Introduction
Mermithid nematodes are obligate invertebrate parasites dating back to the Early Cretaceous. Their fossil records are exceedingly sparse, especially before the Cenozoic era. Thus, little is known about their early host associations (Luo et al. Reference Luo, Poinar, Xu, Zhuo, Jarzembowski and Wang2023). The family Mermithidae Braun, Reference Braun1883, comprises a group of parasitic nematodes with developmental hosts in the Arthropoda and Mollusca (Poinar Reference Poinar1983). Their hosts include species of at least 15 different orders of insects (Nickle Reference Nickle1972).
Integrated pest management approaches aim to avoid causing harm to beneficial organisms in agroecosystems; among them, mermithids can be considered very useful candidates because their potential as entomoparasites has already been realized and documented.
Hexamermis Steiner Reference Steiner1924 has been widely used in studies where agricultural pests are infested by entomopathogenic nematodes (Achinelly & Camino Reference Achinelly and Camino2008). The genus is classified under Mermithidae and characterized by a morphological pattern very similar to what is observed in Agamermis (Cobb et al. Reference Cobb, Steiner and Christie1923), with a very significant difference of presence (vs absence) of tail tip appendage that is always preserved after final molt (Hernández-Crespo & Santiago-Álvarez Reference Hernández-Crespo and Santiago-Álvarez1997). Hexamermis species are generally characterized by their six cephalic papillae; cuticle with distinct cross fibers; mouth central and terminal; small amphids; six hypodermal cords; J, V, or rarely a modified S-shaped vagina; and paired and separate spicules (Poinar & Linares Reference Poinar and Linares1985).
Until now, only one species of Hexamermis, namely Hexamermis albicans (Siebold Reference Siebold1848) Steiner Reference Steiner1923, has been reported from Iran (Nikdel et al. Reference Nikdel, Kaiser and Niknam2011). A new population of this genus was recovered during a recent nematological survey conducted to explore mermithid diversity in the soils of Mazandaran province, north of Iran. The study of the population confirmed that it belongs to an unknown species, whose characterization is the aim of the present contribution.
Material and methods
Sampling, morphological, and morphometric study
Soil samples were collected from the rhizosphere of Diospyros lotus from a natural habitat in Mazandaran province, northern Iran. The modified Brown and Boag (Reference Brown and Boag1988) method was used to extract the nematodes. The extracted nematodes were killed and processed to dehydrated glycerin, according to De Grisse (Reference De Grisse1969), and permanent slides were prepared. Nematodes were measured using an Olympus BX41 light microscope with differential interference contrast optics and a drawing tube. Microphotographs were taken using the same microscope equipped with a DP50 digital camera. Raw photographs were edited using Adobe Photoshop CS. Line drawings were done based on the digital images using CorelDRAW software, v12.
DNA extraction, PCR, and sequencing
Following morphological confirmation, one fresh specimen of Hexamermis zirabi sp. n., was selected for DNA extraction. DNA was extracted using a method from Archidona-Yuste et al. (Reference Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius and Castillo2016). The DNA sample was stored at –20°C until used as a polymerase chain reaction (PCR) template. The D2-D3 expansion segments of 28S rRNA were amplified using forward D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (Nunn Reference Nunn1992). A 25 μL PCR reaction mixture was composed of 10 μL ddH2O, 12.5 μL PCR master mix (Ampliqon), 0.75 μL of each forward and reverse primers, and 1 μL of DNA template. The PCR cycle conditions were as follows: one denaturation cycle of 94°C for 15 min, followed by 35 cycles of 94°C for 45s; an annealing cycle of 55°C for 45s; an extension cycle of 72°C for 45s, and finally, one extension cycle of 72°C for 5 min. The PCR products were purified and sequenced directly for both strands using the same primers with Applied Biosystems 3730/3730xl DNA Analyzer in South Korea. The newly generated sequence of the 28S region was submitted to the GenBank database under accession number OR614376.
Phylogenetic analyses
The recently obtained sequence was aligned with 21 other D2-D3 expansion segments of 28S rRNA gene sequences available in GenBank, using Muscle software implemented in MEGA6 (Tamura et al. Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). Mononchus truncates Bastian, Reference Bastian1865 (AY593064) was chosen as an outgroup taxon. The sequence dataset was analyzed with the Bayesian inference method using MrBayes 3.1.2 (Ronquist & Hulesenbeck Reference Ronquist and Huelsenbeck2003). The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using MrModel test 2.3 (Nylander Reference Nylander2004) with an Akaike-supported model accompanied by PAUP* version 4.0b10 (Swofford Reference Swofford2003). After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The tree was visualized and saved with FigTree v1.4.3.
Results
Hexamermis zirabi sp. n.
Material examined
Six females and one male from one location were well preserved.
Description
These were small-sized mermithids. The body was mostly straight to J-shaped upon fixation, cylindrical, visibly narrowing toward the anterior end and less toward the posterior end since the caudal region is rounded. The color of the trophosome was yellow. Cuticle possessing distinct cross fibers, 4.5 – 6 μm thick in the anterior region, 6 – 9 μm at mid-body, and 8 – 11 μm in the tail region. The stoma was small and thin. The mouth opening was central and terminal. The head was conoid or sharply tapered, with a cephalic cap surrounding the mouth opening. Six cephalic (head) papillae were at the same plane, but there were no lip papillae. Amphids were oval-shaped and small, with an opening behind the lateral head papillae. Their aperture was circular, 5.5 – 7 μm wide, and occupying 19 – 21% lip region diameter. Six hypodermal chords were located at mid-body. Trophosome begins after the nerve ring, narrows at the anterior part, and is extended to near the end of the body in the female, but in the male, continues far beyond the head of the spicules and does not reach the level of the cloaca. The nerve ring encircled the pharynx’s anterior part and was at 2.5 – 2.8% of the body length. Vulva, a longitudinal slit, was located post-equatorially. Vulval lips were lacking or greatly reduced, and the cuticular cone was well developed (sclerotized), 30 – 32 μm long. The vagina was J-shaped, muscular, oriented to the anterior end of body and curved at the distal portion where it meets the branches of the uteri, extending inward 73 – 75% of the body diameter in lateral view. The uterus had Z-organs or sclerotized bodies. The tail was similar in both sexes and was bluntly rounded. Spicules were paired, separate, slightly curved, equal, 11.7 times as long as wide, and shorter than the body width at the cloaca, with rounded tips. The male genital papillae were arranged in five rows: the lateral with 4 papillae as the outer row and 11 papillae in the middle or lateroventral one; the ventral row starts from near the head of the spicules and continues up to tail end, with seven pre-anal papillae: two simples and five triplets; six post-anal: four triplets, one double and one simple, with four genital papillae surrounding cloaca: Post-parasitic juveniles not found (Figs. 1 and 2).
Measurements
Female (n = 6): body length 5.85 – 9.68 (7.54) mm, width of the head at the level of cephalic papillae 29 – 38 (32) μm, body diameter at the level of nerve ring 87 – 106 (98) μm, body diameter at vulval region 140 – 175 (165) μm, distance from head to the nerve ring 169 – 250 (225) μm, body diameter at posterior end of the trophosome 112 – 138 (128) μm, length of vagina 161 – 212 (188) μm, width of vagina 62 – 83 (75) μm, V% = 57 – 60 (59), distance from end of trophosme to tail tip 60 – 80 (74) μm (Table 1).
Main morphometrics of Hexamermis zirabi sp. n. and its three close species of the genus Hexamermis. All measurements in μm (except body length in mm)
a Calculated from Nickle and Grijpma (Reference Nickle and Grijpma1974) illustration.
Male (n = 1): body length 10.20 mm, width of the head at the level of cephalic papillae 43.7 μm, body diameter at the level of nerve ring 125 μm, greatest diameter of the body 158 μm, body diameter at cloaca 142 μm, distance from the head to the nerve ring 260 μm, length of spicules 117 μm, width of spicules 10 μm.
Molecular characterization
A 28S rRNA sequence of Hexamermis zirabi sp. n., was obtained, having 762 bp (OR614376).
Hexamermis zirabi sp. n. (A) Anterior region in lateral median view; (B and H) Anterior region in lateral surface view; (C) Female caudal region; (D) Cross-section, mid-body; (E) Male caudal region in lateral median view; (F) Male caudal region in lateral surface view; (G) Vulval region and vagina.
Diagnosis
Six cephalic papillae; cuticle with distinct cross fibers; head conoid or sharply tapered; mouth central and terminal; amphids small and oval shaped; six hypodermal cords; vulva a longitudinal slit and located post-equatorially; vulval cuticular cone well developed (sclerotized); vagina J-shaped and oriented to the anterior end of body; uterus with Z-organs or sclerotized bodies; tail similar in both sexes, bluntly rounded; spicules paired, separate, slightly curved, shorter than body width at cloaca, with rounded tip; male genital papillae arranged in five rows: the lateral with 4 papillae as the outer row and 11 papillae in the middle or lateroventral one; the ventral row starts from near the head of the spicules and continues up to the tail end, with seven pre-anal papillae: two simples and five triplets; and six post-anal: four triplets, one double and one simple, with four genital papillae surrounding cloaca, separate the new species from previously described members of Hexamermis.
Relationships
Morphologically, Hexamermis zirabi sp. n. is readily recognized and distinguished from its congeners by the peculiar morphology of its uterus, which has Z-organs or sclerotized bodies vs the absence of such structures in the uterus. Additionally, leaving aside H. cavicola (Welch Reference Welch1963), an Australian species, the new species can be separated from other Hexamermis members by its conoid or sharply tapered head region. It differs from H. cavicola by the body length (5.85 – 10 vs 60 – 160 mm), presence of seven pre- and six post-anal vs eight pre- and ten post-anal genital papillae, spicules length (117 vs 470 – 550 μm), vulva location (57 – 60% vs 53 – 56%) and other characters (for more comparison, see Welch, Reference Welch1963). The new species is also comparable to H. albicans, Costa Rican population, which is distinguished by having small body size (5.85 – 9.68 vs 42 – 83.8 mm), narrower lip region (29 – 43.7 vs 51.7 – 53.5 μm), the more posterior position of the vulva (57 – 60% vs 40 – 54%) and the presence of seven pre- and six post-anal vs three pre- and six post-anal genital papillae (Table 1).
Phylogenetically, Hexamermis zirabi sp. n. is related to H. popillae (Mazza et al. Reference Mazza, Paoli, Strangi, Torrini, Marianelli, Peverieri, Binazzi, Bosio, Sacchi, Benvenuti, Venanzio, Giacometto, Roversi and Poinar2017), an Italian species, but can be differentiated by the conoid or sharply tapered vs obtuse head region, amphidial openings small vs large, female body length (5.85 – 9.68 vs 18 – 50 mm), size of the vagina (62 – 83 × 161 – 212 vs 154 – 176 × 224 – 384 μm) and male genital papillae arrangement in a row (vs clustered).
Type locality and habitat
Zirab County, Mazandaran province, north of Iran (GPS coordinates: 36°10´68˝ N 052°57´86˝E, altitude 500 m a.s.l.), in the rhizosphere of Diospyros lotus.
Type material
Holotype female, five paratype females and males were deposited at the Nematode Collection of the Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Etymology
The name of the species refers to the location of the new species, Zirab County, Mazandaran province, north of Iran.
On the phylogeny
The evolutionary relationships of Hexamermis zirabi sp. n., as derived from molecular analysis along with other mermithids, are presented in Figure 3. The average nucleotide composition is as follows: 22.37% A, 21.86% C, 29.97% G, and 25.81% T. The new species forms a highly supported (1.00% Bayesian posterior probability (BPP)) clade with Hexamermis popilliae. The new 28S sequence in the pairwise comparison with Hexamermis popilliae (MF040825) presents 81.89% identity (28 bp differences: insertions, deletions, or substitutions/no gaps). GenBank database is very poor in large subunit sequences deposited from Mermithidae, and more sequences of the species belonging to the family are required to enable a detailed discussion on the molecular phylogeny of the group.
Hexamermis zirabi sp. n. (A) Anterior region in lateral median view; (B) Cross-section, mid-body; (C and G) Anterior region in lateral surface view; (D) Male caudal region in lateral median view; (E) Z-organs; F: Female caudal region; (G) Cross fibers; (H) Spicules; (I and J) Vulval region and vagina. Scale bars: (A, C, E) (H) 20 μm; (B) (J) 40 μm; (D) 35 μm; (F) (I) 40 μm; (G) 15 μm.
Consensus 50% majority rule Bayesian phylogenetic tree of Hexamermis zirabi sp. n. based on 28S rRNA D2-D3 sequences under the GTR+I+G model. Posterior probabilities >50% are given for appropriate clades. The newly obtained sequence is in bold.
Discussion
There has been little study about the mermithids fauna in Iran. The only available taxonomic document is a paper published by Nikdel et al. (Reference Nikdel, Kaiser and Niknam2011) in which they described Hexamermis albicans parasitizing the abdominal cavity of larvae of two lepidopterans, Euproctis chrysorrhoea and Lymantria dispar (Lymantriidae) during a survey conducted to study mermithids in Arasbaran area, northwest of Iran. Although the study on mermithids is still in its early stages in Iran, our recent sampling showed that the group is species-rich in the understudied regions. So, as a part of the survey results, an unknown species of the genus Hexamermis was collected, described, and illustrated in this contribution. Hexamermis zirabi sp. n. was recovered from rhizospheric soil samples of Diospyros lotus from the north of Iran.
The presence of sclerotized bodies in the uterus of the new species and a sharply narrower head region differentiated the species from all the previously described species of the genus. Regarding the function of uterine differentiation, there is no report on the structures in the mermitid’s uterus. Still, several possible functions have been proposed for uterine differentiation in Xiphinema (Cobb Reference Cobb1913), which may also be the case in mermithids. Kruger (Reference Kruger1988) pointed out (p. 250) that ‘These vary from slowing the passage of eggs through the uterus during shell formation, the prevention of sperm reflux from the spermatheca during egg movement, and the secretion of certain substances from the globular structures for eggshell formation’. Z-organs in the understudy population of the new species are triangular to rhomboid and 6.5 – 9 μm long and are seen as a complex structure inside the uterus. Additional histochemical and physiological investigations may elucidate uterine differentiation’s actual function(s) in the future.
Morphological differences of Hexamermis zirabi sp. n. compared with the closely related species were discussed in the earlier parts of the text. We tried several times to find this species’ associated or potential hosts, but unfortunately, it was not possible. According to the insect fauna reported from this area (Table 2), its possible host(s) may be a member of the order Lepidoptera. As mentioned above, Hexamermis albicans was previously isolated and reported from two species of this order in Arasbaran forests of Iran.
Insects reported from Zirab County, Mazandaran province, north of Iran
Adapted from Khayrandish et al. Reference Khayrandish, Ahmadzade and Naderi2011
The present study’s phylogenic analysis was based on one LSU D2-D3 marker. However, the monophyly of the genus was not confirmed, and further sequences of other representatives of the genus are needed to assess their monophyly. In our tree, Hexamermis agrotis with accession number EF617369 was placed in a branch far from the Hexamermis spp. Morphological data of the abovementioned species was not available and its identity may need further confirmation.
As we claimed above, regarding the richness of species of mermithids in Iran, we have identified two new genera and several new species belonging to the different genera of mermithids whose descriptions are in preparation.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interest
The authors have no competing interests.
