Introduction
The Sumatran elephant (Elephas maximus sumatranus) is a subspecies of the Asian elephant that is native to Sumatra Island of Indonesia. The estimated population of this animal had declined to fewer than 1,700 elephants in 2020 (Sukmantoro Reference Sukmantoro2020). The IUCN (2024) categorized the Sumatran elephant as critically endangered and in need of conservation efforts. In Indonesia, elephants are protected by the Ministry of Environment and Forestry Regulation No. P.106/MENLHK/SETJEN/KUM.1/6/2018 concerning Protected Wild Plants and Animals.
Elephants are an important key species that play many roles in the ecosystem. Efforts to conserve this animal are urgently needed for its preservation, one of which is monitoring its parasites. Helminth parasites commonly found in vertebrate animals can cause problems for the welfare, management, and conservation of wildlife populations both living in captivity and in the wild (Pedersen et al. Reference Pedersen, Jones, Nunn and Altizer2007; Zhang et al. Reference Zhang, Daszak, Huang, Yang, Kilpatrick and Zhang2008). It is useful to monitor the helminths of rare and endemic species, as helminth infections can be linked to population declines (Heard et al. Reference Heard, Smith, Ripp, Berger, Chen, Dittmeier, Goter, McGarvey and Ryan2013).
Many gastrointestinal nematodes have been identified in elephants, and are often associated with protein-losing gastroenteropathy that can cause hypoalbuminaemia, anaemia, enteritis, and even death in younger elephants (Chel et al. Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020). Both Asian (Elephas maximus) and African (Loxodonta spp.) elephants are parasitized by a complex of gastrointestinal nematodes belonging to the family Strongylidae. The nematode parasites that infect elephants in Africa and Asia belong to the same genus; however, they are never the same species (Chabaud Reference Chabaud1957; Chel, Reference Chel, Bawm, Htun, Masum, Ichii, Nonaka, Nakao and Katakura2022; Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938). For Asian elephants, there are four species of the genus Murshidia, namely: M. (Murshidia) falcifera, M. (M.) indica, M. (M.) murshida (syn: M. lanei), and M. neveulemairei (syn: M. elephasi), and five species from the genus Quilonia, namely: Q. edentata, Q. guptai, Q. renniei (=Q. quilona), Q. simhai, and Q. travancra that have been reported (Chabaud Reference Chabaud1957; Chel et al. Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020; Fernando & Fernando Reference Fernando and Fernando1961; Gupta & Jaiswal Reference Gupta and Jaiswal1984; Gupta & Trivedi Reference Gupta and Trivedi1984; Khalil Reference Khalil1922; Lane Reference Lane1914; Prahardani et al. Reference Prahardani, Firdausy, Yanuartono and Nurcahyo2019; Van der Westhuysen Reference Van der Westhuysen1938; Witenberg Reference Witenberg1925; Wu Reference Wu1934).
In Indonesia, studies on the nematodes of elephants are very limited. Most previous investigations have focused on prevalence, relying on the detection of eggs and larvae in faecal samples (Juniar et al. Reference Juniar, Rosa and Rustiati2015; Suandhika et al. Reference Suandhika, Dwinata and Arjana2017; Zilfa et al. Reference Zilfa, Fahrimal, Sayuti, Athaillah, Hamzah and Sari2022). However, identifying strongyle species is challenging because eggs and larvae from faeces are often indistinguishable (Bowman et al. Reference Bowman, Lynn, Eberhard and Alcaraz2003). To date, the only taxonomic study of elephant nematodes in Indonesia was conducted by Prahardani et al. (Reference Prahardani, Firdausy, Yanuartono and Nurcahyo2019), who identified a single species, Quilonia travancra, in Sumatran elephants from Way Kambas. Furthermore, molecular data on elephant nematodes in Indonesia are lacking. In this paper, we provide comprehensive redescriptions of Q. renniei, M. (M.) falcifera, and M. (M.) murshida collected from Sumatran elephant in Aceh, Indonesia, using both light and scanning electron microscopy. We also present the first rDNA sequence of Q. renniei and mtDNA sequences of M. (M.) murshida and M. (M.) falcifera, as well as a new mtDNA sequence of Q. renniei. Accurate identification of parasitic nematode species in Sumatran elephants is crucial for understanding their life cycle, biogeography, and epidemiology, and will contribute to the development of effective prevention strategies.
Ethical approval
This study obtained permission from the Ministry of Environment and Forestry, General Directorate of Natural Resources Conservation and Ecosystems: 87/KSDAE/SET.3/ KSA.2/5/2023, and a letter of recommendation for taking research samples (SATS-DN) from the Secretariat of Scientific Authority for Biodiversity, National Research and Innovation Agency number B-2297/IV/KS.00/2/2023.
Materials and methods
Morphological identification
A total of 17 individual adult nematodes deposited in Syah Kuala University and preserved in 70% ethanol were used for this study. The specimens were collected in 2023 from the faeces of Asian elephants maintained in the Sare Conservation Response Unit, Aceh, one day after oral albendazole administration at a dose of 5–7 mg/kg of body weigh by veterinarians of Syah Kuala University.
Specimens were first measured for total body length and width. The midbody of each specimen was used for molecular analysis, while the anterior and posterior parts were processed for morphological identification. For light microscopy, these parts were cleared in lactophenol and observed under an Olympus BX50 upright microscope (Olympus Company, Tokyo, Japan), equipped with a DP26 digital camera (Olympus) and cellSens Entry. Following identification, the anterior and posterior worms were rinsed in cacodylate buffer, post-fixed in glutaraldehyde, treated with tannic acid and then serially dehydrated in ethanol (50%, 70%, 85%, 90%, 100%), and vacuum-dried for 10 minutes using an aspirator (Luchi Seieido Co., Osaka, Japan). Each sample was then mounted on a specimen stub and coated with gold at 5–8 mA for 5 min using an Eiko IB-2 (Eiko Co., Tokyo, Japan) ion coater. The photographs were captured using a scanning electron microscope (SEM), JEOL JSM-IT 200 (5 kV; Tokyo, Japan). The measurements are presented in micrometres unless otherwise indicated. The specimens were identified according to Chabaud (Reference Chabaud1957), Lichtenfels (Reference Lichtenfels, Anderson, Chabaud and Willmott1980), and descriptions of species (Lane Reference Lane1921; Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938; Witenberg Reference Witenberg1925). Voucher specimens have been deposited in the Museum Zoologicum Bogoriense (MZB), Bogor, Indonesia, with accession numbers MZBNa-SEM 1, 2 for Q. renniei, MZBNa-SEM 3 for M. falcifera, and MZBNa-SEM 4 for M. murshida.
DNA extraction, PCR, and sequencing
To determine genetic identities, we amplified each DNA extract at two loci: the rDNA region (including ITS1, 5.8S, and ITS2) and the mitochondrial DNA from the COI region. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA), following the manufacturer’s instructions. The DNA was eluted with 100 μl of the provided elution buffer. PCR amplification of the rDNA region was performed using the forward primer NC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and the reverse primer NC2 (5′-TTAGTTTCTTTTCCTCCGCT-3′) (McLean et al. Reference McLean, Kinsella, Chiyo, Obanda, Moss and Archie2012) to target a ~900 bp amplicon. For mitochondrial DNA from the COI region was amplified using primers HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) and CO3R (5′-GAATTGGCAAAGCCTGGTT-3′) (McLean et al. Reference McLean, Kinsella, Chiyo, Obanda, Moss and Archie2012) to target a ~400 bp amplicon. Each PCR contained 3 μl of extracted DNA, 12.5 μl of PCR Master Mix (MyTaq™ HS Red DNA Polymerase, Meridian Bioscience), 0.75 μl of each primer (10 pmol μl–1), and ddH2O to a final volume of 25 μl. We performed amplification on an Eppendorf Mastercycler Nexus Gradient (Eppendorf, Germany). The rDNA cycling protocol included: 95°C for 10 minutes; 40 cycles of 95°C for 45 seconds, 61°C for 45 seconds, and 72°C for 45 seconds; and 72°C for 5 minutes; and a hold at 4°C. The COI cycling protocol included: 95°C for 10 minutes; 40 cycles of 95°C for 1 minute, 45°C for 1 minute, and 72°C for 1 minute; 72°C for 5 minutes; a hold at 4°C. PCR products were visualized on a 2% agarose gel, and purified amplicons were sent to 1st BASE via PT Genetika Science Indonesia.
Phylogenetic analysis
We attempted to sequence the nuclear DNA region (including ITS1, 5.8S, and ITS2) and mitochondrial COI of Q. renniei, M. (M.) falcifera, and M. (M.) murshida. For mt COI, three newly generated sequences of the mt COI gene were obtained, one of each for Q. renniei, M. (M.) murshida, and M. (M.) falcifera. However, for rDNA, amplification was only successful for Q. renniei (n = 8). DNA sequences of the mitochondrial COI and rDNA were assembled using Geneious® Prime version 2025.0.3 (Biomatters Ltd., Auckland, New Zealand).
Newly generated ITS and COI sequences were aligned separately with representative sequences of the elephant nematodes belonging to subtribe Murshidiinii and Quiloniinii retrieved from GenBank. For both datasets, Cyathostomum tetracanthum was used as the outgroup. Sequence alignments were performed using MAFFT v7.490 (Katoh and Standley Reference Katoh and Standley2013). The ends of the alignments were trimmed to match the shortest sequence, and all sequence gaps were retained in the analyses. Phylogenetic relationships were inferred using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods for the ITS and COI datasets in Geneious® Prime. Maximum Likelihood analyses were conducted using RAxML under the GTR+GAMMA substitution model with 1000 bootstrap replicates to assess nodal support. Bayesian Inference analyses were performed using MrBayes v3.2.6 (Huelsenbeck and Ronquist Reference Huelsenbeck and Ronquist2001) as implemented in Geneious® Prime, with default settings. The HKY85 substitution model with gamma-distributed rate variation among sites (four gamma categories) was used. Markov chain Monte Carlo (MCMC) analyses were run with four heated chains (temperature = 0.2) for 1,100,000 generations, sampling every 200 generations (Larget and Simon Reference Larget and Simon1999). The first 100,000 generations were discarded as burn-in. A random seed value of 25674 was used. Posterior probabilities were used as measures of branch support. The resulting trees were visualized with FigTree v1.4.4.
Result
This study used 17 specimens of adult worm parasites from Sumatran elephant. The examination revealed the presence of the following worms: Quilonia renniei (n = 8), Murshidia (Murshidia) falcifera (n = 4), and M. (M.) murshida (n = 5). Despite being expelled in the faeces following anthelmintic treatment, SEM analysis revealed that the worms’ cuticles remained intact. The absence of wrinkling or surface degradation suggests the medication did not compromise their structural integrity. Detailed descriptions of each worm are as follows:
Description
Quilonia renniei (Railliet, Henry and Joyeux, Reference Railliet, Henry and Joyeux1913) (Figs. 1–3, Table 1)
Anterior region of Quilonia renniei. a. En face view of male showing 18 elements of leaf-crown and buccal teeth, b. Oesophageal region (lateral view), c. Anterior region showing buccal cavity (ventral view), d. Anterior region showing leaf-crown (lateral view), e. Amphid, f. Buccal tooth, g. Sub-median papillae. Scale bars: a, d: 20 μm; b: 100 μm; c: 50; e: 2 μm, f, g: 5 μm.
Posterior regions of Quilonia renniei. a. Bursa (ventral view), b. Dorsal ray (ventral view), c. Genital cone (ventral view), d. Gubernaculum, e. Posterior region of female (lateral view), f. Female tail (lateral view), g. Female reproduction system showing infundibula (in), sphincters (sp), vestibula (vs) (lateral view). Note: 1 is anterior branch, 2 is posterior branch. Scale bars: a: 100 μm; b: 50 μm; c, d: 20 μm: e: 400 μm; f: 200 μm; g: 100 μm.
SEMs of the anterior end of anterior end of female Quilonia renniei specimens bearing same number of coronal leaflets.
Morphometrical data Quilonia renniae from Asian elephants
* Source: Chel et al. (Reference Chel, Bawm, Htun, Masum, Ichii, Nonaka, Nakao and Katakura2022).
Nematode No. 2 from Indian elephant, Evans and Rennie Reference Evans and Rennie1910.
Evansia renniei Railliet, Henry, and Joyeux, Reference Railliet, Henry and Joyeux1913; Lane, Reference Lane1915.
Quilonia quilona Lane, Reference Lane1914.
Evansia renniei Lane, Reference Lane1915.
Nematevansio renniei (Ihle, Reference Ihle1919).
Quilonia renniei Ihle, Reference Ihle1919.
General: Stout worms. Mouth triradiate, terminal, surrounded by four prominent sub-median papillae (Fig. 1A). Oesophagus simple and stout, enlarge posteriorly (Fig. 1B). Amphids situated posterior to level of sub-median papillae. Leaf-crown elements project freely from mouth, thin, long, 18 in male (n = 1, Fig 1A), 20 in female (n = 7, Figs. 3). Buccal capsule wide, shallow, ring-like (Fig. 1C). Two tooth-like structure present at base of buccal capsule anterior to sub-ventral lobes of oesophagus, small cavity at the tip of tooth (Figs. 1A, F). Sub-median papillae elongated, 22.7 long, projecting anteriorly from perioral cuticle; distal segment ovoid, 7.9 long, 37.5 wide; median segment cylindrical, 15.4 long, 7.1 wide; proximal segment short, 7.8 long, 3.7 wide (Fig. 1G).
Male: Copulatory bursa symmetrical (Fig. 2A). Ventral rays stout, parallel, close together. Lateral rays divergent from each other, externo-lateral shortest not reach margin of bursa, medio-lateral almost same length as postero-lateral and reach margin of bursa. Externo-dorsal rays derived from median stem of dorsal ray. Dorsal ray bifurcated for about one-third of its length, each branch divides near distal end into outer and inner branchlets, and each inner branchlet divided again apically (Fig. 2B). Simple genital cone present with one pair of tiny dorsal raylets and one pair of ventral cuticular appendages (Fig. 1C). Spicules slender with blunt tip, 891 long. Gubernaculum long with tapered tip, 69 long (Fig. 2D).
Female (n = 2): Ovejectors Y-shaped (type 1 according to Lichtenfels (Reference Lichtenfels, Anderson, Chabaud and Willmott1980)). Uterine branches initially opposed, posterior branch turns and runs anteriorly (Fig. 2G). Vulva lip salient (Fig. 2E). Tail straight, long, and pointed (Figs. 2E, F).
Murshidia (Murshidia) falcifera (Cobbold, Reference Cobbold1882) (Figs. 4 – 6, Table 2)
Anterior region of Murshidia (Murshidia) falcifera. a. Cephalic end (apical view) showing leaf-crown, b. Cephalic end showing buccal capsule, c. Oesophageal region (lateral view), d. Submedian cephalic papilla, e. Cervical papilla, f. Amphid, g. Excretory pore, h. Leaf-crown tip. Scale bars = a, c: 20 μm; b: 200 μm; d, g, h: 2 μm; e, f: 5 μm.
Posterior region of Murshidia (Murshidia) falcifera. a. Bursa and spicules (lateral view), b. Spicule tip, c. Diagram of dorsal ray, d. Dorsal ray, e. Hair-like projections on genital cone, f. Internal surface of dorsal ray, g. Diagram of ventral and lateral rays. Scale bars = a: 200 μm; b: 20 μm; c, d, g: 100 μm; e: 10 μm; f: 5 μm.
Posterior region of Murshidia (Murshidia) falcifera. a. Bursa (ventral view), b. Single papilla on genital cone, in front of cloacal opening, c. Papilla on the outer lateral branch of bursa, d. Female, posterior end (lateral view), e. Female, posterior end (lateral view), f. Female reproduction system showing infundibula (in), sphincters (sp), vestibula (vs), and uteri (ut) (lateral view). Scale bars: a: 100 μm; b, c: 5 μm; d: 200 μm; e: 50 μm; f: 400 μm.
Morphometrical data of Murshidia (M.) falcifera from Indian elephants
Strongylus falcifer Cobbold, Reference Cobbold1882; Mitter, Reference Mitter1912.
Nematode No. 3, Evans and Rennie, Reference Evans and Rennie1910.
Cylicostomum falciferum Railliet, Henry, and Bauche, Reference Railliet, Henry and Bauche1914.
Murshidia (Murshidia) falcifera Lane, Reference Lane1914.
Description: Body straight, tapering anteriorly, finely striated at intervals of 3.8–4.0. Four tall, conical, sub-median papillae protrude from rounded mouth-collar. External leaf-crown arising from inner surface of mouth collar, consisting of 60 elements (Fig. 4A), elements longer laterally than ventrally or dorsally with longest element 18.5 long, shortest leaflets reach almost to level of mouth margin, each element blunt-tipped (Fig. 4C). Internal leaf-crown absent. Mouth terminal. Oral cavity deep, without teeth; buccal capsule elliptical transversely, width much greater than depth (Fig. 4B). Amphid distinct, dorsal region flower-shaped (Fig. 4D). Papillae consisting of 3 segments; proximal segment rectangular, 22.2 long, 11.9 wide, median segment 3.7 long, 3.4 wide, and distal segment 3.4 long, 2.3 wide (Fig 4E). Oesophagus short, stout, bottle-shaped, with well-defined narrowing at nerve ring (Fig. 4F). Plumose sculpturing of oesophagus absent. Deirids filiform, 37.2 long (Fig. 4G). Deirids and excretory pore (Fig. 4H) posterior to oesophagus.
Male (n = 3): Body widest just anterior of bursa. Bursa distinctly divided into three lobes, with dorsal lobe comparatively short and broad, difficult to unroll (Figs. 5A, 6A). Spicules similar, stout, middle third of each spicule provided with two striated incurved alae that tapper gradually; distal tip of spicule bent dorsally (Fig. 5A, B). Ventro-ventral and ventro-lateral rays apposed, divergent from lateral trunk (Fig. 5C). Medio-lateral and postero-lateral rays parallel, divergent from externo-lateral ray. Externo-dorsal rays derived from base of dorsal ray, close to lateral rays. Dorsal ray deeply cleft in mid-line, and each composed of three subdivisions, unequal in length, first (1) shortest and widest, last (3) longest (Figs. 5D, E). Two papillae present in each outer lateral ray. Internal base of bursa with numerous tiny, long hair-like projections (Fig. 5F). Internal surface of bursa over externo-dorsal and dorsal rays rugose (Fig. 5G). Simple genital cone present with one pair of ventral, cuticular appendages. Single small papilla anterior to cloacal opening (papilla 0 according to Gibbons and Khalil Reference Gibbons and Khalil1982) (Figs. 6A, B). Two small papillae present at each external surface of lateral bursa (Figs. 6A, C)
Female: Tail long, gradually tapering to point (Figs. 6D, E). Vulva immediately anterior to anus. Vagina runs anteriorly to ovejector, two uteri run parallel with one another anteriorly, terminating in ovaries. Ovejectors Y-shaped (type 1 according to Lichtenfels (Reference Lichtenfels, Anderson, Chabaud and Willmott1980)), well developed, muscular vagina short, S-shaped, 207, vestibule 490, sphincter 455, infundibulum 65 (Fig. 6F).
Murshidia (Murshidia) murshida Lane, Reference Lane1914 (Figs. 7–8, Table 3)
Murshidia (Murshidia) murshida. a. Oesophageal region, b.Cephalic end (apical view), c. Cephalic end showing buccal capsule, d. Mouth opening showing leaf-crown, e. Amphid, f. Submedian cephalic papilla, g. Internal surface of dorsal ray. Scale bars: a: 200 μm; b, c, d, e, g: 100 μm; f: 10 μm; h: 20 μm; i: 5 μm.
Murshidia (Murshidia) murshida. a. Bursa and spicule, b. Bursa, c. Hair-like projections on genital cone, d. Posterior end of female, lateral view, e. Female production organs showing infundibula (in), sphincters (sp), vestibula (vs), and uteri (ut) (lateral view). Scale bars: a, d, e: 200 μm; b, c: 100 μm.
Morphometrical data of Murshidia (M.) murshida from Asian elephants
Murshidia lanei Witenberg, Reference Witenberg1925
Description: Body straight with fine striations, 5.0–5.1 apart. Mouth collar well developed, rounded in outline, distinctly separated from rest of body by deep groove (Fig. 7A). Oral aperture bordered by 40 blunt-tipped leaf-crown elements, longer laterally than ventrally or dorsally, longest element 17.4 long, smallest 6.2 long (Fig. 7B). Amphid distinct, dorsal region flower-shaped (Fig. 7C). Oral cavity without teeth; buccal capsule elliptical transversely, width greater than depth (Figs. 7D, E). Four protruding submedian cephalic papillae project freely above head; papillae consisting of 3 segments; proximal segment rectangular, 11.4 long, widest in basal area 10.0, median segment 6.4 long, 4.0 wide, distal segment ovoid, 7.3 long, 3.0 wide (Fig. 7F). Oesophagus short, stout, bottle-shaped, with well-defined narrowing at nerve ring (Fig. 7D). Plumose sculpturing of oesophagus absent.
Male: Internal surface of distal margin of bursa with area of bluntly pointed hairs. Bursa wider than long, distinctly divided into dorsal and 2 lateral lobes, difficult to unroll; internal surface of bursa over externo-dorsal and dorsal rays rugose. Bursa and spicules resemble those of M. (M.) falcifera (Figs. 7D, 8A–C).
Female: Female tail slightly bent ventrally Tail markedly constricted and then tapers gradually to very fine point. Vulva opens immediately anterior to anus (Fig. 8D). Ovejector resembles that of M. (M.) falcifera, muscular vagina short, 412, vestibule 337, sphincter 409, infundibulum 604 (Fig. 8E).
Molecular characterization
Eight ITS sequences of Q. renniei were newly generated in this study and exhibited nucleotide differences ranging from 0% to 0.46%. Eight representative sequences were included in the phylogenetic analyses together with other sequences of cyathostomine nematodes parasitizing elephants available in GenBank. The final alignment comprised 716 bp. The ITS sequences of Q. renniei are deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) (accession numbers PZ318265—PZ318272).
Phylogenetic relationships inferred using Bayesian inference and Maximum Likelihood analyses of the ITS dataset produced congruent topologies (Fig. 9). All Quilonia sequences formed a strongly supported monophyletic clade (100/1). Within this clade, the newly generated sequences of Q. renniei clustered together and were recovered as a sister to Q. africana. Within the Q. renniei clade, the Quilonia sp. sequence from Asian elephants in China was nested, showing sequence identities of 98.30–98.81%. A second major clade in the ITS phylogenetic tree comprised representatives of Murshidia and Khalilia. Murshidia formed a well-supported monophyletic group, including M. africana, M. longicaudata, and M. linstowi. Khalilia sameera occupied a basal position relative to the clade formed by Murshidia and Quilonia.
Relationships between eight newly generated sequences of Q. renniei and other members of elephant nematodes belonging to subtribe Murshidiinii and Quiloniinii based on Bayesian inference (BI) of the ITS datasets. GenBank accession numbers are indicated alongside taxa name. Posterior probabilities from the BI analyses and bootstrap support from the ML analyses are shown above the branches. The scale bar indicated the expected number of substitutions.
Three newly generated sequences of the mt COI gene were obtained, one of each for Q. renniei, M. (M.)murshida, and M. (M.) falcifera. All were aligned with related sequences retrieved from GenBank and yielded a final dataset of 342 bp for the analysis. In the phylogenetic analyses, the newly generated sequences of Q. renniei and M. (M.)falcifera clustered with conspecific sequences from GenBank (LC513783 and LC513776) (Fig. 10), showing 96.5% and 97.5% identity, respectively. No reference for the COI sequence of M. (M.) murshida was available in GenBank. However, the sequence generated here was placed within the Murshidia assemblage. The COI sequences of Q. renniei, M. (M.) murshida, and M. (M.) falcifera are deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) (accession numbers PZ310337, PZ310338, and PZ310336).
Relationships among three newly generated sequences of the COI, Q. renniei, M. (M.) murshida, and M. (M.) falcifera, with other elephant nematodes belonging to subtribe Murshidiinii and Quiloniinii based on Bayesian inference (BI) analyses of the COI datasets. GenBank accession numbers are indicated alongside taxa name. Posterior probabilities from the BI analyses are shown above the branches. The scale bar indicated the expected number of substitutions.
Bayesian inference and Maximum likelihood analyses of the COI alignment produced trees with largely similar overall structure but differed in the placement of several weakly supported nodes (e.g., M. indica and Q. renniei). Neither Murshidia nor Quilonia formed a monophyletic group in either analysis. In addition, both analyses consistently recovered a strongly supported deep split separating a clade comprising K. sameera, Q. africana, M. africana, and M. longicaudata from the remaining taxa. In contrast, relationships among the remaining species were poorly resolved and generally supported by low posterior probability and bootstrap values.
Discussion
Morphological study
During the late Miocene, ~6.6–8.8 million years ago (Mya), the lineages of Asian and African elephants diverged from a common African ancestor (Rohland et al. Reference Rohland, Reich, Mallick, Meyer, Green, Georgiadis, Roca and Hofreiter2010; Kappelhof et al. Reference Kappelhof, Diepeveen, Derks, Madsen, Rogers, Goossens, Sharma, Groenen, Windig and Bosse2019). This probably served as a primary driver for the vicariant speciation in their helminth fauna. As Loxodonta species (African elephants) remained in the African tropics and Elephas (Asian elephant) adapted to the varied landscapes of Eurasia, their respective parasite communities apparently underwent parallel evolution. Today, although both elephant lineages are infected by the same nematode genus, the specific parasite species differ between continents (Chabaud Reference Chabaud1957; Chel et al. Reference Chel, Bawm, Htun, Masum, Ichii, Nonaka, Nakao and Katakura2022; Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938), mirroring the ancient evolutionary divergence of their hosts. As large, long-lived, and highly social animals, elephants provide a stable ecological “island” for parasites, fostering a unique and highly specialized helminth community.
Elephants harbour a diverse community of gastrointestinal nematodes, primarily from the family Strongylidae (specifically the genera Murshidia, Quilonia, and Khalilia) (Lichtenfels Reference Lichtenfels, Anderson, Chabaud and Willmott1980). The current morphological data of many species are far from being complete. Thus, the descriptions for species are scattered; many of them are old papers, and there is no detailed description accompanied by line drawings or photomicrographs presented in a single paper. Therefore, the nematode species belonging to Murshidia and Quilonia are difficult to distinguish based on morphology and measurements as presented in earlier papers. This situation can be resolved only by redescription of new nematode collections.
Within the Asian elephant population, four subspecies are recognized by the International Union for Conservation of Nature (IUCN) Red List: E. maximus indicus on the mainland, E. maximus maximus in Sri Lanka, E. maximus sumatranus on Sumatra, and E. maximus borneensis on Borneo (Williams et al. Reference Williams, Tiwari and Goswami2020; IUCN 2024). In this study, we redescribed three cyathostomine nematodes not previously reported from E. maximus sumatranus: Quilonia renniei, Murshidia (Murshidia) falcifera, and M. (M.) murshida by using both light and scanning electron microscopes.
Murshidia and Quilonia belong to the family Strongylidae because they have Y-shaped ovejectors in the female reproductive tract and 3 rami of the dorsal ray of the male copulatory bursa. They are members of the subfamily Cyathostominae because they have a cylindrical buccal capsule (Lichtenfels Reference Lichtenfels, Anderson, Chabaud and Willmott1980). Quilonia belongs to the tribe Quiloninea by having a small ovejector and completely opposed uteri. Murshidia is a member of the tribe Murshidiinea because it has Y-shaped ovejectors and the uteri are parallel from their origin. The genus Quilonia is characterized by buccal capsule walls not being attached to the lining of the buccal cavity, the vulva is in the posterior third of the body, but the vagina is very short. Whilst the genus Murshidia has a long female tail and the spicule tips are simple (Lichtenfels Reference Lichtenfels, Anderson, Chabaud and Willmott1980). The morphology and the measurements of the present specimens are nearly identical to those of Q. renniei, M. (M.) falcifera, and M. (M.) murshida as currently described (Figs. 1–8; Tables 1–3). The measurements of Q. renniei and M. (M.) falcifera males are smaller compared to other records. However, the female measurements are within the range of previous descriptions. M. (M.) murshida in this study is longer than in other descriptions; however, the other measurements were within the range of previous descriptions.
The genus Murshidia was described by Lane (Reference Lane1914), with M. murshida as the type species. The same species was reported by Witenberg (Reference Witenberg1925) under the name M. lanei, from a single male specimen. Van der Westhuysen (Reference Van der Westhuysen1938) provided data for a single male specimen that shared no distinguishable traits with M. murshida. Chabaud (Reference Chabaud1957) conducted a careful analysis of the genera Quilonia and Murshidia, reviewing and re-examining the specimen of Neveu-Lemaire. He pointed out that many were teratological specimens, leading to errors in the identifications by Neveu-Lemaire. He agreed with Van der Westhuysen (Reference Van der Westhuysen1938) and designated M. lanei as a junior synonym of M. murshida. For Asian elephants, he also synonymized M. elephasi with M. neveu-lemairei. Chabaud (Reference Chabaud1957) also separated Murshidia parasitizing elephants into two subgenera: so called ‘primitive forms’, which have 20 to 40 external leaf-crown elements, and the oral capsule rounded in cross-section in the sub-genus Pteridopharynx, and ‘evolved forms’, which have an external leaf-crown of 40 to 80 elements, a laterally compressed oral capsule, and a dorsoventrally elongated oral opening in the subgenus Murshidia.
The number of leaf-crown elements of Murshidia and Quilonia is an important character in their diagnosis, which is often counted in lateral views in the original description and other publications (Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938; Witenberg Reference Witenberg1925). Chabaud (Reference Chabaud1957) and Chabaud and Rousselot (1956) stated that the number of these elements can only be determined with certainly in apical views. Mistakes can easily be made in lateral views. Khalil (Reference Khalil1922) described 28 leaf-crown elements in M. linstowi in lateral views, while Chabaud and Rousselot (1956) re-determined the number of elements as 46 in apical views.
An error was made by Van der Westhuysen (Reference Van der Westhuysen1938), who gave special emphasis to the presence of a second leaf-crown in Murshidia although this structure is not mentioned in the previous papers by Lane (Reference Lane1914), Khalil (Reference Khalil1922), and Witenberg (Reference Witenberg1925). Chabaud (Reference Chabaud1957) refuted that statement and noted that the second leaf-crown mentioned by Van der Westhuysen was an artifact. Based on the SEM images of M. (M.) falcifera and M. (M.) murshida, it is also clear that there is no second leaf-crown. The SEM observations of the apical views of M. (M.) falcifera and M. (M.) murshida also revealed that the number of the external leaf-crown elements is 60 and 40 elements in number, respectively. They are longer laterally than ventrally or dorsally. All previous papers noted that the number of elements of the external leaf-crown in M. (M.) falcifera was 80 elements (Chel et al. Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020; Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938; Lane Reference Lane1914), and 80–85 elements (Chabaud Reference Chabaud1957); while M. (M.) murshida had 60 elements (Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938; Lane Reference Lane1914), and 58 elements (Chabaud Reference Chabaud1957).
Chabaud (Reference Chabaud1957) stated that the number of external leaf-crown elements in Quilonia is constant for each species. However, Chel et al. (Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020), in their observation of the apical view of Q. renniei using SEM, reported that the number of elements in Q. renniei varies between 16 and 19 in males and 19 and 20 in females. In the present study, the number of elements was 18 (n = 1) in males and consistently 20 in females (n = 7) (Fig. 3; only six presented in the figure). Therefore, further research is required to determine whether the number of external leaf-crown elements in each species of Murshidia and Quilonia is truly constant.
The use of a light microscope to observe the number of external leaf-crown elements is still possible in Quilonia spp., which have a smaller number and larger elements. However, due to its larger number and smaller elements, it is challenging to distinguish one element from another using a light microscope in apical views of the external leaf-crown elements in the subgenus Murshidia (40 to 80). Additionally, the long elements on the lateral side occasionally bend, making it possible to count elements twice or for them to obscure the bases of adjacent elements. Therefore, we recommend observing the apical view with the aid of SEM to determine precisely the number of leaf-crown elements.
Van der Westhuysen (Reference Van der Westhuysen1938) constructed a key for the identification of Quilonia species. He placed Q. renniei in the group which has no buccal teeth. The recent publication of Chel et al. (Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020) also did not mention that this species has buccal teeth. Chabaud (Reference Chabaud1957) noted that two sub-ventral buccal teeth are specific characteristics of Quilonia. The detailed observations of Q. renniei made here show two tooth-like structures at the base of the buccal capsule anterior to the sub-ventral lobes of the oesophagus. Chabaud (Reference Chabaud1957) also described for the first time curious filamentous structures on the genital cone of Murshidia males in all species, which seem to be able to be useful for specific diagnoses. In the current study, this character was also seen clearly in both M. (M.) falcifera and M. (M.) murshida by light and scanning electron microscopy. The present SEM observations also revealed the presence of ‘cells’ attached in the dorsal region of amphidial pores in M. (M.) falcifera and M. (M.) murshida which may secrete mucus. It needs to be proven whether this structure exists only in M. falcifera and M. murshida or all species of members of the genus Murshidia.
Molecular study
The current nematode classification by Hodda (Reference Hodda2022) and Lichtenfels (Reference Lichtenfels, Anderson, Chabaud and Willmott1980), Murshidia and Quilonia are classified within the tribe Cyathostomini, which comprises six subtribes. Murshidia is placed in the subtribe Murshidiinii, along with Khalilia, while Quilonia belongs to the subtribe Quiloniinii. However, this arrangement is not supported by molecular evidence from the ITS region. In the present study, K. sameera was placed as a distinct basal lineage relative to the clade formed by Murshidia and Quilonia. A similar pattern was also reported by McLean et al. (Reference McLean, Kinsella, Chiyo, Obanda, Moss and Archie2012), whose ITS phylogeny failed to place Khalilia with Murshidia. Instead, Khalilia appeared as a sister to Q. africana, although with low support. These results raise questions about the current placement of Khalilia within Murshidiinii and highlight the need for reassessment based on molecular phylogenetic evidence.
A representative ITS sequence of Quilonia sp. from China was included in the present dataset. Zhu et al. (Reference Zhu, Hou, Zhang, Yan and Tang2025) reported that their Quilonia sp. sequences showed close BLAST matches to Q. africana from African elephants in Kenya (percent identity: 96.28–96.59%; query coverage: 98–99%). However, in the present ITS phylogenetic tree, a sequence of Quilonia sp. clustered with the eight newly generated Q. renniei sequences with high sequence identity (98.30–98.81%). Given that Q. africana is a parasite of African elephants and that nematode parasites from African and Asian elephants have never been reported to be conspecific, even though they belong to the same genera (Chabaud Reference Chabaud1957; Khalil Reference Khalil1922; Van der Westhuysen Reference Van der Westhuysen1938). This strongly suggests that Quilonia sp. sequence represents Q. renniei rather than Q. africana. A similar discrepancy is reported by Hota et al. (Reference Hota, Sahoo, Dash, Pahari, Mohanty and Sahoo2020), who identified Murshidia linstowi from an Indian elephant calf using rDNA sequences (ITS1, 5.8S, and ITS2). However, the phylogenetic tree of this study and Hamad et al. (Reference Hamad, Junsiri, Sumpanpae, Narapakdeesakul and Taweethavonsawat2025) confirmed that Murshidia sequences from Asian elephants form a distinct clade, clearly separated from the true African M. linstowi sequences reported by McLean et al. (Reference McLean, Kinsella, Chiyo, Obanda, Moss and Archie2012).
In the present study, nucleotide diversity among eight Q. renniei specimens based on ITS datasets ranged from 0% to 0.46%. Using a different genetic marker (COI), Chel et al. (Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020) likewise observed a similar level of intraspecific variation in Q. renniei with a mean nucleotide diversity of 0.99% based on 29 specimens. In the present COI analyses, however, the newly generated sequence of Q. renniei showed 96.5% identity to a representative sequence from Chel et al. (Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020). A comparable pattern has also been reported in cyathostomine nematodes by Louro et al. (Reference Louro, Kuzmina, Bredtmann, Diekmann, de Carvalho, von Samson-Himmelstjerna and Krücken2021), who showed a wide range of COI sequence identify (86.7–100%) within morphologically defined species, indicating the presence of deeply divergent mitochondrial lineages within a single species. In addition, all specimens of Q. renniei herein examined by SEM showed a consistent number and arrangement of coronal leaflets, whereas Chel et al. (Reference Chel, Bawm, Htun, Masum, Ichii, Nonaka, Nakao and Katakura2022) reported some variation in this character. This difference likely reflects population-level morphological variability and/or differences in geographic origin and sampling.
In contrast to the ITS results, phylogenetic analyses based on the mitochondrial COI gene did not support the monophyly of either Murshidia or Quilonia. In both Bayesian inference and Maximum Likelihood trees, species of these two genera were interspersed among each other, and neither genus formed a single, well-supported clade. Although a deeply divergent lineage comprising K. sameera, Q. africana, M. africana, and M. longicaudata was consistently supported, relationships among the remaining taxa were poorly resolved and characterized by low posterior probability and bootstrap values. A similar pattern has been reported in cyathostomine nematodes, where COI-based analyses were able to discriminate species but frequently placed species from different genera closer together than species within the same genus (Louro et al. Reference Louro, Kuzmina, Bredtmann, Diekmann, de Carvalho, von Samson-Himmelstjerna and Krücken2021). Even in the COI phylogeny of Chel et al. (Reference Chel, Iwaki, Hmoon, Thaw, Soe, Win, Bawm, Htun, Win, Oo, MdA, Ichii, Nakao, Nonaka and Katakura2020), where Murshidia and Quilonia appeared as separate clusters, the relevant intergeneric nodes were supported by low bootstrap values, indicating weak support for deeper relationships. Together, these results indicate that mitochondrial COI provides limited resolution for inferring generic-level relationships in cyathostomines, and that the lack of monophyly observed here for Murshidia and Quilonia likely reflects limitations of the marker rather than true generic-level non-monophyly.
Conclusions
Three cyathostomine nematodes, Quilonia renniei, Murshidia (Murshidia) falcifera, and M. (M.) murshida were redescribed from Sumatra elephant. All identified specimens belong to the Strongylidae family that can cause damage to the intestinal lining and microvasculature to utilize host nutrients, ultimately causing nutritional deprivation. This study has enabled the observation of previously unreported morphological features of taxonomic significance and corrected some errors. Molecular analyses based on ITS and COI confirmed the identity of Quilonia renniei, Murshidia murshida, and M. falcifera and showed that the ITS marker provides better phylogenetic resolution than the COI gene in cyathostomine nematodes.
Acknowledgements
This study part of the ‘Rumah Program Pengungkapan dan Pemanfaatan Biodiversitas Nusantara’ of the Research Organization for Life Sciences and Environment-BRIN, year 2023–2024, and ‘Riset dan Inovasi Indonesia Maju (RIIM)’ sponsored by the Indonesia Endowment Fund for Education Agency (LPDP), year 2024, with contract numbers B-1116/II.7.5/FR.06/3/2024 and B-949/III.5/FR.06.00/3/2024. The authors thank Prof. (Em.) Ian Beveridge for helpful suggestions on earlier versions of this text. We also thank Dr. Lynda Gibbons for the confirmation of Murshidia’s amphid.
Author contribution
KD: designed and conducted the study, prepared the manuscript draft, conducted light and scanning electron microscopy, performed molecular analysis, and species identification. GA: performed molecular analysis and prepared the manuscript draft. MH, AS, YF: provided resources and contributed to study design. YSF: provided molecular methods and conducted laboratory work for molecular studies. YA: conducted laboratory work for molecular studies. RF, FF, RR, RRW, and RM: provided resources. All authors have read, reviewed, and approved the final manuscript.
