syphaciinae and Enterobiinae ranged from 11.6%
between R. eutamii (Tiner, 1948) and E. macaci
Yen, 1973, to 17.9% between P. ambiguus
Rudolphi, 1819 and E. vermicularis (mean =
14.5%).
The genetic distance between H. freitasi and W.
siamensis (i. e. within the tribe Protozoophagini)
ranged from 11.5–11.6% (mean=11.5%). The
distance between Protozoophagini and Hilgertini
ranged from 12.7% between H. freitasi and R.
eutamii, to 16.5% between W. siamensis and R.
eutamii (mean = 14.3%). The distances between
Protozoophagini and Syphacini ranged from
12.7% between H. freitasi and S. stroma, to 17.5%
between W. siamensis and S. agrarian (mean =
14.7%). The distances between Protozoophagini
and Passalurini ranged from 12.7% between H.
freitasi and P. ambiguus, to 15.3% between W.
siamensis and P. ambiguous (mean = 13.7%).
Neotropical Helminthology, 2019, 13(2), jul-dic Mitochondrial DNA and morphology data of Helminthoxys
141
ML and BI phylogenies resulted in similar
topologies with little variation in nodes and support
values, as shown in Figure 4. All analyses agreed
with. H. freitasi haplotypes forming a
monophyletic group, sister to W. siamensis with
strong support (aLRT=99%, BP-ML=100%,
B P P = 1 0 0 % ) . T h e t r i b e s H i l g e r t i i n i ,
Protozoophagini, and Syphaciini formed a
monophyletic group with strong support only in the
aLRT (99%) and the BPP (99%). Passalurini was a
sister group to the other tribes. The subfamily
Syphaciinae thus formed a monophyletic group,
including all four tribes represented in our sample,
although with weak to moderate support (aLRT =
89%, BP-ML = 51%, BPP = 63%). The subfamily
Enterobiinae also formed a monophyletic group,
although with weak to moderate support (aLRT =
77%, BP-ML = 39%, BPP = 62%).
Table – 1 Subfamilies, tribes of Syphaciinae, species, GenBank accession numbers, and references of specimens
used in this study.
Subfamily Syphaciinae
Tribes Species Genbank accession
number Reference
Heteroxynematinae Aspiculuris tetraptera KT764937 Wang et al. (2016)
Enterobiinae Enterobius macaci
Enterobius vermicularis
AB626858
EU281143
Hasegawa et al. (2012)
Kang et al. (2016)
Syphaciinae Passalurini Passarulus ambiguus
Passarulus ambiguus
KT879302
KF472059
Liu et al. (2016)
Sheng et al. (2014)
Hilgertiini Rauschtineria eutamii
Rauschtineria eutamii
KT875323
KT875241
Bell et al. (2016)
Bell et al. (2016)
Shypaciini Syphacia frederici MF142425 Stewart et al. (2016)
Syphacia montana AB282581 Okamoto et al. (2007)
Syphacia obvelata KT900946 Wang et al. (2016)
Syphacia agraria AB282589 Okamoto et al. (2007)
Syphacia emileromani AB282590 Okamoto et al. (2007)
Syphacia ohtaorum AB282592 Okamoto et al. (2007)
Syphacia stroma MF142420 Stewart et al. (2016)
Protozoophagini Helminthoxys freitasi A MF212135 This study
Helminthoxys freitasi B MF212136 This study
Wellcomia siamensis GQ332427 Park et al. (2011)
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
142
Neotropical Helminthology, 2019, 13(2), jul-dic Mitochondrial DNA and morphology data of Helminthoxys
143
Table 2 - Measurements, in micrometers, of male and female of all species of genus Helminthoxys, plus the specimens in study.
Male
H.
caudatus
(H.pujoli)
H.
tiophila
H.
eflatus
(H.velizy)
H.
urichi
H.
quentini
H.
gigantea
H.
abrocomae
H. freitasi H. freitasi
Body length (L)
5.500
6.550
6.800
3.000
5.240
6.320
11.672
5.030 3.725
Body width (W)
330
340
360
200
-
-
367
230 230
Nervous ring
190
240
200
120
200
180
306
170 183
Excretory pore
-
1.400
1.250
900
1.200
1.200
1.151
930 840
Oesophage (L)
660
860
800
350
730
700
957
370 546
Bulb
(L x W)
170x160
200x150
290x200
130x110
220x160
200x115
285x153
150x120 172x124
Tail (L)
930
650
550
410
490
900
1.365
475 390
Tip of tail (L)
830
-
420
350
-
850
1.243
430 319
1stmamelon to tip tail
3.100
-
3.400
-
-
3.400
5.938
2.450 2.325
2ndmamelon to tip tail
3.500
-
3.880
-
-
3.900
7.099
2.730 2.565
Spicule
320
420
295
533*Hugot,1986
175
234
866
630 580
Gubernaculum
Body
size/spicules (%)
45
5.8
120
6.4
50
4.3
45
17.6
38
3.3
80
3.7
152
7.4
60
12.5
42.5
15.5
Female
Body length (L)
11.620
19.200
20.660
8.640
12.480
13.500
21.211
13.000 10.325
Body width
(W)
530
800
800
600
790
420
654
525 407
Nervous ring
270
200
400
110
270
270
512
215 249
Excretory pore
-
2.250
2.470
1.400
1.170
1.830
3.023
1.700 1.200
Oesophage (L)
710
1.250
1.300
580
990
1.150
1.383
650 697
Bulb
(L x W)
250x190
350x-
380x250
180x180
290x270
270x180
359x205
225x200 219x179
Tail (L)
1.150
1.600
3.130
1.220
1.310
1.900
3.381
1.720 562
Vulva to tip tail
4.160
8.000
7.680
5.570
5.300
5.000
8.146
8.000 4.657
Anus to tip tail
212
-
-
-
-
-
-
--
Eggs (L x W)
104x41
90x40
115x65
-
-
-
77x33
88x38
Host
Microcavia
australis
Mysateles
prehensilis
Lagidium
viscacia
Dasyprocta
leporina
Capromys
pilorides
Octodon
degus
Abrocoma
cinerea
Thrichomys
laurentius
Mesomys
hispidus
Locality Argentina Cuba Argentina Trindade Cuba Argentina Andes da
Bolívia Brazil Brazil
Author/Year
Anus to tip tail Freitas
-Texeira et al.
(1937)
Vigueras
(1943)
Schuurmans
-Stekhoven
(1951)
Cameron&
Reesal (1951)
*Hugot (1986)
Barus
(1972) Quentin Hugot &
Gardner
(2000)
Quentin
(1969) Present
study
* Measurements in micrometers.
et al.
(1975)
144
Figure 3. Helminthoxys freitasi A) Adult female, general view of the anterior part of the body and alae lateral (thin arrow); B)
Cephalic plate, apical view, amphid (asterisk) and two papillae (head arrow); C) Bursal cauda, one pair of papillae (head arrow)
and one papillae pedunculate (arrow); D) Detail of the cloaca (C) showing one pair of sessile papillae (large arrow), accessory
hook of gubernaculum A); E) Area of the rugosa posterior to the second mamelon; F) Detail of the mamelon surface showing
longitudinal striations; G) Anus of the female, ventral view.
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
145
Figure 4. Phylogenetic relationships of Helminthoxys freitasi isolates from this study and other oxyuroids using the MT-CO1. Bayesian 50% majority rule consensus tree after
burn-in. Support values are shown at the following nodes, respectively: aLRT, ML-BP, and BPP. Branch lengths are proportional to the mean posterior probabilities of the branch
lengths of the sampled trees (scale bar, substitutions per site).
Neotropical Helminthology, 2019, 13(2), jul-dic Mitochondrial DNA and morphology data of Helminthoxys
267 g) (Patton et al., 2015).
The subfamily Syphaciinae Railliet, 1916
comprises five tribes, including the tribe
Protozoophagini composed of three genera, which
include the following: Helminthoxys Freitas, Lent,
and Almeida, 1937; Wellcomia Sambon, 1907; and
Protozoophaga Travassos, 1923, based on
morphological characteristics of the genital
structures of males and cephalic plaques, according
to some synapomorphies that both genera share
(Hugot, 1988). Studies based on molecular
phylogenies have shown the evolutionary affinity
between Wellcomia and Protozoophaga genera
(Nadler et al., 2007). There is, however, no
phylogenetic study based on DNA sequences
including the genus Helminthoxys. In this work, in
spite of a limited number of GenBank sequences
available, we inferred the phylogenetic
relationships of representatives of the tribes
Hilgertiini, Passalurini, Syphacinii and
Protozoophagiini based on the MT-CO1 gene. The
phylogenetic reconstructions obtained by ML and
BI revealed Wellcomia siamensis as a sister to H.
freitasi, with high support in all analyses, thus
providing support for the tribe Protozoophagiini as
a natural group. Our phylogenetic data thus
confirms previous works based on morphology
(Hugot, 1988; Hugot et al., 2013).
In conclusion, the present study details some
morphological characteristics of H. freitasi using
SEM and light microscopy and contributed with
additional taxonomic characters of male and
female. This study also contributed the first genetic
information for Helminthoxys. Our new DNA data
suggests a close relationship of H. freitasi with the
genus Wellcomia, corroborating the morphological
phylogeny proposed by Hugot (1988).
Additionally, H. freitasi was recorded for the first
time in the Amazon region and parasitizing a new
host, M. hispidus. This work expands the
geographic distribution of the species H. freitasi
both occurring in a new biome and reported in a
new host.
This study was established under a partnership
between the Instituto Federal do Acre (IFAC) and
the Instituto Oswaldo Cruz (IOC). We are grateful
DISCUSSION
The taxonomic characteristics of the genus
Helminthoxys were the presence of two mamelons
in the sub-ventral region of the body, ventral
ornamentation, size of the spicule, presence of
gubernaculum, and cervical and lateral alae
according Quentin, 1973. Morphological
characteristics that identify H. freitasi are the size
of the spicule in male and the position of the vulva
in relation to the body length in the female, situated
at the posterior part of the body.
In comparison, H. urichi is the closest species it
resembles based on the proportion of the spicule
length to the body length. This characteristic
corroborates the evolutionary hypothesis proposed
by Hugot (1986), which states that the opening of
the posterior vulva in the female body is associated
with the elongation of the spicules in males of such
species (Hugot, 1986). These two species may be
separated by the position and size of the mamelons,
in which the males of the species H. urichi are
further developed.
The genus Helminthoxys have a wide distribution
within the Neotropical region, extending
throughout Central and South America, with each
species found associated to a family of host. The
species H. freitasi was first described infecting the
echimyid rodent Thrichomys laurentius
(Trouessart, 1880). The genus Thrichomys is found
in open and forested areas in the Caatinga, Cerrado,
Chaco, and Pantanal biomes in Brazil, Bolivia, and
Paraguay (Patton et al., 2015). The present study
reports a new host, the rodent, Mesomys hispidus
(Desmarest, 1817) 18 also belonging to family
Echimyidae, and a new geographical distribution
in the Amazon Forest in Brazil. Our findings
suggest an association of H. freitasi and hosts of the
family Echimyidae, enable that this specie could
also parasitize others echimyids.
However, the differences in the measurements of
morphological structures of the specimens studied
compared tothe original descriptions may be
associated with intraspecific variations relative to
adaptive modification to its hosts, and may also be
associated with the difference between the body
mass of host Mesomys hispidus (140–202mm,
160g) and Thrichomys laurentius (197–209mm,
ACKNOWLEDGMENTS
Neotropical Helminthology, 2019, 13(2), jul-dic Andrade-Silva et al.
146
to the DNA-Sequencing Genomic Platform
(PDTIS/FIOCRUZ), to the Rudolf Barth Electron
Microscopy, and thank Ricardo Baptista Schimidt
for the image services, and the Venicio da Costa
Ribeiro Junior for the draw enhancement Instituto
de Comunicação e Informação Científica e
Tecnológica em Saúde- ICICT/Multimeios. This
study received financial support from
Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior –CAPES and Instituto Oswaldo
Cruz (FIOCRUZ). AMI is financially supported by
CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico) fellow shipgrant number
307932/2014-1, and FAPERJ (Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro),
fellow shipgrant number CNE2/2016.
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