The super family Thelastomatoidea is one of the two super families of the order
Oxyurida. They are the nematodes parasitic or commensal in gut of terrestrial
arthropods and usually feed upon the bacterial microfauna found there (Jex
et al., 2005). Adamson and Van Waerebeke (1992a-c)
divided the superfamily into five families viz., Thelastomatidae, Protrelloididae,
Hystrignathidae, Travassosinematidae and Pseudonymidae which is followed by
subsequent workers. The family Thelastomatidae is by far the largest family
of the superfamily Thelastomatoidea reported that the family Thelastomatidae
is represented by 28 genera. Recently, three more genera to the family were
added and hence the family now represented by 31 genera. So far most of these
genera are characterized on morphological basis which often creates taxonomic
Recently, molecular markers have often been used for taxonomic identification
and phylogenetic analyses in different species groups (Abdul
Majeed et al., 2001; Li et al., 2011).
Molecular methods have been used to assess the relationships of the major species
and to examine intraspecific variations. It has been demonstrated that the 28S
rRNA gene and nuclear ribosomal DNA containing the first internal-transcribed
spacer (ITS1), 5.8S rRNA and second internal transcribed spacer (ITS2) regions
are informative for molecular diagnostics of nematodes and in the phylogenetic
relationship analyses between nematodes from population to class level (Adams
et al., 1998; Kampfer et al., 1998;
Iwahori et al., 1998; Beckenbach
et al., 1999; De Giorgi et al., 2002;
Wang et al., 2005; Nadler
et al., 2006).
During the survey of insect parasitic nematodes from Meerut, U.P., India, investigator
came across, a known species of the family Thelastomatidae, the Leidynema
appendiculata (Leidy, 1850; Chitwood,
1932) having minor variations harbouring alemntary canal of common cockroach.
In this study, the 28S rRNA gene sequences were used to investigate the identification,
validity and phylogenetic affinities with different geographical isolates in
order to provide molecular evidence of this nematode in India along with detailed
morphological and morphometric analysis as original description lacks some morphological
and morphometric details. Reason behind selecting the 28S ribosomal RNA gene
has been made because it represents a well-conserved gene that evolves relatively
slowly. Moreover, sequences of this gene have recently been used in the studies
resolving the phylogenetic relationships between nematode and other animals
and this gene was shown to be a suitable marker for barcoding of nematodes (Barthelemy
et al., 2006). Besides this, RNA secondary structure has also bee
used for phylogenetic reconstruction.
MATERIALS AND METHODS
Hosts , P. americana were caught from Meerut, U.P., India, alive with the help of nets and brought to the laboratory in live condition in insect cages and were kept in these cages till surgical procedures. On dissection, nematodes were removed from the digestive tract, placed in 0.6% saline and fixed in hot 70% alcohol solution. For light microscopical examination, the nematodes were cleared gradually in glycerin. A light microscope equipped with differential interference contrast, digital image analysis system (Motic digital microscope for Windows) was used for morphological and morphometric analysis and drawings were made by camera lucida.
Initially methods to extract genomic DNA were following from the work of Oboh
et al. (2009) and Al-Saghir (2009). Genomic
DNA was extracted from ethanol-preserved parasite using the DNeasy Tissue Kit
(Qiagen). 28S rDNA was amplified using the Master Cycler Personal (Eppendorf)
in a final volume of 25 μL PCR reaction. Each amplification reaction contained
10X PCR buffer, 0.4 mM dNTP, 1 U Taq polymerase (Biotools) and 10 pM of each
primer- specifically designed forward (5-TTGGCGTCTCAGTGTGAAAG-3)
and the reverse primer (5-TTCACCATCTTTCGGGTCTC-3). PCR was carried
out with the following steps: an initial denaturation at 94°C for 3 min,
35 cycles of 94°C for 30 sec, 56°C for 45 sec and 72°C for 1 min,
and a final extension at 72°C for 10 min. PCR products were separated on
1.5% agarose gel. The products were then purified by Chromous PCR cleanup kit
(# PCR 10), according to manufacturers instructions. Both DNA strands
were sequenced using a Big Dye Terminator vr.3.1 cycle sequencing kit in an
ABI 3130 Genetic Analyzer using same primers. Sequences were analyzed using
the MEGA 5.01 (Tamura et al., 2011; MEGA: Molecular
Evolutionary Genetics Analysis), using neighbor-joining, minimum evolution,
maximum likelihood and maximum parsimony methods. Bootstrap confidence values,
reported as percentages, were calculated based on 1,000 replicates (Felsenstein,
1985). Distances (base substitutions per site) were computed using the Kimura
2-parameter method (Kimura, 1980). The ME tree was searched
using the Close-Neighbor-Interchange (CNI) algorithm (Nei
and Kumar, 2000).
RNA secondary structure for 28S rRNA was predicted by using the Sriobo program
(Ding et al., 2004) in Sfold (Statistical and
Rational Design of Nucleic Acids). Sfold predicts RNA structure by identifying
suboptimal structure using the free energy optimization methodology at a default
temperature 37°C. The dynamic programming algorithm used in Sfold
is based on the work of Zuker and Stiegler (1981). GC
percentage was determined using CG calculator (http://www.genomicsplace.com/gc_calc.html).
RESULTS AND DISCUSSION
Leidynema appendiculata (Leidy, 1850; Chitwood,
||Periplaneta americana L.
||Meerut (29° 01' N, 77° 45' E), U.P., India
|Site of infection
||The holotype and paratype slides have been deposited in the museum of
Department of Zoology (Voucher number Nem/2009/01), Ch. C.S. University,
Meerut, U.P., India.
||L. appendiculata female, (a) Whole mount, (b) Anterior
end of female, (c) Posterior end of female and (d) Eggs
The worms are small. Males are with evenly tapering anterior end and very short tail. The caudal appendage in female is elongated. Mouth is surrounded by very large sub median labial papillae bearing amphids or lateral organs in the form of circular protuberances. Body of the worm in both sexes is provided with well developed lateral alae. The cuticular covering of body bears transverse striations which are coarse in the anterior region in either sex. The excretory pore is marked in post oesophageal part of body in both the sex (Fig. 1, 2).
Female: Body of female is elongated, measuring 2.70-2.75 mm in length.
Maximum width 0.25-0.28 mm recorded in pre-gonadal region of the body. Mouth
leads into well developed, rectangular buccal cavity measuring 0.012-0.014 x
0.015-0.017 mm. Oesophagus is long measuring 0.35-0.42 mm in total length and
is divisible into three parts: corpus, isthmus and oesophageal bulb.
||L. appendiculata male, (a) Whole mount, (b) Anterior
end of male, (c) Posterior end of male and (d) Spicule
Anterior most corpus measures 0.25-0.28 x 0.03-0.04 mm. Middle oesophageal
part, isthmus is smaller in comparison to corpus and is highly muscularized,
measuring 0.021-0.024 x 0.028-0.032 mm. Posterior rounded valvular oesophageal
bulb measuring 0.08-0.12 mm in diameter. Nerve ring is located at 0.12-0.14
mm from anterior end of the body. Excretory pore is post-oesophageal in location,
at 0.55-0.58 mm from anterior end of the body. Intestine is well developed and
equipped with prominent cardia and a diverticulum at its anterior end but posteriorly
it is cylindrical and simple. Anus is located slightly anterior to posterior
most part of the alae at 0.65-0.68 mm. Ovaries are two, uteri are amphidelphic.
Vulva is more or less equatorial, located at 1.50-1.80 mm away from anterior
end of the body. Vagina is non-muscular and straight. Uteri filled with large
number of eggs which are oval in outline, elongated and slightly flattened on
one side measuring 0.065-0.068 x 0.038-0.034 mm. Tail is filiform, elongated
measuring 0.14-0.16 mm in length (Table 1).
Male: Males are much smaller than females measuring 0.82-0.85 mm in
length. Maximum width, 0.09-0.12 mm recorded in the post oesophageal region
of the body. Mouth leads into rectangular buccal cavity measuring 0.013-0.015x0.012-0.014
|| Phylogenetic tree of the relationships between the L.
appendiculata and related species
Oesophagus is elongated measuring 0.08-0.094 mm in length and comprises of
well a developed corpus, isthmus and oesophageal bulb. In the original description
measurement of oesophagus is possibly miscalculated for the reasons best known
to the authors. Length of oesophageal corpus is 0.05-0.06 mm and its width is
0.015-0.017 mm. Isthmus measures 0.005-0.007x0.012-0.016 mm. Oesophageal bulb
is oval in outline measuring 0.025-0.027x0.024-0.028 mm in diameter. Nerve ring
is located at 0.062-0.065 mm from the anterior end of the body. Excretory pore
is located at 0.11-0.13 mm form anterior end of the body. Iintestine is simple,
without any diverticulum and it is provided with a well developed cardia. Diameter
of intestine is more or less double as compared to the oesophageal bulb. Anus
is located at 0.021-0.025 mm from the posterior end of body. Testis elongated,
occupying two third posterior part of the body. Tail is pointed, very small
and measures 0.009-0.012 mm in length. Spicule is single, slightly curved, measuring
0.049-0.052 mm in length. Papillae are five pairs (one pair pre-anal, one pair
ad-anal and three pairs post-anal). Pre-anal papillae are protruded whereas;
ad-anal and first pair of post-anal papillae is sessile (Table
The fragment of 28S rDNA is 220 bp in length (sequence of L. appendiculata was deposited in GenBank database accession number GQ925910). Significant alignment was produced in BLAST with sequences of different nematodes species. Close similarity between the sequence of L. appendiculata and other nematode sequences included in alignment, ranged between 90 and 99% with the proportion of gaps ranging from 0 to 5% (Fig. 3). On the basis of base pair sequences, BLAST clustered present worm together with L. appendiculata from Russia (accession number EU365630) and showed that worms at the disposal of authors is 99% related. Phylogenetic tree of the relationships between the L. appendiculata and related species with high bootstrap values in a minimum evolution tree. Similar results were obtained using neighbor-joining, maximum likelihood and maximum parsimony maximum parsimony.
Details of the secondary RNA structure prediction are: G+C content for the
28S region of rDNA of L. appendiculata is 51.4%. The minimum free
energy is estimated by summing individual energy contributions from base pair
stacking, hairpins, bulges, internal loops and multi-branch loops. RNA secondary
structure consists of stems and loops (Fig. 4). From the data
we were able to draw the rRNA secondary structure where each residue is identified
by a base pair, the backbone and the hydrogen bonds are represented as dots
between the base pair. Mainly five types of loops are present in RNA secondary
structure viz., interior, hairpin, exterior, multi and bulge (Fig.
5). Each residue is represented on the abscissa and semi-elliptical lines
connect bases that pair with each other. In a centroid diagram, bases are positioned
along a circle, in a clockwise orientation. An arc connecting two bases across
the circle indicates pairing between the bases.
|| Predicted 28S RNA secondary structure of L. appendiculata
The lack of pseudoknots in the secondary structure is reflected by the absence
of intersecting lines in the centroid structure (Fig. 6).
From the two-dimensional histogram (2Dhist), the patterns of base pair frequencies
are nearly identical for the sample (Fig. 7). It contains
base pair frequencies for constructing 2D histogram. A two-dimensional histogram
(2Dhist) displays base pair probabilities computed from a statistical sample
of structures. In the 2Dhist, base pair probabilities are shown by solid squares
in the upper left triangle, with the nucleotide positions on both axes. The
areas of the solid squares are proportional to the frequencies of the base pairs
in the sampled structures.
|| Histogram showing types of loops in 28S region of L. appendiculata
|| Centroid structure of predicted 28S RNA structure of L.
The probability profile displays predicted accessible sites on the target RNA.
For prediction of target accessibility, a complete probability profile of single-stranded
regions is generated for the entire target RNA. Sites with high probabilities
of being single-stranded are predicted to be accessible. The probability profiles
(W = 4) for the sample are also computed and in a plot, by plotting against
nucleotide position (Fig. 8). On a profile for fragment width
W, the probability that W consecutive bases are all unpaired is plotted against
the first base of the segment. This approach has proved to make substantially
better predictions than the MFE structure. The significance of assigning probability
as a measure of confidence in prediction is highlighted (Fig.
8). A single-stranded region predicted by both the MFE structure and the
ss-count has low probabilities on the probability profile (W = 4 bases, as described
in Ding and Lawrence, 2001).
|| The two-dimensional histogram of base pairs in the L.
|| Line plot representing secondary structure of 28S RNA of
The ss-count statistic gives the propensity of a base to be unpaired, as measured
by the frequency with which it is unpaired in a group of the optimal and suboptimal
foldings within a specified increment of the MFE. At nucleotide position i,
the probability that nucleotide i, i + 1, i + 2, i + 3 (i.e., fragment width
W = 4) are all single stranded is plotted against i. This probability is computed
by MFE structure and by ss-count from mfold for the nucleotides 1-220 of
L. appendiculata. A 3D energy landscape plot of the sampled ensemble and
representative structures (Fig. 9). The optimal number of
clusters determined by our software is 2. Structures belonging to the clusters
are marked as solid dots of two different colors. The MFE structure and the
ensemble centroid are both in the largest cluster (light blue color), with a
probability of 0.767. The coordinates for a structure are (MDS axis 1, MDS axis
2, free energy), where the horizontal axes are from MDS and the vertical axis
is the free energy of a secondary structure. The coordinates are for the MFE
structure (-14.68, 1.03, -84.90), for the ensemble centroid (3.49, -1.99, -85.10),
for the centroid of cluster 1, (3.49, -1.99, -85.10) and for the centroid of
cluster 2, (-14.60, 0.96, -84.60).
||The energy landscape of the sampled ensemble and representative
structures for L. appendiculata of 220 nt
Clusters are sorted in descending order of cluster size. The cluster containing
the MFE structure is marked using a red asterisk. The size of a cluster is the
sampling estimate for the probability of the cluster i.e., the sample frequency
of the cluster. The MFE structure is excluded from the calculation of cluster
sizes and the sizes of all clusters sum to one. Multi-Dimensional Scaling (MDS)
plot of the sampled ensemble and representative structures MDS is a technique
for representing high-dimensional objects in typically two dimensions (Kruskal
and Wish, 1977). For RNA secondary structures, base-pair distances are used
as an input to MDS. Members of all other clusters are plotted as small circles.
The genus Leidynema was established by Schwenk (Travassos,
1929). The study of the worms at the disposal of authors places it under
the genus Leidynema Schwenk (Travassos, 1929).
Morphologically this worm was found closest to Leidynema appendiculata
(Leidy, 1850; Chitwood, 1932)
with minor variations in the measurement of various body parts (Table
1). Variations were noticed mainly in the measurements of various body parts.
That could be due to the presence of parasite in different hosts and ecological
In India, molecular phylogenetic studies of nematodes are still at an early
stage. The position of L. appendiculata in the phylogenetic trees
reconstructed in BLAST confirms its placement of the worm at our disposal within
genus Leidynema Schwenk (Travassos, 1929). Moreover,
it shows that the position in the molecular phylogenetic tree corresponds well
to the morphological similarity with the representative of Oxyuroidea. Genetic
relatedness between the studied species of Leidynema indicates closest
similarity with L. appendiculata from Russia i.e., 99%. The reasons for
differences in genetic similarity between same species from different continents
are due to their geographical distribution or due to adaptation with their host
in different continents. Present phylogenetic analysis based on the LSU rRNA
gene sequence, L. appendiculata from India and Russia are same species.
Therefore, we concluded that L. appendiculata may be the same species
from different region and different hosts. Genetic comparison with further specimens
documented from other parts of the world may alter our taxonomical concept of
this group and provide further clues to the understanding of the evolution of
In the present study, we propose that L. appendiculata from India and Russia are same species. The minor molecular biological differences might be due to the following reasons- (1) these two species are presently undergoing a speciation that precedes the molecular difference of 1%. (2) as these two morphological types are exclusively and respectively found on two different allopatric hosts, this molecular differentiation (i.e., 1%) could be host-induced. (3) This might also be due to the presence of this parasite in two different zoo-geographical regions which are separated way back by the mighty barriers.
Beside this, we also inferred the secondary structures of the long subunit
(LSU) in L. appendiculata. The structure prediction method we propose
presents a promising approach to reconstruct secondary structures of non-coding
genes in taxa that have not been studied so far but has significant taxonomic
and phylogenetic value. The consideration of taxon-specific secondary structure
models helps to improve the inference. Different RNA folding algorithms also
take into account the structural energy as the major determinant in furnishing
RNA secondary structure models and conformation which will definitely add meaningful
dimensions to our understanding of the relationships among the sequence features
and structural parameters that come into play in determining the structural
energy. This approach can be further fine-tuned in resolving ambiguities using
differences at the RNA structural level for identification of sibling species
complexes. Representative structures, including the MFE structure, ensemble
centroid and centroids of all colored clusters, are drawn as large dots in the
graph. The units on the axes of the MDS plot only serve the purpose of indicating
the relative positions of the objects (Chan et al.,
The present study demonstrates use of secondary structure on phylogenetic analyses of rRNA sequences. This effect was remarkable in sequence alignment and tree reconstruction of both simulated and empirical data. However, the favorable performance of structural alignments partly vanished in tree reconstructions, an aspect which clearly needs further investigations.
This study of the Leidynema appendiculata (Leidy,
1850; Chitwood, 1932) from P. americana represents
the first study for phylogenetic purposes and concludes that sequences of large
subunit are suitable to complement morphological data for the reconstruction
of phylogenetic relationships. RNA secondary structure analysis could be a valuable
tool for distinguishing species and completing L. appendiculata systematics,
more so because 28S secondary structure contains more information than the usual
primary sequence alignment. The analysis of morphological and molecular-taxonomic
characters supported the independent species status of Leidynema appendiculata.
The authors are thankful to Head, Department of Zoology, C.C.S. University, Meerut, for providing laboratory facilities. This work was funded by the Department of Science and Technology, (grant number SR/SO/A543/2005) awarded to HSS.