Amphimermis enzoni n. sp. (Nematoda: Mermithidae) parasitizing damselflies and dragonflies in Argentina
Published Online: May 18, 2020
Page range: 1 - 9
Received: Mar 10, 2020
DOI: https://doi.org/10.21307/jofnem-2020-051
Keywords
© 2020 José Matias Rusconi et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Mermithids are obligate parasites of invertebrates. Most of them have been found to be parasites of insects, even though they are also found in spiders, crustaceans, leeches, and molluscs (Poinar, 1975; Poinar and Stockwell, 1988; Poinar and Ćurčić, 1992). In their life cycle, infective juveniles (preparasitic second-stage juveniles [J2]) hatch from eggs and actively seek and penetrate the host. The third-stage juvenile (J3) develops in the host, at which time the postparasitic fourth-stage juvenile (J4) emerges, killing the host. The J4 develops into adults, which mate and lay eggs in the substrate. Juveniles hatched from these eggs will penetrate a new host and begin the cycle anew (Becnel and Johnson, 1998).
While conducting an extensive survey for parasites of mosquitoes, nymphs of dragonflies and damselflies from a stream were found containing mermithid nematodes.
There are few articles about mermithid infections on Odonata worldwide. Artykhovsky and Negrobov (1967) discovered an undescribed mermithid in naiads of dragonflies in Usman, Russia, and Willis (1970) found an
In Argentina, the genus
In the present study, a new species of the genus
Nymphs of dragonflies and damselflies were found in the stream Cajaravilla, Magdalena (34° 47´ 25˝ S; 58° 08´ 55˝ W), Buenos Aires Province, Argentina. The insects were collected near flooded reeds with a scoop (400 ml), placed in individual recipients with water from the environment and taken to the laboratory. Nematodes were obtained from the emergence of parasitized insects and maintained on 9 cm diameter Petri dishes with sterilized soil (in oven at 220°C during 1 hr) and 40 ml of water from the same stream.
For the morphological description, the mermithids were fixed in T.A.F. (2% triethanolamine, 7.5% formaldehyde in distilled water). All measurements are in micrometers unless otherwise specified and are presented as the range followed by the mean in parentheses. Specimens for molecular studies were fixed in absolute ethanol. Nematodes were measured using an ocular micrometer in a Leica DM 500 microscope. Photographs were taken with an Olympus DP-71 camera and a Leica DM 500 microscope; drawings were made using an Olympus CX-31 with drawing tube. For the examination of the hypodermal chords, a 0.3 to 0.5 cm portion of the midbody region of two specimens was removed and slide-mounted in a glycerin-lactic acid stain for observation at 10-100 x according to Tripodi and Strange (2018).
Voucher specimens have been deposited in the Museo de Ciencias Naturales de La Plata, Buenos Aires, Argentina.
For the scanning electron microscopy (SEM) study, specimens were dehydrated through a graded series of ethyl alcohols and critical point dried with carbon dioxide; then specimens were mounted on metal stubs with silver paste, coated with gold, and examined in a Philips 505 scanning electron microscope equipped with a digital imaging program (Philips Electron Optics BV, Eindhoven, Netherlands).
To confirm the nematodes identification, a molecular approach was performed. Genomic DNA was extracted using 100 µl of a 5% suspension of Chelex in deionized water and 2 µl of proteinase K, followed by overnight incubation at 56°C, boiling at 90°C for 8 min and centrifugation at 14,000 rpm for 10 min. An aliquot of 1 µl of the supernatant was utilized as template for Polymerase Chain Reaction (PCR). The 18S rRNA partial sequences were amplified using the primers Merm F 18S (5´-CAAGGACGAAAGTTAGAGGTTC-3´) and Merm R 18S (5´-GGAAACCTTGTTACGACTTTTA-3´) according to Kobylinski et al. (2012) with the Go Taq Master Mix (Promega Corporation, Madison, USA). The thermocycle conditions were as follows: 94°C for 15 min; 35 cycles of 94°C denaturation for 30 s, annealing 52°C for 40 s and extension 72°C for 60 s; a single final extension period of 72°C for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gels and visualized by staining with ethidium bromide. The amplicons were sequenced in Macrogen Inc. (Korea) and edited with the platform GENEIOUS (Biomatters, Auckland, NZ,
In total, 17 specimens of
As the nematodes matured, they oriented themselves longitudinally in the abdominal and thoracic regions of the host and could be seen with the naked eye during the late stages of parasitic development (Fig. 1A). The nematodes emerged from the hosts in the regions of the anus or mouth (Fig. 1B) and this escape did not always immediately kill the host. Some hosts (
Figure 1:
A. Nematode inside
(Figs. 2A-E, 3A-F, 4A-D, 5A-D).
Figure 2:
Female (A-C). A. Head. B. Vulva (arrowhead). C. Tail. Post-parasitic juvenile (D, E). D. Head. E. Tail with appendage (arrowhead). Scale bars: 50 µm.
Figure 3:
Male (A-F). A. Head showing cephalic papillae (arrowheads) and amphids (arrows). B. Tail showing the spicule. C. Proximal part of the spicule (white arrowheads showing the beginning of the spicule and black arrowhead showing a section of the proximal twisted part). D. Untwisted part of the spicule. E. Distal twisted part of the spicule (arrowhead). F. Tip of the spicule (arrowhead). Scale bars: 50 µm (A-F), 200 µm (B).
Figure 4:
Male tail (A, B). A. First papilla separated from the rest (arrow) of the cephalic papillae. B. Cloaca (arrow). Female (C, D). C. Vulva. D. Tail.
Figure 5:
Male (A-D). A. Schematic arrangement of genital papillae. B. Tail, ventral view showing arrangement of genital papillae. C. Scheme of the spicule: 1 (proximal twisted part), 2 (untwisted part), 3 (distal twisted part), 4 (tip). D. Cross section, mid-body. d, dorsal chord; l, lateral chord; sv, subventral chord; v, ventral chord. Scale bars: 50 µm (A-C), 9 µm (D).
Female (
Male (
Post-parasitic juvenile (
They are white medium size nematodes vary from 3 to 10 cm. Cuticle has cross fibers. Head is rounded. Mouth is terminal. There are six cephalic papillae around the mouth. Opening of lateral cephalic papillae is at the level of or slightly anterior to the level of sub-median cephalic papillae. Amphids are cup-shaped. There are six hypodermal cords.
In female, opening of the vulva is a transverse slit at the middle of the body. Vulval flap is present. Vulval cone is absent. Vagina is S shaped, long, muscular and posterior loop is slightly smaller in length than anterior loop and is bended rounded. The junction of vagina and uterus is slightly posterior to vulva. Tail is conical and slightly flattened on the ventral surface.
They are similar in size to the adults. Head is rounded. Cuticle has cross-fibers and tail has appendage.
In male, tail is curled, conoid, and bluntly rounded. Spicules are paired; proximal part is twisted for 34% of its length, then untwisted for 12%, twisted for 30%, and finally untwisted for 24%. First genital papillae are located at the level of the first untwisted part of the spicule, separated from the rest. Spicule length is approximately 8 × body width at cloaca. Genital papillae are arranged in three rows; medial row marginally longer than sub-medial rows; medial row is bifurcate immediately anterior and posterior to cloaca. There are a total of 111 genital papillae (73 pre-anals and 38 post-anals).
Following is the taxonomic summary:
Type host:
Type locality/collection dates: stream Cajaravilla, Magdalena (34° 47´ 25˝ S; 58° 08´ 55˝ W), Buenos Aires Province, Argentina; October to February 2018-2019; 2019-2020.
Site of infection: body cavity.
Prevalence: 9.94% in
Specimens deposited: all types have been deposited in the Colección Helmintológica del Museo de Ciencias Naturales de La Plata with the following accession numbers: Holotype male No. MLP-He 7639, allotype female No. MLP-He 7640, and one paratype (postparasitic juvenile) No. MLP-He 7641.
Etymology: this species is named after Enzo Rusconi, nephew of JMR.
The 18S rDNA sequence of
Figure 6:
Phylogeny of the Mermithidae family based on 18S rDNA data including the new species,
The male of
Female of
Genetically, 18S sequences from these specimens matched known representatives of
This paper contributes the first molecular characterization of a species of the genus