image/svg+xml191New species of UrocleidoidesNeotropical Helminthology (Lima). Vol. 17, Nº2, jul - dic 2023Neotropical HelminthologyNeotropical Helminthology, 2023, vol. 17 (2), 191-195ORIGINAL ARTICLE / ARTÍCULO ORIGINALNEW SPECIES OF PROPARASITYLENCHUSWACHEK (TYLENCHIDA, ALLANTONEMATIDAE) PARASITIZING HARMONIA AXYRIDIS PALLAS, 1773 (COLEOPTERA, COCCINELLIDAE) IN ARGENTINANUEVA ESPECIE DE PROPARASITYLENCHUSWACHEK (TYLENCHIDA, ALLANTONEMATIDAE) PARÁSITO DE HARMONIA AXYRIDISPALLAS, 1773 (COLEOPTERA, COCCINELLIDAE) EN ARGENTINANora B. Camino1,2,3*, Sergio Rodríguez-Gil1,4, Guillermo R. Reboredo1,4& Sandra E. González1,3ISSN Versión Impresa 2218-6425 ISSN Versión Electrónica 1995-1403DOI: https://dx.doi.org/10.24039/rnh20231721648Volume 17, Number 2 (jul - dec) 2023Este artículo es publicado por la revista Neotropical Helminthology de la Facultad de Ciencias Naturales y Matemática, Universidad Nacional Federico Villarreal, Lima, Perú auspiciado por la Asociación Peruana de Helmintología e Invertebrados Af nes (APHIA). Este es un artículo de acceso abierto, distribuido bajo los términos de la licencia Creative Commons Atribución 4.0 Internacional (CC BY 4.0) [https:// creativecommons.org/licenses/by/4.0/deed.es] que permite el uso, distribución y reproducción en cualquier medio, siempre que la obra original sea debidamente citada de su fuente original.1 Centro de Estudios Parasitológicos y de Vectores, CEPAVE, Argentina.2 Universidad Nacional de La Plata, UNLP, Facultad de Ciencias Naturales y Museo, Argentina.3 Investigador CIC, Comisión de Investigaciones Científ cas de la provincia de Buenos Aires, CIC, Argentina.4 Consejo Nacional de Investigaciones Científ cas y Técnicas, CONICET, Argentina.* Corresponding author: nemainst@cepave.edu.ar Nora B. Camino: https://orcid.org/0000-0003-4910-3986Sergio Rodríguez-Gil: https://orcid.org/0000-0003-4608-1961 Guillermo R. Reboredo: https://orcid.org/0000-0002-1214-316XSandra E. González: https://orcid.org/0000-0002-0609-0516ABSTRACTA new species Proparasitylenchus inalienatussp. n. is described and illustrated, as a parasite of the coccinelid Ha rmonia axyridisPallas, 1773in a locality near Gran La Plata, Colonia Urquiza, Argentina. Of the 13 species described, this species is characterized by having a cephalic end projecting, faint stylet in its normal place, vulva posterior, tail appendage conoid, with a bifurcated tip in both sexes, male with two spicules and a tiny gubernaculum, and leptoderan bursa. We obtained the 18S rDNA partial sequence.Keywords: Allantonematidae – bursa leptodera – Coccinellidae – ArgentinaRESUMENUna nueva especie Proparasitylenchus inalienatus sp. n., se describe parasitando al coccinélido Harmonia axyridis Pallas, 1773, de una localidad próxima al Gran La Plata, Colonia Urquiza. De las 13 especies descriptas, esta se caracteriza por tener en el extremo cefálico una proyección, estilete tenue en su lugar normal, vulva posterior, apéndice caudal conoide,
image/svg+xml192Neotropical Helminthology (Lima). Vol. 17, Nº2, jul - dic 2023Camino et al.con punta bifurcada en ambos sexos, macho con dos espículas y un diminuto gubernáculo y bursa leptodera. Obtuvimos la primera secuencia parcial 18S rDNA para el género.Palabras clave: Allantonematidae – bursa leptodera – Coccinellidae – ArgentinaINTRODUCTIONIn search of parasitic nematodes of insects of horticultural interest in the surroundings of the city of La Plata, Argentina, the genus Proparasitylenchuswas found parasitizing the coccinelid Harmonia axyridis Pallas, 1773.At present, the following genera: ProparasitylenchusWachek, 1955; Parasitylenchoides Wachek, 1955, NeoparasitylenchusNickle, 1967, and ProtylenchusWachek, 1955 are poorly defned and they would need a better and expanded description. Te species of the genus Proparasitylenchusare parasites of Coleoptera. Staphylinidae in Europe and North America. At this moment seven species have been described from the genus Proparasitylenchus, P. athetae (Wachek, 1955) Nickle, 1967; P. platystethi(Wachek, 1955) Nickle, 1967; P. medonis(Wachek, 1955) Nickle, 1967; P. myrmedoniae (Wachek, 1955) Nickle, 1967; P. oxyteli (Wachek, 1955) Nickle, 1967; P. trogophoei (Wachek, 1955) Nickle, 1967 from Europe; and P. californicusPoinar, G.O. Jr. et al., 2015 from USA, all parasites of Coleoptera, Staphylinidae, except for P. myrmedoniaewhich parasitizing ants. In this paper we describe a new species P. inalienatus n. sp., a parasite of coccinellids from Argentina. MATERIAL AND METHODSInsects: adult coccinellid insects were collected by hand from September 2021 to April 2022 on plant species Cucurbita maximavar. zapallito(Carrière) Millán, 1947 (round green zucchini), at a locality near Gran La Plata, Colonia Urquiza (34°96’72”S, 58°04’96”W), Argentina. Te beetles were distributed in individual plastic containers. In total, 260 adults of Harmonia axyridis(Pallas, 1773) (Coleoptera, Coccinellidae), were sampled and dissected. Te coccinellids adults were dissected in Petri dishes flled with distilled water under a stereomicroscope. Nematodes: living nematodes were removed from the hemocel of adult host, and then they were killed by placing them in distilled water at 60°C for 2 pure TAF (Poinar, 1975). For the molecular study, 20 specimens were fxed in absolute alcohol. All the specimens were used for photographing in Olympus BX51 microscope with Olympus DP71 camera. Measurements of common nematode body features were performed on 16 fxed nematode specimens of each nematode stage of life cycle. Mean and in parenthesis minimum and maximum.Total genomic DNA was extracted from four specimens using PURO-Genomic DNA (PB-L Productos Bio-Logicos, Buenos Aires, Argentina) according to the manufacturer’s protocol. Te partial fragment of the 18S rDNA gene was amplifed from individual worms. Amplifcation was conducted using polymerase chain reaction (PCR) on an Eppendorf Mastercycler thermal cycler (Hamburg, Germany) using forward primer Nem 18SF (5’- CGCGAATRGCTCATTACAACAGC-3’) and Nem 18SR (5’-GGGCGGTATCTGATCGCC-3’) according to Singh et al.(2013). Te thermocycler conditions were: 94ºC for 5 min; 94ºC for 30 s, 35 cycles of 60°C for 40 s and 72ºC for 60 s; and 72ºC for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gels and visualized by staining with ethidium bromide. Te amplicons were sequenced in Unidad de Genómica del Instituto de Biotecnología del Instituto Nacional de Tecnología Agropecuaria, Argentina (INTA). Te 18S rDNA partial sequence generated from this study were submitted to the National Center for Biotechnology Information (NCBI) GenBank database (http:// www.ncbi.nlm.nih.gov) and can be accessed using the GenBank accession numbers: OP889000 Ethic aspect:Te ethical biosafety standards established for laboratory animals and good animal experimentation practices were followed.RESULTSProparasitylenchusWachek, 1955 (Nickle, 1967)Proparasitylenchus inalienatussp.n.Description (Figs. 1-5)Entomoparasitic female: body cylindroid and arcuate, yellow. Body surface neither wavy nor with constrictions
image/svg+xml193New species of Proparasitylenchusin CoccinellidaeNeotropical Helminthology (Lima). Vol. 17, Nº2, jul - dic 2023and swellings. Cephalic end projecting (Fig. 1), faint stylet in its normal place. Excretory pore is behind the nerve ring. Anus present. Vulva posterior (Fig. 2). Te postvulval uterine sac is absent. Tail appendage conoid, with bifurcated tip (Fig. 3). 13452Figures 1-5.Proparasitylenchus inalienatussp. n. 1. Head of the female; 2. Posterior end of female; 3. Posterior end of the female; 4. Posterior end of the male; 5. Posterior end of the larva. Bars = 50 µm.21 5
image/svg+xml194Neotropical Helminthology (Lima). Vol. 17, Nº2, jul - dic 2023Camino et al.Female (n = 12): diameter of head at level of cephalic papillae: 40.90 µm (38.65-45.23); width of body at level of nerve ring: 65.60 µm (62.40-68.32); maximum body diameter: 84.35 µm (82.35-90.12); width of body at level of posterior end: 35.42 µm (32.50-40.10); distance anterior end to nerve ring: 135 µm (132-142); distance from anterior end to excretory pore: 170.80 µm (164.32-175.90); vagina length: 38.55 µm (32.42-40.22); V (distance anterior end to vulva/body length x 100): 86% (82-90); tail appendage length: 8.41 µm (7.98-9.25).Male (n = 10): smaller than female. Weak, dorsal and paired subventral pharyngeal glands. Two spicules short and a tiny gubernaculum, narrow, subterminal and small leptoderan bursa (Fig. 4).Male (n = 19): diameter of head at level of cephalic papillae: 6.25 µm (5.82-7.12); width of body at level of nerve ring: 10.41 µm (9.35-14.26); maximum body diameter: 20.50 µm (18.32-24.12); width of body at level of posterior end: 130 µm (125-142); distance anterior end to nerve ring: 20.85 µm (18.30-22.24); distance from anterior end to excretory pore: 43.75 µm (39.45-45.60); spicule length: 14.12 µm ( 12.34-18.50); gubernaculum length: 3.97 µm (3.25-4.43); tail appendage length: 4.41 µm (3.92-5.35)Juveniles (n= 30): free living form very small and transparent, with sytlet distinctly knobbed, excretory pore at less than 100 µm from anterior end. Tail appendage ends forked into two points (Fig. 5). Type host: Harmonia axyridisPallas, 1773 (Coleoptera, Coccinellidae)Type locality: near Gran La Plata, Colonia Urquiza (34°56’21.199”S, 58°6’56.902”W), Buenos Aires, Argentina.Etymology: inalienatus means unspoiled.Type material: Holotype y Allotype deposited in Helmintológica collection of Museo de La Plata, and Paratypes in CEPAVE TY 156. ADN CharacterizationMassey (1974) grouped four related genera: Proparasitylenchus, Parasitylenchoides, Protylenchus and Neoparasitylenchus,proposing further study of them. Tere is no molecular study of these genera so we cannot make a comparison or discussion, we only contribute to this paper the 18S rDNA partial sequence. Future studies are needed to fll in this unknown. REMARKS AND DISCUSSIONTere are 13 species of the genus Proparasitylenchus, which are parasites of Coleoptera - Staphylinidae, and one species P. myrmedoniae (Wachek, 1955) Nickle, 1967, which parasites ants, that we will not be considered in this paper. All are from Germany, Europe and USA. Our new species shares with the described species the characters of the genus: body cylindroid and arcuate, yellow, excretory pore is behind the nerve ring, vulva posterior, two spicules short, gubernaculum small, and leptoderan bursa. P. athetae (Wachek, 1955) Nickle, 1967 the head of the parasitic female is sunk into the body, the tail appendage is stumpy, and the free-living stages have blunt tail tips and short stylets. P. platystethi(Wachek, 1955) Nickle, 1967 can be separated by the cuticle anullata, and the tail appendage in mucron shaped. P. medonis(Wachek, 1955) Nickle, 1967 has narrow head in the parasitic female. P. oxyteli (Wachek, 1955) Nickle, 1967 has the spicules thick with irregular edges and the stylet is very short. P. trogophoei (Wachek, 1955) Nickle, 1967 is distinguished by the structure of the spicule, pear-shaped spicule body and long, slender tip. P. californicusPoinar, G.O. Jr., et al., 2015 has the head of the parasitic female is sausage-shaped, not have knobbed but not cleft stylets.Although the species is identifed morphometrically, the phylogenetic tree could not be represented, this is because there are few genetic data deposited in the genus GenBank and these references have been geographically isolated.Author contributions: CRediT (Contributor Roles Taxonomy)NBC= Nora B. CaminoSRG= Sergio Rodríguez Gil GR= Guillermo Reboredo SG= Sandra GonzálezConceptualization: NBC, SRG, GR, SGData curation: NBC, SRG, GR, SGFormal Analysis: NBC, SRG, GR, SGFunding acquisition: NBC, SRG, GR, SGInvestigation: NBC, SRG, GR, SGMethodology: NBC, SRG, GR, SGProject administration: NBC, SRG, GR, SGResources: NBC, SRG, GR, SG
image/svg+xml195New species of Proparasitylenchusin CoccinellidaeNeotropical Helminthology (Lima). Vol. 17, Nº2, jul - dic 2023Software: NBC, SRG, GR, SGSupervision: NBC, SRG, GR, SGValidation: NBC, SRG, GR, SGVisualization: NBC, SRG, GR, SGWriting – original draft: NBC, SRG, GR, SGWriting – review & editing: NBC, SRG, GR, SGBIBLIOGRAPHIC REFERENCESMassey, C.L. (1974). Biology and taxonomy of nematode parasites and associates of bark beetles in the United States.Agriculture Handbook nº 446, USDA, Forest Servove.Nickle, W.R (1967). On the classifcation of the insect parasitic nematodes of the Sphaerulariidae Lubbock, 1861 (Tylenchoidea: Nematoda). Proceedings of the Helminthological Society of Washington, 34, 72-94.Poinar, G.O.Jr., Datlen, N., Espinoza, M., & Mclaughlin, J. (2015). Proparasitylenchus californicusn. sp. (Tylenchida: Allantonematidae), parasitic in the intertidal rove beetle Tarphiota geniculata(Mäklin) (Coleoptera: Staphylinidae) in California, USA. Systematic Parasitology,92, 57-64. Singh, N., Chaudhary, A., & Singh, H.S. (2013). Morphological redescription and molecular characterization of three species of Travassosinematidae (Nematoda: Oxyurida: Telastomatoidea) from Gryllotalpa africanaBeauv (Orthoptera: Gryllotalpidae). Bioinformation, 9, 818-823. Wachek, F. (1955). Die entomoparasitischen Tylenchiden.Parasitologische Schriftenreihe, 3,1-119.Received September 2, 2023.Accepted December 4, 2023.