Morphological and molecular characteristics of Malayfilaria sofiani Uni, Mat Udin & Takaoka n. g., n. sp. (Nematoda: Filarioidea) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia

The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia). We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing. Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews. The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.


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Results: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews. Conclusions: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.
Recently, there has been a worldwide increase in the incidence of zoonotic infections in humans caused by filarial nematodes that parasitise other animals, such as dogs, cats, cattle and wild boars. It is believed that global warming, deforestation and human demographics are affecting the transmission of parasites by bridging the gaps between vectors, host animals and humans [13]. For example, the number of cases of zoonotic onchocercosis has increased in Europe, the USA and Japan, with infections expanding into areas where the diseases had not previously been reported [14][15][16]. In Malaysia, B. pahangi (Buckley & Edeson, 1956), a parasite of cats, dogs and wild carnivores, has recently been found to cause zoonotic diseases in humans [17,18].
A total of 34 species of filarioids from 21 genera have previously been recorded from vertebrates in Malaysia [3,19]. Species in the genera Wuchereria Silva Araujo, 1877 and Brugia Buckley, 1960 have biological similarities with regard to the vectors used and their location in the definitive hosts [4]. Recent molecular analyses have indicated that they are closely related, forming a single clade [20][21][22][23][24][25][26].
Based on their geographical distribution, Bain [27] suggested that W. bancrofti in humans spread from the Oriental Pacific area through the tropical belt and that Wuchereria and Brugia may have diversified in Southeast Asia. However, the origin and evolution of these species remain to be clarified [23].
During this investigation, we found filarial nematodes in common treeshrews captured in Kelantan, on the east coast of Peninsular Malaysia. None of the specimens collected match any of the hitherto described species or genera of filariae [2,3,19]. A new genus and species are, therefore, proposed to accommodate the present nematodes, which are similar to representatives of Wuchereria and Brugia, but distinct from them on the basis of several morphological characters. Analyses of the cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes and the internal transcribed spacer 1 (ITS1) region supported our findings that the new species differs from W. bancrofti and Brugia spp. at the genus level, and phylogenetic tree topologies depicted it as sister species of the Wuchereria-Brugia clade.

Collection of hosts and parasite material
Between June 2012 and March 2016, we captured 81 common treeshrews from primary and secondary (second-growth) forests in 14  Kuala Lumpur, the Federal Territory. All animals were captured with permission of the Department of Wildlife, Malaysia, using box cage traps baited with palm oil kernels or bananas. The common treeshrews were anaesthetised and sacrificed in accordance with the policy and protocols approved by the Animal Care Committee, University of Malaya, Kuala Lumpur, Malaysia. To obtain adult filariae, we dissected the lymphatic tissues, peritoneal cavity and subcutaneous connective tissues of common treeshrews under a stereomicroscope.

Newly collected material
Thick blood smears were made and stained with 3% Giemsa solution (pH 7.4), and skin snips were taken from the face, ears, back, abdomen and tail, as described by Uni et al. [35]. We then searched for microfilariae in the blood smears and skin snips under a compound microscope. We also recorded the length and width of microfilariae taken from the uteri of adult worms.
Isolated adult worms were then transferred into either 70% ethanol for the examination of fixed worms from one common treeshrew (group A) or into phosphatebuffered saline (pH 7.4) for the examination of dead unfixed worms from another individual (group B). The fixed adult worms in group A were cleared in lactophenol solution (R & M Chemicals, Essex, UK) and drawn using a compound microscope equipped with a camera lucida (Olympus U-DA, Olympus, Tokyo, Japan). The midbody region of a fixed female was embedded in paraffin and sections were stained with haematoxylin and eosin. Dead worms that had neither been fixed nor cleared (group B) were also drawn using the same apparatus. For each worm in groups A and B, we recorded body length, body width, distance between anterior end and nerve ring, distance between anterior end and vulva, length of oesophagus, spicules and tail. Metrical data are presented as the range and measurements are in micrometres unless otherwise indicated.

Additional material examined
We also examined specimens of B. malayi (taken from a human in Pahang, Malaysia, in 1999) and B. pahangi (taken from a domestic cat at Kampung Kerinchi, Kuala Lumpur, in 1999), archived at the Department of Parasitology, Faculty of Medicine, University of Malaya, Malaysia, for comparative purposes. One female (ID no. M1) and one male (ID no. M2) of B. malayi and one female (ID no. M3) and one male (ID no. M4) of B. pahangi were fixed in hot 70% methanol and immersed in glycerine. Microfilariae of each species were examined on thick-blood-film slides stained with Giemsa solution (ID no. MS1 for B. malayi and ID no. MS2 for B. pahangi).

Molecular methods
Three females (F0, F1 and F2) were transferred directly into 80% ethanol. DNA extraction, PCR amplification and sequencing were performed as described previously [36][37][38] to determine the partial sequences of the mitochondrial cox1 and 12S rRNA genes. We also cloned the PCR products of the ITS1 region into pGEM-T vectors and determined the sequences of the recombinant plasmid [39]. Filaria martis Gmelin, 1790 was selected as the outgroup for cox1 and 12S rRNA gene analyses, and Onchocerca spp. were selected for analyses based on ITS1. Sequence data were deposited in the GenBank database (see taxonomic summary). The corresponding GenBank accession numbers of other species used to compare the present specimens and to determine their phylogenetic relationships, are provided in the figures. We used the Kimura 2-parameter model [40] to estimate evolutionary distances between species and calculated K2P distances between species of filarial parasites in MEGA6 as an estimate of the accumulated number of nucleotide substitutions per site [41].
Phylogenetic trees of the nucleotide sequences of the genes were constructed using neighbour-joining (NJ) and maximum-likelihood (ML) methods [41,42]. Analyses were performed based on 569 bp of the cox1 gene, 319 bp of the 12S rRNA gene and 495 bp of the ITS1 region using MEGA6. Analyses were also performed based on the concatenated alignment (cox1 + 12S rDNA, 888 bp).

Diagnosis
Anterior extremity slightly expanded into cephalic bulb not set-off from body. Head papillae comprising four labial and four cephalic papillae. Buccal cavity narrow, with pre-oesophageal cuticular ring. Nerve-ring situated at level of muscular oesophagus. Oesophagus divided into short muscular and long glandular parts. Salient cuticular annules present in region of midbody. Female. Vulva at level of anterior part of glandular region of oesophagus. Vagina with two bends. Ovejector long, muscular. Uteri didelphic and opisthodelphic. Tail extremity with two small ventrolateral lappets. Male.
Area rugosa precloacal. Caudal alae absent. Spicules unequal in length and dissimilar in shape; left spicule long and slender, divided into handle and simple blade, right spicule short and robust. Gubernaculum present. Caudal papillae and two small lappets on tail end present. Tail long, its length almost twice (1.9×) width of body at anus. Microfilariae sheathed, with one terminal nucleus; present in peripheral blood. Type-species: Malayfilaria sofiani Uni, Mat Udin & Takaoka n. sp. Etymology: The generic name is derived from Malaya, the former name of Malaysia.

Description
General. Adult worms small, slender, tapering toward both ends. Anterior extremity slightly dilated, not set-off from remainder of body (Fig. 1a). Labial and cephalic papillae arranged in a circle of four each (Fig. 1b). Amphids lateral, on level of labial papillae. Mouth opening small, followed by pre-oesophageal cuticular ring. Nerve ring surrounding oesophagus on level of muscular part. Deirids not observed. Oesophagus clearly divided into short muscular and long glandular parts. Intestine narrower than glandular oesophagus. Cuticle in midbody region with distinct annules comprising several transverse striations (Fig. 1d); in transverse section, cuticle thickened at lateral chords. Tail moderately long in both sexes.

Prevalence and intensity
We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Microfilariae of M. sofiani n. g., n. sp. were present in the blood of five individuals (6.2%) collected from a rubber plantation (secondary forest) in Jeram Pasu, Kelantan, Malaysia. From two of the treeshrews harbouring microfilariae, adults were recovered as well (see taxonomic summary). Only a small number of microfilariae were found in skin snips from the two hosts that Fig. 2 Micrographs of Malayfilaria sofiani n. g., n. sp. Females (a-c), males (d-e) and microfilariae (f-g). a Adult female (arrow) in tissue surrounding the lymph nodes of the neck of a treeshrew (Tupaia glis). b Pre-oesophageal cuticular ring (arrow). c Annules (arrows) at midbody region. d Bulbous head with pre-oesophageal cuticular ring (arrow). e Annules (arrows) at midbody region. f Anterior part with cephalic space (*) and nerve ring (arrow) (Giemsa staining). g Posterior part with anal pore (*) and terminal nucleus (arrow) (Giemsa staining). Scale-bars are in micrometres harboured adult worms, whereas large numbers were found in blood smears from these two individuals. Microfilariae from both blood smears and skin snips were very similar in size. Since we also found a small number of blood cells in the skin snips, the microfilariae here appeared to have originated from the blood. We, thus, conclude that microfilariae of the new species circulate in the blood of their hosts. Microfilariae of B. tupaiae and M. (T.) dunni were not found in the blood smears examined.  [2] on the basis of the following characters: an oesophagus with a well-developed glandular part, and a long tail in both sexes; vulva is located in the anterior region of the body in females, and, in males, spicules differ markedly in size and morphology, sessile caudal papillae are present, whereas caudal alae are absent.

Supplementary information on
Comparison of morphological characteristics of the present specimens with those of other onchocercine genera revealed close similarities with Wuchereria and Brugia [2, 3, 6, 7], i.e. presence of a buccal cavity with a pre-oesophageal cuticular ring, a dilated cephalic extremity, and a non-protuberant vulva as well as a rounded caudal extremity in females. Furthermore, males have a long tail, with a length almost twice the width of the body at the anus, microfilariae are sheathed and circulate in the blood. However, Malayfilaria n. g. can be distinguished from members of both Wuchereria and Brugia on the basis of a number of morphological characters (Additional file 1: Table S1).
Malayfilaria n. g. differs from Wuchereria as modified by Buckley [7] and characterised by Anderson & Bain [2] in having fewer caudal papillae (13 vs c. 24) and microfilariae with a terminal nucleus near the tail tip. In addition, the diameter of the cephalic bulb in M. sofiani n. g., n. sp. is 1.5 times the size of that recorded in the currently two species included in the genus Wuchereria, W. bancrofti and W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 [44][45][46], and its glandular oesophagus is six to seven times longer than that in Wuchereria spp. Unlike in Wuchereria spp., salient cuticular annules are present in the midbody region in the new species, but no cuticular bosses were found on the posterior extremity of females. While the morphology of the left spicule is similar in both Wuchereria spp. and M. sofiani n. g., n. sp. in that the blade is simple, spicules differ in length and are shorter in the new species [44,45].
In a recent comprehensive molecular phylogeny of the Onchocercidae by Lefoulon et al. [26], species of Wuchereria and Brugia formed a strongly supported clade (ONC5) consisting of two further genera of the Onchocercinae, three genera of the Dirofilariinae Sandground, 1921 and four of the Splendidofilariinae Chabaud & Choquet, 1953. Hence, we compared the morphological characteristics of M. sofiani n. g., n. sp. to the generic diagnoses of the respective genera as well.
(T.) dunni, described from the same host as the new species described here, the structure of the right spicule is complex and differs from that in the present specimens [34]. Other than Malayfilaria n. g., microfilariae of species of Breinlia York & Maplestone, 1926 (Onchocercinae) have no sheath [2,19,55,56].
In contrast with the new genus, males of species of Loa Stiles, 1905, Pelecitus Railliet & Henry, 1910 and Foleyella Seurat, 1917 (Dirofilariinae) have strongly developed caudal alae with large, pedunculate papillae [1,2]. In addition, the oesophagus is undivided in species of both Loa and Foleyella, and species of Pelecitus possess well-developed lateral alae [2]. Furthermore, adults of Loa spp. parasitise primates [2], adults of Pelecitus spp. live among the tendons and muscles near the joints of the legs and feet of birds and mammals (North American lagomorphs and Australian marsupials) [1][2][3], and adults of Foleyella spp. occur in the subcutaneous connective tissues and body cavity of agamid and chamaeleonid reptiles [1,3].
Contrary to Malayfilaria sofiani n. g., n. sp., species of Cardiofilaria Strom, 1937, Aproctella Cram, 1931, Rumenfilaria Lankester & Snider, 1982 and Madathamugadia Chabaud, Anderson & Brygoo, 1959 (Splendidofilariinae) have subequal spicules [2,3,57]. Cardiofilaria and Aproctella can be further distinguished from the new genus by caudal papillae that are arranged in a circle or semi-circle around the cloaca of males (sometimes irregularly distributed in Cardiofilaria), and members of both genera are found in the body cavity of birds [1,2,[58][59][60]. Adult worms of Rumenfilaria spp. occur in the subserosal connective tissue between folds of the ruminal wall of moose, Alces alces (Linnaeus, 1758), in Canada and further differ from the new genus in having a smooth Fig. 3 Taxonomic position of Malayfilaria sofiani n. g., n. sp., inferred using the maximum-likelihood method, based on cox1 nucleotide sequences. The tree was based on the Tamura-Nei model, which was selected as the best model (MEGA6) with 500 replicates. The percentage of replicate trees in which the associated taxa clustered together is shown next to the branches. Values > 50% are shown. The tree is drawn to scale, with branch lengths corresponding to the number of substitutions per site. There were a total of 569 positions in the final dataset. The scale-bar below the diagram indicates the number of changes inferred as having occurred along each branch. The red triangles indicate the sequences generated in this study cuticle and nine pairs of caudal papillae [3]. Species of Madathamugadia are found in lizards in the Palaearctic and Afrotropics [59][60][61].

Molecular results
To elucidate the molecular characteristics of M. sofiani n. g., n. sp., we compared the nucleotide sequences of the cox1 and 12S rRNA genes and the ITS1 region with those of related filarial parasites that were available in the GenBank database. As shown in Additional file 2: Table S2, the K2P distance between sequences of the cox1 gene of M. sofiani n. g., n. sp. and other known species was 11.8% for W. bancrofti, 13.8% for B. malayi and 14.1% for B. pahangi. Phylogenetic ML trees based on the cox1 and 12S rRNA genes placed M. sofiani n. g., n. sp. as sister species to the clade formed by W. bancrofti, B. malayi and B. pahangi, indicating that the new species is closer to W. bancrofti than to Brugia spp. (Figs. 3, 4 and 5). These findings are supported by the tree inferred from the ITS1 region (Fig. 6). The NJ trees for the cox1 and 12S rRNA genes and the ITS1 region yielded results similar to the ML trees (see Additional file 3: Figure S1; Additional file 4: Figure  S2; and Additional file 5: Figure S3).
The K2P distance between the sequences of the cox1 gene of M. sofiani n. g., n. sp. and W. bancrofti was larger than that between W. bancrofti and B. malayi (10.6%). The genetic distances between M. sofiani n. g., n. sp. and other related species were 13 Fig. 4 Taxonomic position of Malayfilaria sofiani n. g., n. sp., inferred using the maximum-likelihood method, based on 12S rDNA nucleotide sequences. The tree was based on the Tamura-Nei model, which was selected as the best model (MEGA6), with 500 bootstrap replicates. Values > 50% are shown. Gblocks (version 0.91b, 2002) was used to eliminate poorly aligned positions and divergent regions of the alignment, making the data more suitable for phylogenetic analysis [70]. There were 319 positions in the final dataset. The scale-bar below the diagram indicates the number of changes inferred as having occurred along each branch. The red triangles indicate the sequences generated in this study host data and predilection sites, we conclude that the new species is well distinct from previously described genera and we, thus, propose the new genus Malayfilaria in order to accommodate it.
As detailed above, M. sofiani n. g., n. sp. differs from all of these in having an extremely long glandular oesophagus, annulation in the midbody region and small lappets at the terminal end of the tail. In contrast, the pre-oesophageal cuticular ring is a character shared between the three genera. However, despite being mentioned in the description of Wuchereria sp. from a monkey [6] and the inclusion of this character in the generic diagnosis of Brugia by Buckley [7], detailed descriptions of the preoesophageal cuticular ring are so far available for W. bancrofti [6,45,46], B. malayi [47] and B. patei [48] only. In this study, we provide metrical data for the pre-oesophageal cuticular ring in B. pahangi for the first time. Among the Onchocercidae, Acanthocheilonema spp. also have a salient pre-oesophageal cuticular ring and a stout glandular oesophagus [62,63], while Cercopithifilaria spp. have a small pre-oesophageal cuticular ring and no glandular oesophagus [62]. Chabaud & Bain [64] suggested that Cercopithifilaria was derived from the Acanthocheilonema lineage, and a stout glandular oesophagus is considered to be one of the ancestral characteristics in Onchocerca spp. [65]. Based on this, we infer that the new species has ancestral morphological characteristics when compared with Wuchereria spp. and Brugia spp.
In the present study, we carefully extracted specimens of M. sofiani n. g., n. sp. from the tissues of treeshrews under a stereomicroscope and placed them in phosphate-buffered saline (pH 7.4) in Petri dishes. Regardless of whether or not the worms were fixed in alcohol, we usually observed ornamentation in the midbody region of both sexes of the filarioid (Figs. 1d, 1n, 2c, 2e). Based on the definition provided in the CIH Keys to the Nematode Parasites of Vertebrates [66], we diagnosed this ornamentation as annules. To the best of our knowledge, the presence of annules is a specific characteristic of the new filarioid described here, although various cuticular ornamentations such as transverse ridges, longitudinal ridges and bosses have been identified previously in filarial parasites [2,46,67].
Previous phylogenetic trees based on 12S rDNA and cox1 sequences [21,22], as well as 16S rDNA sequences of the endosymbiont bacteria Wolbachia in the host filarioids [21,24], have shown W. bancrofti to be close to Brugia spp. On the basis of multi-locus sequence analyses, Lefoulon et al. [26] separated taxa of the Onchocercidae into five clades; in one of these, ONC5, W. bancrofti, B. pahangi, B. timori and B. malayi formed a monophyletic group. Tree topologies based on the cox1 and 12S rRNA genes and the ITS1 region obtained in the present study, placed M. sofiani n. g., n. sp. as a sister taxon to the Wuchereria-Brugia clade, represented by W. bancrofti on the one hand and B. malayi and B. Fig. 6 Taxonomic position of Malayfilaria sofiani n. g., n. sp., inferred using the maximum-likelihood method, based on ITS1 nucleotide sequences. The tree was based on the Tamura 3 model, with 1,000 bootstrap replicates. Values > 50% are shown. Gblocks (version 0.91b, 2002) was used to eliminate poorly aligned positions and divergent regions of the alignment, making the data more suitable for phylogenetic analysis [70]. There are 495 positions in the final dataset. The scale-bar below the diagram indicates the number of changes inferred as having occurred along each branch. The red triangles indicate the sequences generated in this study pahangi on the other hand. Based on the K2P distance between cox1 gene sequences, the new species is closest to W. bancrofti.
A total of 81 common treeshrews from various areas in Peninsular Malaysia were examined for M. sofiani n. g., n. sp. However, the parasite was present in five host individuals only, all of them captured in Jeram Pasu, Kelantan, a locality on the east coast of Peninsular Malaysia. We conclude that the prevalence of M. sofiani n. g., n. sp. is very low and that it parasitises common treeshrews inhabiting a limited geographical area.
Insects belonging to the family Culicidae are known to act as vectors for W. bancrofti and Brugia spp., as well as species of Aproctella and Foleyella  [1,26,59,68]. Therefore, although we did not find any potential vectors for M. sofiani n. g., n. sp. in the present study, we speculate that the new species is also transmitted by haematophagous arthropods.
Wuchereria bancrofti and W. kalimantani parasitise humans and monkeys, while Brugia spp. parasitise a wide range of hosts, including primates (humans and monkeys), lagomorphs, carnivores and treeshrews [6,8,31,[48][49][50][51][52][53]. Thus, there are no close relationships between the host species of the three filarial genera, Malayfilaria n. g., Wuchereria and Brugia. Phylogenetically, treeshrews belong to the order Scandentia, which is considered to be more closely related to Primates than to Rodentia and Lagomorpha [30]. The common ancestor diverged into Scandentia, Dermoptera and Primates during the Cretaceous (approximately 90 million years ago), and the genus Tupaia Raffles, 1821 arose at the end of the Miocene (approximately 10 million years ago) [69]. Roberts et al. [28] suggested that Miocene tectonic events, volcanism and geographical instability drove treeshrew diversification in Southeast Asia; while according to Bain [27], the radiation of nascent filarial lineages may have occurred with the expansion of mammals between the Paleocene and the Pleistocene (66 to 2.5 million years ago).
With regard to diversification of filariae in the Wuchereria-Brugia clade, Morales-Hojas [23] suggested that co-speciation between the hosts and parasites was the most plausible driving factor, because some species of Brugia and Wuchereria parasitise humans and monkeys. However, he emphasised that more information on the phylogenetic relationships between the species within this clade was required to discuss their evolution. Since M. sofiani n. g., n. sp. appears to display more ancestral morphological characteristics than either MNHN: Muséum National d'Histoire Naturelle; CIH: Commonwealth Institute of Helminthology