Journal of Helminthology Morphological and molecular characterization

cambridge.org/jhl
of Aporcella femina sp. n. (Dorylaimida,
Aporcelaimidae) from Nigeria
M. Rashidifard1 , T.T. Bello1,2, H. Fourie1, D.L. Coyne2 and R. Peña-Santiago3
Research Paper
1
Cite this article: Rashidifard M, Bello TT, Unit for Environmental Sciences and Management, North-West University, Private Bag X6001, Potchefstroom
Fourie H, Coyne DL, Peña-Santiago R (2021). 2520, South Africa; 2International Institute of Tropical Agriculture, IITA, PMB 5320, Ibadan, Nigeria and
3
Morphological and molecular characterization Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén,Campus ‘Las Lagunillas’ s/n,
of Aporcella femina sp. n. (Dorylaimida, Edificio B3, Jaén 23071, Spain
Aporcelaimidae) from Nigeria. Journal of
Helminthology 95, e7, 1–8. https://doi.org/
10.1017/S0022149X20001042
Abstract
A new species of the genus Aporcella collected from a watermelon field in Nigeria is described,
Received: 19 November 2020 including its morphological and molecular (small subunit (SSU) and large subunit (LSU)
Revised: 29 December 2020
Accepted: 30 December 2020 ribosomal DNA (rDNA)) characterization. Aporcella femina sp. n. is distinguished by its
3.21–3.64 mm-long body, inner cuticle layer with fine but distinct transverse striation, lip
Key words: region offset by deep constriction, 22–25 μm broad, odontostyle 20–26 μm, neck 661–811 μm
Description; LSU; morphology; morphometry; long, pharyngeal expansion occupying 52–56% of the total neck length, female genital system
phylogeny; SSU
didelphic–amphidelphic, uterus 191–350 μm or 1.9–3.3 mid-body diameters long, V = 52–57,
Author for correspondence: tail short and convex conoid (35–48 μm, c = 72–98, c′ = 0.7–0.9) and males absent.
M. Rashidifard, Phylogenetic analyses based on the partial sequence of SSU and LSU (D2–D3) rDNA revealed
E-mail: [email protected] a close relationship of A. femina sp. n. with other Aporcella species, confirming the monophyly
of the genus as well as its association to a clade made of several taxa characterized by the absence
of pars refringens vaginae.

Introduction
The genus Aporcella Andrássy, 2002 is a widely distributed dorylaimid (Dorylaimida) taxon,
inhabiting soils of all zoogeographical realms/regions except Antarctica (Álvarez-Ortega et al.,
2013; Vazifeh et al., 2020). In Africa, it occurs in the southern part of the continent (Botswana
and South Africa), where four species have so far been recorded: Aporcella adriaani (Botha &
Heyns, 1990) Álvarez-Ortega, Subbotin & Peña-Santiago, 2013; Aporcella debruinae Álvarez-
Ortega, Subbotin & Peña-Santiago, 2013; Aporcella parapapillata (Botha & Heyns, 1990)
Andrássy, 2009; and Aporcella tropica (Jana & Baqri, 1981) Álvarez-Ortega, Subbotin &
Peña-Santiago, 2013.
Since its original description by Andrássy (2002), the taxonomy of Aporcella has been sub-
jected to a number of changes, including a new definition (Álvarez-Ortega et al., 2013), the
description of new species (Andrássy, 2012; Álvarez-Ortega & Peña-Santiago, 2016; Naghavi
et al., 2019, 2020) and a recent update of its taxonomy (Vazifeh et al., 2020). Molecular ana-
lyses have repeatedly supported the monophyly of Aporcella. Surprisingly, the closer evolution-
ary relationship of Aporcella with non-aporcelaimid taxa (tylencholaims, discolaims) than
with other aporcelaims has also been reported by various researchers (Álvarez-Ortega &
Peña-Santiago, 2016; Imran et al., 2019; Naghavi et al., 2019; Vazifeh et al., 2020).
From a 2016 survey of nematode fauna associated with watermelon in Nigeria, specimens
of an Aporcella population that belong to an undescribed species were recovered. This would
represent a novel geographical record for the genus, and, by means of its molecular analysis,
offers the chance to confirm previous findings about its monophyly and phylogeny.

© The Author(s), 2021. Published by Materials and methods

Cambridge University Press. This is an Open
Access article, distributed under the terms of Extraction and processing of nematodes
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/), Nematode specimens were recovered from rhizosphere soil samples that were collected from
which permits unrestricted re-use, distribution, watermelon fields in south-west Nigeria during 2016. Extraction of nematodes from soil fol-
and reproduction in any medium, provided the lowed a modified pie pan method (Coyne et al., 2007). Nematodes were fixed in a hot 4% for-
original work is properly cited. maldehyde solution for morphological observations (Nico et al., 2002) and subsequently
mounted in anhydrous glycerine as permanent slides (De Grisse, 1963), while others were
stored in dimethyl sulphoxide, disodium ethylenediamine tetra-acetic acid and saturated
sodium chloride (abbreviated here as DESS) solution for molecular analysis. Morphological
features of nematodes were observed, measured and photographed using an Eclipse 80i

https://doi.org/10.1017/S0022149X20001042 Published online by Cambridge University Press

 2 M. Rashidifard et al.

Fig. 1. Aporcella femina sp. n. (female): (a, b) anterior region in lateral median view; (c) anterior region in lateral surface view; (d) vagina region; (e) neck region; (f)
posterior genital branch; (g) pharyngo–intestinal junction; (h) posterior body region; (i) caudal region. Scale bars: (a, g, i) 20 μm; (b–d) 10 μm; (e, f) 100 μm; (h)
50 μm.

microscope (Nikon, Tokyo, Japan) equipped with differential SSU ribosomal DNA (rDNA), as well as D2A (5′ -ACAAGTA
interference contrast optics, a drawing tube (camera lucida) and CCGTGAGGGAAAGTTG-3′ ) and D3B (5′ -TCGGAAGGAA
a DS digital camera (Nikon, Tokyo, Japan). CCAGCTACTA-3′ ) (Subbotin et al., 2006) for D2–D3 large sub-
unit (LSU) rDNA. The polymerase chain reaction (PCR) amplifi-
cation process was followed as reported by Rashidifard et al.
Molecular identification (2020). Four microliters of PCR products were loaded on 1%
agarose gel and visualized by an ultraviolet transilluminator to
Fixed specimens in DESS were rinsed using double-distilled water, check the quality of DNA. The DNA was sequenced in both for-
and two specimens were transferred to individual 1.5 ml tubes ward and reverse directions by the Inqaba Biotec™ genomic com-
containing 15 μl nuclease-free water. DNA were extracted using pany (South Africa; www.inqaba-southafrica.co.za).
chelex-100 by adding 20 μl of chelex (5% w/v) and 5 μl proteinase
K to each tube, followed by incubation at 57°C for 120 min and at
95°C for 10 min (Rashidifard et al., 2019).
Phylogenetic analyses
DNA amplification was carried out on a Vacutec thermocycler
(www.vacutec.co.za) using the primers: small subunit (SSU) The new SSU and LSU sequences obtained for the new species
F04 (5′ -GCTTGTCTCAAAGATTAAGCC-3′ ), and SSU R26 were compared to other available sequences in the GenBank
(5′ -CATTCTTGGCAAATGCTTTCG-3′ ) (Blaxter et al., 1998) for using the BLASTN search tool. One data set was prepared for

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 Journal of Helminthology 3

Fig. 2. Aporcella femina sp. n. (female: light microscopy): (a–c) anterior region in lateral median view; (d) cuticle; (e) anterior region in lateral surface view; (f) vagina
region; (g) posterior body region; (h) pharyngo–intestinal junction; (i) neck region; ( j) posterior genital branch; (k) caudal region. Scale bars: (a, h, k) 20 μm; (b–f)
10 μm; (g) 50 μm; (i, j) 100 μm.

each of the SSU and LSU regions and taxa selection was carried invariable sites and a gamma distribution (GTR + I + G) was
out according to Rashidifard et al. (2020). The available sequences selected as the most appropriate model for the LSU dataset.
as well as outgroups in the SSU data set were aligned using the Bayesian inference was conducted in MrBayes v3.1.2 (Ronquist
MUSCLE alignment tool (Edgar, 2004) in Geneious Prime® & Huelsenbeck, 2003) implemented in Geneious Prime® 2020.2.3
2020.2.3 (www.geneious.com), while the LSU data set was aligned (www.geneious.com). The chain was running 3 × 106 generations
using the E-INS-I algorithm in the MAFF alignment tool (Katoh for both the SSU and LSU data sets, and after discarding the 25%
& Standley, 2013). According to the jModeTest 2.1.10 (Darriba burn-in samples, the remaining samples were used for further
et al., 2012), the Hasegawa–Kishino–Yano with a gamma distribu- analyses. The Markov chain Monte Carlo (MCMC) algorithm
tion (HKY + G) was the best substitution model for the SSU data- was implemented to calculate the posterior probabilities
set. However, the General Time Reversible with proportion of (PB) for each of the phylogenetic trees (Larget & Simon, 1999)

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 4 M. Rashidifard et al.

Fig. 3. Bayesian tree with 50% majority rule of Aporcella femina n. sp. from Nigeria using partial SSU rDNA sequences under the HKY + G model. The sequence of
new species is indicated by bold font. Scale bar shows the number of substitution per site.

using the 50% majority rule. The mononchid taxa were used as Description
outgroups for both the SSU and LSU phylogenies. Female. Moderately to slender (a = 28–38) nematodes of large
size, 3.21–3.64 mm long. Body cylindrical, tapering towards
both ends but substantially towards the anterior region, while
Results
the tail is short and rounded. Upon fixation, habitus regularly
Aporcella femina sp. n. curved ventrad, C- to G-shaped. Cuticle two-layered, very thick
throughout the entire body, 4–7 μm in anterior region, 7.5–
Material examined
9.5 μm at mid-body and 7.5–11.5 μm on tail, consisting of a
Ten females in variable but generally good state of preservation thin outer layer with nearly smooth surface, and a thicker inner
(figs 1–3). layer that displays a fine but distinct transverse striation (fig.
2g). Lateral chords narrow, 6–17 μm wide or 6–19% of mid-body
Measurements diameter. Ventral and dorsal pores restricted to cervical region,
All measurements are presented in table 1. two or three in number at each side at odontostyle–odontophore

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 Journal of Helminthology 5

Table 1. Morphometrics of Aporcella femina sp. n. from Nigeria and two other African species of the genus.

A. femina sp. n. A. pseudospiralis

Species A. parapapillata
Nigeria
Population South Africa South Africa Botswana
Present paper
Referencea 1 1 2
Holotype Paratypes Type material Type material

Characteristics n ♀ 9♀♀ 4♀♀ 3♀♀ 3♀♀

L 3.50 3.47 ± 0.14 (3.21–3.64) 3.64–4.42 2.11–2.51 2.57–2.79

a 31 33.1 ± 2.9 (28–38) 31–35 29–33 31–38
b 4.8 4.7 ± 0.4 (4.2–5.4) 4.5–5.1 3.9–4.4 3.9–5.1
c 74 84.3 ± 8.2 (72–98) 58–73 62–75 68–95
V 54 54.5 ± 1.6 (52–57) 50–53 52–56 52–55
c’ 0.76 0.73 ± 0.12 (0.61–0.94) 0.93–1.15 0.8–1.0 0.6–0.9
Lip region diameter 23 24.0 ± 1.1 (22–25) 23–27 19–20 20–23
Odontostyle length
Ventral side 20 21.9 ± 1.9 (19–24) ? ? ?
Dorsal side 22 23.7 ± 1.9 (20–26) 23–26 18–19 19–22
Odontophore length 49 47.9 ± 1.2 (46–50) 47–52 32 37–38
Neck length 721 736 ± 53 (661–811) 810–880 530–590 510–650
Pharyngeal expansion length 379 397 ± 34 (349–451) ? ? ?
Body diameter
at neck base 98 97.0 ± 7.9 (87–113) ? ? ?
mid-body 111 106 ± 12 (89–129) 117–130 65–84 69–89b
anus 62 57.0 ± 4.8 (50–63) 57–61 27–39 ?
Distance vulva–anterior end 1901 1890 ± 76 (1780–1975) 1910–2270 1360–1730 1362–1509b
Prerectum length 196 153 ± 41 (88–203) 219–280 136–173 88–244
Rectum length 63 63.8 ± 6.7 (50–75) 65–85 46–48 50–58
Tail length 47 41.4 ± 4.0 (35–48) 57–68 32–36 27–41
Measurements in μm except L in mm, and in the form average ± standard deviation (range).
a
1: Botha & Heyns (1990); 2: De Bruin & Heyns (1992).
b
Calculated from other morphometrics.

level; lateral pores small. Lip region offset by a distinct constric- didelphic–amphidelphic, with well-developed genital branches,
tion, 3.3–3.8 times wider than high and less than one-third the anterior 346–565 μm or 11–15% of body length, the posterior
(22–29%) of body diameter at neck base; lips separate, with mod- 381–638 μm or 12–18% of body length. Ovaries comparatively
erately protruding papillae. Amphidial fovea stirrup-shaped, its small, mostly not reaching the oviduct–uterus junction, the anter-
opening 9–10.5 μm, less than one-half (38–46%) of lip region ior 103–259 μm and posterior 106–231 μm long. Anterior oviduct
diameter. Cheilostom broad, 10.5–14 μm long, with thick walls. 127–200 μm or 1.4–1.8 body diameters long, posterior oviduct
Odontostyle strong, 3.8–4.3 times longer than wide, barely shorter 133–203 μm or 1.4–1.9 body diameters long, both consisting of
(0.9–1.0 times) than lip region diameter and 0.58–0.73% of total a slender distal portion made of prismatic cells and a distinct
body length, with aperture 13–17 μm long or c. two-thirds (58– pars dilatata with perceptible lumen. Oviduct and uterus sepa-
76%) of total length. Guiding ring simple but distinct, plicate. rated by a distinct sphincter. Anterior uterus 191–325 μm or
Odontophore linear, rod-like, 1.9–2.4 times longer than odontos- 1.9–3.1 body diameters long, posterior uterus 204–350 μm or
tyle. Pharynx entirely muscular, enlarging very gradually, with its 2.1–3.3 body diameters long, often appearing as a simple, tube-
basal expansion 5.8–9.0 times as long as wide, 3.1–4.5 times the like structure, but with some (well-preserved) specimens having
body diameter at neck base and occupying 52–56% of total a longer uterus, which enables the appreciation of a wider prox-
neck length; pharyngeal gland nuclei located as follows (n = 1): imal uterine region and a more slender distal region, both of
DO = 47–52, DN = 48–56, DO-DN = 3–5, S1N1 = 61–66, S1N2 = about similar length. No uterine egg observed. Vagina extending
73–75, S2N = 85–88. Cardia short and rounded conoid, 21–32 × inwards 40–54 μm, occupying c. one-half (41–45%) of body
20–34 μm, surrounded by intestinal tissue that forms a short diameter: pars proximalis 28–41 × 19–28 μm, with distally conver-
extension projection into the intestinal lumen. Genital system gent walls and surrounded by a relatively weak musculature; pars

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 6 M. Rashidifard et al.

Fig. 4. Bayesian tree with 50% majority rule of Aporcella femina n. sp. from Nigeria using partial LSU (D2–D3) rDNA sequences under the GTR + I + G model. The
sequence of new species is indicated by bold font. Scale bar shows the number of substitutions per site.

distalis well-developed, 10–14 μm long; pars refringens absent. vegetable production; specimens were collected from a field culti-
Vulva a slightly posterior transverse slit. Prerectum 2.4–4.0, rec- vated with watermelon (Citrullus lanatus) on sandy-loam soil
tum 0.8–1.5 times the anal body diameter long. Tail short, convex (sand = 66%, silt = 13%, clay = 21%, organic matter = 11.18%, pH
conoid, its ventral side weakly straighter than the dorsal side; cau- 6.22).
dal pores two pairs, close together, at the posterior half of tail, one
lateral, another sublateral. Molecular characterization
Male. Unknown. Females do not contain sperm cells. After sequencing and editing, two totally (100%) identical
sequences per each locus (SSU and LSU) were obtained; therefore,
Type locality and habitat only one sequence was used for analysis and deposited into the
South-west Nigeria, Odeda (7°10′ 59.999′′ N, 3°26′ 42.01′′ E), an agrar- NCBI GenBank: a 745 bp-long LSU sequence (accession number
ian locality with a history of cassava (Manihot esculenta Crantz) and MW237838) and another 855 bp-long SSU sequence (accession

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 Journal of Helminthology 7

number MW237832). The evolutionary relationships of the new which, when available, would clarify any ambiguity. However, the
species are presented in figs 3 and 4. available morphological information clearly distinguishes the three
species from each other, supporting a separate status for them.
Type material The evolutionary relations of the new species, as derived from
Female holotype and nine female paratypes, deposited in the the molecular analyses based on the partial sequence of SSU and
nematode collection of the University of Jaén, Spain. LSU rDNA, are represented in figs 3 and 4. According to the SSU
phylogeny, the new species is in a highly supported (BPP: 97%)
Etymology sister relation with Aporcella vitrinus. Furthermore, Bayesian
The specific epithet is a Latin term meaning woman or female, and tree using partial LSU sequence shows that the new species
refers to the existence of only females in this species. forms part of a fully supported (BPP: 100%) subclade with
Aporcella charidemiensis and A. vitrinus, the former being the
Diagnosis and relationships closest relative, which is recorded from Spain. A highly supported
The new species is characterized by its 3.21–3.64 mm-long body, (BPP: 94%) Aporcella clade is made of this subclade along with
inner cuticle layer with fine but distinct transverse striation, lip three Aporcella sequences belonging to A. simplex. Therefore,
region offset by deep constriction and 22–25 μm broad, odontos- the present results confirm previous findings (for instance, see
tyle 20–26 μm at its dorsal side and 19–24 μm at its ventral side, Naghavi et al., 2019; Vazifeh et al., 2020) about both the mono-
neck 661–811 μm long, pharyngeal expansion 349–451 μm long phyly of Aporcella, and the belonging of Aporcella sequences to
or 52–56% of total neck length, female genital system a large clade morphologically characterized by the absence of
didelphic–amphidelphic, uterus 191–350 μm or 1.9–3.3 body dia- pars refringens vaginae, which also includes discolaims in the
meters long, V = 52–57, tail short and convex conoid (35–48 μm, tree provided herein.
c = 72–98, c′ = 0.7–0.9) and males absent.
Financial support. One author (R.P.S.) is grateful for the financial support
In having a comparatively long body (more than 3.0 mm long),
received from the scientific project (ref.: Action 1-PAIUJA 2019-2020:
Aporcella femina sp. n. resembles Aporcella magna Andrássy,
EIRNM02-2017), University of Jaén. Infrastructure and partial funding by
2012 and Aporcella pseudospiralis (Botha & Heyns, 1990) the Nematology Unit at North-West University of South Africa is also
Andrássy, 2012, and significantly differs from its other congeners acknowledged.
(body 3.21–3.64 vs up to 3.08 mm long) (see compendium by
Álvarez-Ortega et al., 2013 and Vazifeh et al., 2020). It can be dis- Conflicts of interest. None.
tinguished from A. magna, a species occurring in Chile, in its
Author contributions.
smaller general size: body 3.21–3.64 versus 4.48–5.00 mm long, M.R. and T.T.B. contributed equally.
n = 5; neck 661–811 versus 888–1020 μm long; tail 35–48 versus
50–60 μm. It is also smaller than A. pseudospiralis, which is
only known to occur in South Africa, with a smaller general References
size (body 3.21–3.64 vs 3.64–4.42 mm long, n = 4; neck 661–811
vs 810–880 μm long), shorter prerectum (122–203 vs 219– Álvarez-Ortega S and Peña-Santiago R (2010) Studies on the genus
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