Zoosyst. Evol. 100 (4) 2024, 1315-1331 | DOI 10.3897/zse.100.125466 yee BERLIN Synonymy, redescription, molecular characterisation, and new distribution data of species of Stilestrongylus and Guerrerostrongylus (Nematoda, Heligmonellidae) parasitic in sigmodontine rodents from Argentina and Uruguay: a collection- based survey Maria Celina Digiani!*, Paula Carolina Serrano! 1 CONICET-Consejo Nacional de Investigaciones Cientificas y Técnicas, La Plata, Argentina 2 Divisién Zoologia Invertebrados, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, 1900 La Plata, Argentina https://zoobank. org/DE37DFA9-F0D9-41AD-B021-7F990C241FA3 Corresponding author: Maria Celina Digiani (mdigiani@fcnym.unlp.edu.ar) Academic editor: Andreas Schmidt-Rhaesa # Received 17 April 2024 # Accepted 8 August 2024 # Published 24 September 2024 Abstract In this study, we review the taxonomic status of the parasitic nematodes Stilestrongylus oryzomysi and Guerrerostrongylus uruguay- ensis (Heligmonellidae), which had been described only once from sigmodontine rodents from Argentina and Uruguay, respectively. To this aim, we examined 38 complete helminth sets deposited in the Helminthological Collection of the Museo de La Plata (MLP- He), Argentina, including type material of G. uruguayensis, S. oryzomysi, and a closely related species, Stilestrongylus azarai. We also examined voucher rodent specimens deposited in the Mammal Collection of the Museo de La Plata (MLP-Mz) to assess the identity of the symbiotypes and other rodent hosts. Based on these observations, S. oryzomysi is proposed as a junior synonym of S. azarai; the identity of the symbiotype of S. oryzomysi and Trichofreitasia lenti (Heligmonellidae) is emended from Oligoryzomys flavescens to Akodon azarae, G. uruguayensis is proposed as a junior synonym of Guerrerostrongylus zeta; S. azarai is redescribed based on type and voucher material; and the latter species and G. zeta are molecularly characterised using the ITS+ gene. We extend the geographic distribution of S. azarai to include Uruguay and provide a new host record for 7: /enti (A. azarae, type host species), a new host record for G. zeta in Argentina (O. flavescens), and the first record of helminths for Oligoryzomys nigripes in Uruguay. Key Words Akodon azarae, Guerrerostrongylus uruguayensis, Guerrerostrongylus zeta, integrated collections, ITS+, Oligoryzomys spp., Stilestrongylus azarai, Stilestrongylus oryzomysi, Trichofreitasia lenti Introduction Heligmonellidae is the most speciose family of the Trichostrongylina (Strongylida), with ca. 350 species distributed worldwide, most of which are parasites of rodents. Within this family, the cosmopolitan Nippos- trongylinae includes the largest number of species (ca. 230), with rodents of the superfamily Muroidea as main hosts (Beveridge et al. 2014; Durette-Desset et al. 2017). In South America, muroids are mostly represented by the Sigmodontinae, which includes ca. 380 species (D' Elia and Pardifias 2015). In Argentina, ca. 29 species of Nip- postrongylinae have been described from sigmodontines mainly inhabiting the Rio de la Plata Basin region (Du- rette-Desset and Sutton 1985; Sutton and Durette-Desset 1991; Digiani and Durette-Desset 2003a, 2003b; Digiani etal. 2003; Navone et al. 2009; Digiani and Kinsella 2014; Panisse et al. 2017; GOmez-Mufioz et al. 2020; Serrano et Copyright Digiani, M.C. & Serrano, P.C. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1316 al. 2021). In particular, four of the latter species were de- scribed in 1991 from yellow pygmy rice rats, Oligoryzo- mys flavescens (Waterhouse) (commonly known as “co- lilargo del Plata”), which were collected at two localities in Argentina and Uruguay, distant ca. 400 km from each other and separated by the Uruguay River. These species are Stilestrongylus oryzomysi Sutton & Durette-Desset, 1991; Trichofreitasia lenti Sutton & Durette-Desset, 1991, from the Argentinean locality; Stilestrongylus fla- vescens Sutton & Durette-Desset, 1991; and Guerreros- trongylus uruguayensis Sutton & Durette-Desset, 1991, from the Uruguayan locality. Subsequently, S. flavescens was reported from its type host species and other sigmodontine rodents in the Rio de La Plata Basin region in Argentina (Navone et al. 2009; Hancke and Suarez 2018; Serrano 2024). Trichofreitasia lenti was found in different eco-regions in Argentina and Brazil, though never from its type host species (Digiani et al. 2007; SimGes et al. 2011, 2012b; Panisse et al. 2017). Stilestrongylus oryzomysi and G. uruguayensis were not recorded again (see, however, Digiani et al. 2007 for re- cords of Guerrerostrongylus spp.), despite the intensive sampling effort used in different studies (Navone et al. 2009; Panisse et al. 2017; GOmez-Mufioz et al. 2020: Serrano et al. 2021). Both species were described from a very small number of specimens, and their differential di- agnoses against known species were mainly based either on morphometric characters of soft organs or on unspecif- ic or rather ambiguous morphological features. Stilestrongylus oryzomysi was differentiated from S. azarai Durette-Desset & Sutton, 1985, parasitic in Akodon azarae (Fischer) from Buenos Aires province, by having a more posterior excretory pore and a more de- veloped genital cone (Sutton and Durette-Desset 1991). However, the authors only provided measurements of the holotypes and allotypes, and therefore no measurement ranges are available for any of the two species. The remarkable similarity between S. azarai and S. oryzomysi, together with the fact that the latter has only been reported in its original description, led us to suspect that the specimens described as S. oryzomysi could be an infrequent acquisition of S. azarai by O. flavescens. Similarly, Guerrerostrongylus uruguayensis was differ- entiated from Guerrerostrongylus zeta (Travassos, 1937), parasitic in various sigmodontines from Brazil (concern- ing the use of the spelling zeta, see Digiani et al. 2024), by its larger body size and a widening of the body just anterior to the tail in females, and by details of the bursal rays in males. However, the diagnostic characters pro- posed for females are taxonomically unreliable because they may vary with age and reproductive status, whereas those for males are ambiguous and subjected to individual variation. In addition, the description of G. uruguayensis did not account for morphometric variability since it was based solely on the holotype and allotype. Within this framework, the main aim of the present study was to review the taxonomic status of S. oryzomysi and G. uruguayensis based on type material examination and comparison with closely related species. Additionally, zse.pensoft.net Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection our results allowed us to provide a morphological rede- scription of S. azarai based on type and voucher material, as well as a molecular characterisation of this species and of G. zeta based on the internal transcribed spacer (ITS) region. Finally, we shed some light on the host spectrum and geographic distribution of the species concerned. Materials and methods Type material examined from the Helminthological Collection of the Museo de La Plata, Argentina (MLP-He) Stilestrongylus azarai ARGENTINA * <, holotype; Buenos Aires Province, Balcarce; 1977-1978; Sutton leg.; Akodon azarae; MLP-He 0687-1* &, allotype; same data as for holo- type; MLP-He 0687-2 * 6 3, 6 29, paratypes; same collection data as for preceding; MLP-He 0687-3. Stilestrongylus oryzomysi ARGENTINA * 2 3, 10 2, paratypes; Buenos Aires Province, Campana, National Route 12 (RN12) Km 100-101; [34°00'27.432"S, 58°58'29.28"W]; Apr. 1989; Sutton leg.; Akodon azarae, MLP-Mz 3076, “Oryzomys flavescens, 1914”; MLP-He 1914-3. Guerrerostrongylus uruguayensis URUGUAY * <6, holotype; Artigas Department, Bella Union, Colonia Espafia; [30°21'52"S, 57°38'34"W]: Jul. 1989; Sutton leg.; “Oryzomys flavescens”, MLP-He 2046-1 * 9, allotype; same data as for holotype; MLP- He 2046-2 * 2 4 (distal fragments), 3 2 9, paratypes; same collection data as for preceding; MLP-He 2046-3. Type material of G. zefa was not examined; instead, data were drawn from two published redescriptions, which included the study of syntypes and voucher specimens housed in the Helminthological Collection of the Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, Brazil (Sim6es et al. 2012a) and of voucher specimens from Brazil and Argentina (Misiones province) (Digiani et al. 2012). Other material examined Additionally, we examined 35 sets of parasitic intestinal helminths in A. azarae and Oligoryzomys spp. housed in MLP-He for identification. These were selected because they had been collected from the type localities of S. aza- rai, S. oryzomysi, and G. uruguayensis and from the same sampling events as the type material of these species. The sets were complete, and the worms remained unidenti- fied. Therefore, each set examined corresponded to an Zoosyst. Evol. 100 (4) 2024, 1315-1331 infracommunity of intestinal helminths of either A. azarae or Oligoryzomys spp. from each of the localities concerned. From Locality I (type locality of S. azarai): MLP-He 674, 675, 676, 683, 686. From Locality I] (type locality of S. oryzomysi): MLP- He 1905, 1906, 1907, 1911, 1913, 1915, 1933, 1940. From Locality III (type locality of G. uruguayensis): MLP-He 2047, 2048, 2049, 2050, 2051, 2052, 2054, 2055, 2056, 2057, 2058, 2059, 2061, 2062, 2063, 2064, 2066, 2067, 2068, 2069, 2070, 2072. Prevalence (P) and mean intensity of infection (MI) were calculated by host and locality according to Bush et al. (1997). We followed Beveridge et al. (2014) for high-level taxonomy (Order to Subfamily). Hosts Hosts from Loc. I (Table 1) were collected and identified by mammalogists of the Museo Municipal Lorenzo Scaglia, Mar del Plata, Buenos Aires province, Argentina. Their viscera were submitted for parasitological examination to the Division Zoologia Invertebrados (Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata) in the framework of an agreement between both institutions in the 1980’s. Voucher host specimens of all helminth sets from Loc. II and several sets from Loc. II were deposited in the Mammal Collection of Museo de La Plata (MLP- Mz), thus allowing us to confirm or emend previous host identifications (Tables 2, 3). Table 1. Helminth sets from Locality I (Balcarce, Argentina, type locality of Stilestrongylus azarai). In parentheses, intensity of infection. Accession HostID Heligmonellid Other intestinal N° MLP-He species parasites 674 Akodon _ Stilestrongylus — Rictulariidae undet. (3) azarae azarai (187) Cestoda undet. (3) Trichuris sp. (23) Syphacia sp. (6) 675 A. azarae __ §S. azarai (5) - 676 A. azarae _ S. azarai (14) Syphacia sp. (3) 683 A. azarae _ §S. azarai (31) Syphacia sp. (98) 686 A. azarae _ S. azarai(7) Syphacia sp. (15) 687T A. azarae _ S. azarai (13) Trichuris sp. (44) ttype host of S. azarai. Morphological study Worms were studied in toto under a light microscope (Leica DM2500 equipped with a drawing attachment). Measure- ments are provided in micrometres unless stated otherwise, with the range followed by the mean (in parentheses) and the coefficient of variation expressed as a percent value. Transverse body sections were made and mounted for syn- lophe examination. The sections are oriented with the dor- sal side of the worm towards the top of the page and the left side of the worm towards the left of the page. L317 Table 2. Helminth sets from Locality Il (RN 12 Km 100, Cam- pana, Argentina, type locality of Stilestrongylus oryzomysi). In parentheses, intensity of infection. The host’s identity was con- firmed by the re-examination of the voucher specimen. No other taxa were present in the intestine. Accession HostN° in HostID Heligmonellid species N° MLP-He MLP-Mz 1905 3072 Oligoryzomys Guerrerostrongylus zeta (10) flavescens __ Stilestrongylus flavescens (23) 1906 3090 _—O. flavescens G. zeta (3) S. flavescens (76) 1907 3073 = Oligoryzomys G. zeta (1) S. flavescens (26) nigripes 1913 3075 O. nigripes G. zeta (1) S. flavescens (12) Stilestrongylus lanfrediae (1) 1911 3074 Akodon Stilestrongylus azarai (93) azarae 1914 3076t A. azarae S. azarai (9) Trichofreitasia lenti (16) 1915 3077 A. azarae S. azarai (93) T. lenti (1) 1933 3078 A. azarae S. azarai (13) 1940 3079 A. azarae S. azarai (7) tsymbiotype of S. oryzomysi and Trichofreitasia lenti. Molecular study All attempts to obtain DNA from the aged material de- posited in the MLP-He were unsuccessful, and DNA had to be extracted from specimens collected in lo- calities other than the type localities (Table 6). These were recovered from A. azarae and Oligoryzomys ni- gripes (Olfers) and identified as S. azarai and G. zeta, respectively, on morphological criteria. The posterior ends of ethanol-fixed males were cut and individually stored in 70% ethanol as voucher specimens (accession numbers MLP-He 8101 to 8104). Genomic DNA was extracted from the remaining part of the body using a commercial kit (Promega) following the manufacturer’s instructions. A region of nuclear rDNA including 18S 3’-terminus, ITS1, 5.8S subunit, ITS2, and 28S 5’-ter- minus (ITS+) was amplified using the primer sets ITS-F (5’-TTG AAC CGG GTA AAA GTC G-3’) and ITS-R (5’-TTA GTT TCT TTT CCT CCG CT-3’) (Stock et al. 2001), a PCR master mix (Productos Bio-Logicos, Ar- gentina), and 6 ul of DNA for a final volume of 50 ul under the following PCR conditions: an initial dena- turation at 94 °C for 10 min, followed by 45 cycles at 94 °C for 30 s, 54 °C for 40 s, 72 °C for 80 s, anda final extension at 72 °C for 10 min. PCR products were analysed on 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualised on a UV tran- silluminator. Amplicons were submitted for sequencing to Macrogen Inc. (South Korea) with the same primers used for amplification. The obtained sequences ranged between 995 and 1010 bp in length; they were manually edited and compared with those in GenBank using the BLASTn tool (Altschul et al. 1990) to determine their phylo- genetic position. Alignments were made with MUS- CLE through MEGA v10.2.6 (Kumar et al. 2018) and zse.pensoft.net 1318 Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection Table 3. Helminth sets from Locality II (Bella Union, Uruguay, type locality of Guerrerostrongylus uruguayensis). In parentheses, intensity of infection. In bold, the host’s identity was confirmed by a voucher specimen. Otherwise, host identification is based on Heligmonellid species Guerrerostrongylus zeta (5) Stilestrongylus flavescens (5 G. zeta (14) S. flavescens (1 G. zeta (9) S. flavescens (16 G. zeta (7) S. flavescens (17 G. zeta (19) S. flavescens (51 G. zeta (48) S. flavescens (45) G. zeta (7) S. flavescens ( G. zeta (18) S. flavescens G. zeta (8) G. zeta (20) S. flavescens (5) G. zeta (40) S. flavescens (38) G. zeta (13) S. flavescens (7) G. zeta (25) S. flavescens (3) G. zeta (47) S. flavescens (12) G. zeta (27) S. flavescens (47) G. zeta (9) S. flavescens (20) Hassastrongylus hoineffae (1) G. zeta (21) S. flavescens (41) 7 6) —~ WY O1 Ww Other intestinal parasites Syphacia sp. (1) Syphacia sp. (5) Strongyloides sp. (1) Syphacia sp. (3) Syphacia sp. (7) Syphacia sp. (7) Syphacia sp. (3) field notes. Accession N° __— Host N° in Host ID MLP-He MLP-Mz 2046 - “Oryzomys” T 2047 3080 Oligoryzomys nigripes 2048 3081 Oligoryzomys flavescens 2049 3082 O. nigripes 2050 - No field data 2052 3083 O. nigripes 2054 - No field data 2056 ~ “Oryzomys” 2057 - “Oryzomys” 2058 3084 O. nigripes 2059 - “colilargo” 2062 ~ “Oryzomys” 2063 3085 O. nigripes 2064 3086 O. flavescens 2067 - “Oryzomys” 2068 3088 O. flavescens 2069 - “Oryzomys” 2051 ~ “Akodon” 2055 ~ “Akodon” 2061 ~ “Akodon” 2066 3087 Akodon azarae 2070 3089 A. azarae 2072 - No field data S. azarai (66) Rictulariidae undet. (2) Cestoda Hymenolepididae (1) Physalopteridae undet. (stomach) (1) Pterygodermatites sp. (1) Syphacia sp. (2) Rictulariidae undet. (1) S. azarai (315) S. azarai (2) S. azarai (23) S. azarai (67) Syphacia sp. (1) S. azarai (17) - ttype host of Guerrerostrongylus uruguayensis and Stilestrongylus flavescens. further edited with GBlocks (Castresana 2000). The genetic distances were calculated using the p-distance method as implemented in MEGA v10.2.6 with default parameters. The nucleotide substitution model was se- lected using jModelTest v2.1 based on the best-fitting model indicated by the Bayesian Information Criteri- on (BIC) (Darriba et al. 2012). Maximum likelihood analysis was performed in MEGA with 1,000 bootstrap replicates. Posterior probabilities of all branches were calculated using Bayesian-based inference as imple- mented in MrBayes v3.2, and analyses were run for 20,000,000 generations (Ronquist et al. 2012). Unci- naria lucasi Stiles, 1901 (Ancylostomatidae) was used as an outgroup. The resulting phylogenetic trees were edited in FigTree v1.4.4. It is worth mentioning that, despite several attempts, we failed to amplify the mitochondrial cytochrome c oxidase subunit 1 (COI) gene using the primer sets COlintF (5’-TGATTGGTGGTTTTGGTAA-3’) — and COlintR (5’-ATAAGTACGAGTATCAATATC-3’) (Ca- siraghi et al. 2001), LCOI1490 (5’-GGTCAACAAAT- CATAAAGATATTGG-3’), and HCOI2198 (5’-TA- AACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). zse.pensoft.net Results Morphological study Order Strongylida (Railliet & Henry, 1913) Suborder Trichostrongylina (Leiper, 1908, family) Durette-Desset & Chabaud, 1993 Family Heligmonellidae (Skrjabin & Schikhobalova, 1952 tribe) Durette-Desset & Chabaud, 1977 Subfamily Nippostrongylinae Durette-Desset, 1971 Genus Stilestrongylus Freitas, Lent & Almeida, 1937 Figs 1, 2, Table 4 Stilestrongylus azarai Durette-Desset & Sutton, 1985 Redescription. Based on type material (see Material and Methods) and 27 voucher specimens from the same sam- pling event (14 males, 13 females). General. Worms small to medium-sized, varying from loosely to tightly coiled, usually with 2-3 spires in ante- rior portion of body. Head. Observed in two voucher specimens. Round- ed buccal opening surrounded by thin ring; 2 amphids; Zoosyst. Evol. 100 (4) 2024, 1315-1331 1319 Figure 1. Stilestrongylus azarai. A, B. Synlophe in transverse section at midbody: A. Male; B. Female; C. Male caudal bursa, ventral view showing papillae 2; D. Right lobe of bursa, dorsal view; E. Left lobe of bursa, dorsal view; F. Dorsal lobe of bursa, dorsal view; G. Genital cone, left lateral view. 21-61 - left bursal rays 2 to 6; 81 - left ray 8; 2r-6r - right bursal rays 2 to 6; 8r - right ray 8; p0 - single papilla 0; p7 - paired papillae 7. Scale bars: 20 um (A, B, E); 50 um (C, D, F, G). C, G from paratypes. A, B, and D-F from other specimens in A. azarae from the type locality (Balcarce). 4 externo-labial (2 dorsal, 2 ventral); and 4 cephalic papillae visible; lateral externo-labial papillae probably fused with amphids; each cephalic papilla connected with its contiguous externo-labial papilla by thickening, shaped as arc of circle (see description in Digiani and Durette-Desset 2003c). Synlophe. Studied in 1 male and 1 female. Identical to that illustrated by Durette-Desset and Sutton (1985). With 25 subequal ridges in both sexes. Double axis of orientation of ridges: right axis inclined at 60° to sagittal axis, left axis at 80°. Twelve dorsal and 13 ventral ridges with respect to axis of orientation (Fig. 1A, B). Males. Measurements in Table 4. Bursa large, bell- shaped, and dissymmetrical, with dorsal lobe well de- veloped and right lobe markedly larger than left one. Prebursal papillae not observed. Bursa characterised by papillae 2 (ventral) very close (45—50 um) to each oth- er (Fig. 1C), implying that lobes were studied separately zse.pensoft.net 1320 Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection Figure 2. Stilestrongylus azarai. A, B. Female posterior end: A. Ventral view; B. Right lateral view; C, D. Anterior end: C. Curved and wrinkled, right lateral view (male); D. Stretched, left lateral view (female); E. Female synlophe in transverse section at mid- body; F, G. Male: F. Genital cone, right lateral view; G. Right lobe of caudal bursa, dorsal view; H, I. Female, posterior end: H. Ventral view; I. Right lateral view. 2r-6r - right bursal rays 2 to 6; pO - single papilla 0; p7 - paired papillae 7. Scale bars: 50 um (A-D, G—I); 20 um (E, F). A—C from paratypes of S. azarai. D from another specimen in A. azarae from the type locality (Bal- carce). E, H, and I from a paratype of S. oryzomysi from Campana. F and G from other specimens in A. azarae from Campana. because spreading out the bursa would break them. Right lobe (Fig. 1D): pattern of type 1-4, with ray 2 arising first from long common trunk of rays 3-6. Ray 3 aris- ing at mid-length of common trunk, but distance between papillae 2 and 3 larger than that between papillae 3 and 4. zse.pensoft.net Rays 4-6 diverging nearly at same level at distal quarter of common trunk, forming the characteristic “lateral tri- dent”. Ray 6 curved posteriorly, may be slightly longer than rays 4 and 5. Rays 3, 4, 5, and 6 reaching bursal mar- gin. Left lobe (Fig. 1E): pattern of type 2-2-1. Rays 2 and Zoosyst. Evol. 100 (4) 2024, 1315-1331 1321 Table 4. Comparison of measurements of Stilestrongylus azarai from Balcarce (type locality), Campana (including paratypes of Stilestrongylus oryzomysi), and San Luis City. Measurements are in micrometres, unless otherwise stated. Locality (Province) Balcarce (Buenos Aires) Campana (Buenos Aires) San Luis Capital City (San Luis) Host Akodon azarae Akodon azarae Graomys griseoflavus Source This work This work Digiani & Durette-Desset (2003c) Body length (mm) (BL) @ (n=-23) 2 (n = 19) 4 (n = 16) 2 (n = 20) 4 (n = 10) 2 (n = 10) 2.12-2.75 (2.35) 2.3-3.8(3.1) 2.03-3.39 (2.67) 1.88-5.63 (3.33) 3.00-4.00 (3.33) 4.5-5.7 (5.1) 6% 14.2% 19.4% 40.9% Body width 50-90 (78) 15% 70-100 (82) 50-120 (72) 75-130 (106) 120-150(132) 100-140 (120) 11.4% 34.4% 19.1% Cephalic vesicle length 45-60 (53) 8.4% 55-60 (57) 4.6% 42-60 (54) 40-65 (52) 55-65 (61) 50-75 (65) (n = 6) 10.6% 13.5% Cephalic vesicle width 18-30 (24) 20-28 (25) 20-28 (23) 14-45 (24) 30-40 (33) 35-40 (38) 15.1% 11.6% (n = 6) 14.4% 28.8% Oesophagus length (OeL) 230-310(277) 280-330(298) 245-295 (273) 210-340(276) 290-310(300) 320-360 (344) 6.8% 5.8% 6.7% 13.6% Nerve ringt 100-135 (113) 110-135(118) 110-150(130) 90-170(124) 140-175(155) 120-170 (150) 9.7% (n = 8) 9.8% (n = 5) 10.4% 14.4% Excretory poret (EP) 170-195 (176) 195-220(203) 185-245 (214) 157-250(197) 220-260 (239) 190-240 (220) 11% (n = 5) 7.1% (n = 3) 9.7% (n = 6) 16% EP/OeL (%) 59.6-70.9 (64) 59.1-73.3 (66.6) 72.5-83.1 (77.8) 61.3-76.9 (69.7) 71.0-88.1 (79.2) 54.3-66.7 (61.8) LOR etne=-5)e” 1.7 el = 3) 5.1% (n = 6) 8.3% Deiridst 140-205 (177) 195-220 (206) 185-245 (219) 160-245(200) 220-260 (239) 190-240 (220) 12.9% (n=10) 6.4% (n = 4) 9.3% (n = 7) 16.2% Spicule length (SpL) 310-460 (380) - 290-445 (366) - 440-480 (456) - 10.2% 13.2% SpL/BL (%) 14.4-18.9 (16.5) - 11.7-16.5 (13.8) - 11.3-16.0 (13.8) = 8.7% 10% Genital cone length 70-125 (88) - 60-135 (109) - = = 16.3% 20.7% Genital cone width 27-50 (40) - 32-50 (42) - - - 16.7% 14.9% Vulvat - 55-80 (68) - 32-88 (62) - 70-100 (82) 11.6% (n = 9) 27.5% Vestibule length - 30-65 (48) 22% - 35-65 (54) - 55-75 (62) 16.3% (n = 9) Sphincter length - 25-30 (27) 7.9% - 20-30 (28) Ss 30-40 (38) 12.9% Infundibulum length - 65-85 (77) 8.7% ~ 70-80 (78) 6.5% - 90-110 (104) (n = 10) (n = 4) Uterus length (UtL) - 270-880 (460) - 270-1000 (575) - 800-980 (874) 31.2% 45.4% UtL/BL (%) - 11.2-23:2 (15.5) - 11.3-21.1 (14.8) - 15-19 (17) 22.7% 22.2% Tail length - 20-35 (28) - 22-45 (34) - 20-25 (23) 16.7% (n = 7) 25.4% (n = 9) Egg number - 3-24 (9) 61.1% - 3-12 (7) 42.4% - 24-43 fdistance from apex. tdistance from posterior extremity. 3 diverging in “‘V” proximally. Very short common trunk of rays 4-6, with rays 3 and 6 diverging at same level. Rays 4 and 5 diverging last. All rays reach bursal margin. Distance between papillae 2 and 3 slightly larger than that between papillae 5 and 6. Papillae 3, 4, and 5 approxi- mately equidistant from each other. Rays 2 thickest and 6 thinnest. Dorsal lobe well developed, displaced to the left, compensating the reduced size of left lobe. Dorsal ray very long, divided at about its distal quarter into two branches, each one bifurcated distally into two papillae, external rays 9 and internal rays 10. Arising and path of rays 8 markedly dissymmetrical (Fig. 1F). Left ray 8 aris- ing proximally from dorsal ray, nearly at its base; then running close and parallel to left trunk, approaching to and ending near left papilla 6. Right ray 8 arising more distally on dorsal ray, at about its proximal quarter, run- ning between right trunk and dorsal ray, then heading distally towards right ray 6 but ending far from it. Geni- tal cone long, well developed, bulbous at base, strongly curved ventrally in distal part. Distal half of cone with sclerotised walls. Dorsal lip bearing two rounded con- spicuous papillae 7; ventral lip with single lappet-shaped papilla 0 (Fig. 1G). Gubernaculum conspicuous. Spicules thin and alate, ending in single sharp tip. zse.pensoft.net 1322 Females. Measurements in Table 4. Reproductive tract monodelphic. Uterus less than 20% of body length; eggs few (3-9). Infundibulum slightly longer than vestibule. Posterior extremity straight or barely curved. Slight cu- ticular inflation from level of distal uterus up to vulvar aperture. Tail short and stout, ending in mucron (Figs 2A, B). Posterior end moderately retractile. Voucher material examined. ARGENTINA * 2 33, 2 9: Buenos Aires Province, Balcarce; 1977-1978; Sut- ton leg.; Akodon azarae; MLP-He 0674-1 * 3 d@; same collection data as for preceding; MLP-He 0675-1 ° 1 3, 1 9; same collection data as for preceding; MLP-He 0676- 1°5 do, 6 PY; same collection data as for preceding; MLP-He 0683-1 * 3 33’, 4 92: same collection data as for preceding; MLP 0686-1. Prevalence and mean intensity. P=100% (n=6), MI=42.8 (range 5-187) (Table 1). Remarks. Worms of the type series showed strong contraction and curvature of the anterior body (Fig. 2C). Indeed, the body portion between the cephalic vesicle and the oesophageal-intestinal junction often appeared considerably wrinkled, hindering measurements of soft organs in this region (Table 4). On the contrary, the poste- rior body was relaxed, stretched, and easily accessible for measurements. The scarcity of material led us to include additional specimens from other A. azarae hosts collected in the same sampling event. We examined a total of five complete helminth sets, all of which were composed of worms assignable to S. azarai. Among these, we selected worms with relaxed and stretched anterior ends (Fig. 2D) to take measurements of the proximal part. Specimens identified as Stilestrongylus oryzomysi Sutton & Durette-Desset, 1991 Fig. 2, Table 4 Considerations on the type host species. The rodent with field number 1914, designated as the type host of S. oryzomysi and T: lenti by Sutton and Durette-Des- set (1991), was originally attributed to “Oryzomys fla- vescens” (current name= Oligoryzomys flavescens). After re-examination of the voucher (now designated as symbi- otype) in MLP-Mz (accession N° 3076), it was found to belong to A. azarae (Table 2, Fig. 3). Therefore, A. azarae is the actual type host species of S. oryzomysi. Examination of the type series. Head. Observed in one female paratype. Identical to that illustrated by Sut- ton and Durette-Desset (1991). Synlophe. Based on one female paratype. With 25 subequal ridges, regularly spaced, mostly oriented from right-ventral to left-dorsal quadrant, with axis of orien- tation inclined at about 60° to sagittal axis. Right-ventral ridges smallest (Fig. 2E). Males. Proximal part of body loosely coiled. Geni- tal cone long, well developed, strongly curved ventrally in distal part. Distal half of cone with sclerotised walls. Dorsal lip bearing two papillae 7; ventral papilla O not zse.pensoft.net Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection observed. Gubernaculum conspicuous. Spicules thin and alate, ending in single sharp tip (Fig. 2F). Females. Body varying from loosely coiled in 2-3 tr- regular spirals to tightly coiled in up to 5 spirals. Mono- delphic. Uterus less than 20% of body length; eggs few (3-9). Infundibulum longer than vestibule. Posterior ex- tremity mostly straight, sometimes slightly curved ven- trally. Cuticular inflation present from level of ovejector up to vulvar aperture. Posterior end retractile into infla- tion, never completely invaginated. Tail short and stout, ending in mucron (Fig. 2H, I). Remarks. The worms in the type series of S. ory- zomysi were relaxed and stretched, and diaphonisation was unnecessary for taking measurements. However, the males showed excessive flattening of the body, hin- dering the study of the bursa. Additional worms had to be included from the same sampling event due to mate- rial scarcity. We examined the complete helminth sets recovered from two O. flavescens, two O. nigripes, and five A. azarae. None of the Oligoryzomys spp. harboured worms assignable to S. oryzomysi. Instead, they were parasitised by S. flavescens (P=100%, MI=34.2) and to a lesser degree, by Stilestrongylus lanfrediae Souza, Digiani, Sim6es, Rodrigues-Silva & Maldonado, 2009 (Table 2). On the other hand, specimens of A. azarae were consistently parasitised by worms assignable to S. azarai (P=100%, MI=43) (Fig. 2G). Both A. azarae and Oligoryzomys spp. were also parasitised by nippostron- gylines of other genera: two A. azarae specimens har- boured 7: /enti, and all Oligoryzomys spp. harboured G. zeta (P=100%, MI=3.7) (Table 2). Voucher material examined. ARGENTINA * 11 34, 10 9 9; Buenos Aires Province, Campana, National Route 12 (RN12) Km 100-101; [34°00'27.432"S, 58°58'29.28"W]; Apr. 1989; Sutton leg.; Akodon azarae, MLP-Mz 3074; MLP-He 1911-1 * 3 4; same collection data as for pre- ceding; MLP-Mz 3079; MLP-He 1940-1. Genus Guerrerostrongylus Sutton & Durette-Desset, 1991 Fig. 4, Table 5 Guerrerostrongylus zeta (Travassos, 1937) Longistriata zeta Travassos, 1937 Hassalstrongylus zeta (Travassos, 1937): Durette-Desset, 1971 Guerrerostrongylus zeta (Travassos, 1937): Sutton & Durette-Desset, 1991 Main diagnostic characters. Body large to very large (males 4.20-8.40, females 5—13.7 mm), uncoiled or coiled irregularly; cephalic vesicle relatively short and stout. Synlophe with numerous (>35) continuous, sub- equal ridges, oriented from right to left (right, dorsal, and ventral ridges) or perpendicular to body surface (left ridges). Males: 37 to 44 ridges, caudal bursa subsymmet- rical, elliptical to rectangular, pattern 2-2-1 tending to 1-3-1, dorsal lobe well developed; rays 6 longest, arising at same level as rays 2; rays 8 arising at proximal 1/4 or Zoosyst. Evol. 100 (4) 2024, 1315-1331 1323 Table 5. Comparison of measurements of G. zeta from different sources (including types of Guerrerostrongylus uruguayensis). Measurements are in micrometres except otherwise stated. Travassos 1937 Digiani et al. 2012 Simoes et al. 2012a Werk et al. 2016 This work (description) (redescription, on _(redescription, on syntypes (new locality voucher material) and voucher material) records) S(n=?) 9 (n=?) 3 (n=23) 9 (n=27) J (n=10) 2 (n= 10) 3 (n= O(n= G(n=22) 9° (n= 21) 10) 10) N° of ridges ? if 40-44 35-48 37 (syntypes, 53 (syntypes, 40-46 (n not 38-53 40-50 at midbody (n = 4) (n= 7) n=1)36-42 n=1) 38-42 specified) (n = 4) (n = 4) (vouchers, (vouchers, =?) n = 2) Body length 6400 6800- 44-84 5.5-13.7 4.28-6.90 5.06-12.67 6.6-10.7 10.2- 4.35-8.9 4.95-14.9 (mm) (BL) 7300 (6.75) (9.17) (5.2) (8.41) (8.5) 17.9 (7.0) 18.3% (9.47) (14.5) 21.8% Body width 150 = 140-150 140-290 100-290 80-180 100-320 83-157 157-230 90-250 200-350 (197) (197) (176) 24.6% (247) 16.1% Cephalic 45-52 45-52 35-70(56) 35-65 (55) 43-70 40-74 55-64 46-64 40-60(50) 40-70 (49) vesicle length 12.2% 17.8% Cephalic - 32-40 30-60 (44) 35-60 (43) 20-56 36-67 46-55 46-64 30-70(47) 30-55 (41) vesicle width (36) 23.4% 19.6% Oesophagus 340-470 340-470 345-495 350-500 340-716 260-390 369-498 500-672 315- 330-430 length (OeL) (401) (411) 375(348) — (383) 9.9% 8.2%(n=5) (n= 6) Nerve ringt - - 190-295 130-285 70-233 100-250 182-314 434 120-145 135-210 (234) (169) (132) 9.6% (178) 21.8% (n = 3) (n = 3) Excretory 200 200 250-345 345-380 229-633 221-402 323-425 455.470 185-225 205-270 pore (EP) (305)) (203) 9.9% (237) 13.7% (n = 3) (n = 3) EP/OeL (%) - - 66-84 75-81 (n= - 58.8-73.9 - - - 58.7-77.1 12) (66.1) (n = 5) (66) 14.8% (n = 3) Deiridst - - 250-380 235-275 235-327 200-268 - - 230(n=1) 330(n=1) (315) (253) (267) (n =4) (233) (n = 3) Spicule length 877 - 750-1420 - 580-1160 - 800- - 810-1130 - (SpL) (1115) 1300 (963) 10.7% SpL/BL (%) 137 - 10.6-24.4 - - - - 11.8-18.6 - (15.5) (15) 14.9% Vulvat - 112-135 - 112-255 - 105-233 - 157-277 - 105-145 (176) (130) 10.9% (n = 6) Uterus length - - - 800-1560 - 1350-2540 - - - 1400-1950 (UtL) (1230) (1622) 14.1% UtL/BL (%) - - - 9.5-22.4 - - - - 15.4-21.9 (16.1) (19.2) 11.8% (n = 6) Tail length - 43-45 - 40-100 - 43-97 - 42-61 - 35-60 (70) (49) 18.7% (n = 6) Egg number - - - 6-50 (28) - - - >50 - 41-130 (80) 40.1% (n = 6) fdistance from apex. {distance from posterior extremity. 1/3 of dorsal trunk. Genital cone moderately developed, telamon V-shaped, spicules long and thin with undulating or winding pattern. Females: 35 to 53 ridges; vestibule Fig. 4, Table 5 as long as infundibulum; uterus ca. 20% of body length; Specimens identified as Guerrerostrongylus uruguayensis Sutton & Durette-Desset, 1991 Considerations on the type host species. The rodent with field number 2046, designated as type host of G. uruguayensis and Stilestrongylus flavescens by Sutton and Durette-Desset (1991), was originally attributed to eggs numerous. Posterior extremity straight; tail conical, not retractile. Tail length about 1/3 of distance from vulva to posterior extremity (from Travassos 1937; Digiani et al. 2012; SimGes et al. 2012a). zse.pensoft.net 1324 Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection Figure 3. Akodon azarae MLP-Mz 3076 (field number 1914), symbiotype of Stilestrongylus oryzomysi and Trichofreitasia lenti. Figure 4. Guerrerostrongylus zeta. A. Male synlophe in transverse section at midbody; B. Male caudal bursa, dorsal view (left lobe omitted); C. Spicules, ventral view; D. Gubernaculum and genital cone, ventral view; E. Female, posterior end, right lateral view. 81 - left ray 8; 2r-6r - right bursal rays 2 to 6; 8r - right ray 8. Scale bars: 50 um (A, D); 100 um (B, C, E). A, B, D, E from specimens of Oligoryzomys nigripes from Bella Union. C from a paratype of G. uruguayensis. zse.pensoft.net Zoosyst. Evol. 100 (4) 2024, 1315-1331 “Oryzomys flavescens” (current name= Oligoryzomys fla- vescens). Unfortunately, its specific identity could not be established because no voucher was found in MLP-Mz or in other zoological collections. A symbiotype for these species is therefore lacking. Examination of the type series. Most of the inter- nal characters of the specimens could not be observed or measured because of their opaque bodies, even after using strong clearing agents. The low number of material prompted us to include more specimens from the same sampling event, and as a result, 16 complete helminth sets from hosts assigned to Oligoryzomys spp. were exam- ined. All of them harboured several specimens of Guer- rerostrongylus sp., which could be studied and measured. General. Worms large to very large, uncoiled. Head. Cephalic vesicle short and stout. Apical struc- tures hardly visible. Oral aperture large, triangular, with rounded corners, and surrounded by thick ring. Four ex- terno-labial papillae (dorsal and ventral) observed in one female. Synlophe. Based on four males and four females. With 38—53 ridges in males (Fig. 4A), 40—50 in females. Ridg- es continuous, subequal, oriented from right to left (right, dorsal, and ventral ridges), or perpendicular to body sur- face (left ridges). Two prominent lateral cuticular dila- tations, left larger than right one. Synlophe identical to those described by Digiani et al. (2012, figs 3, 4) and (Sim6es et al. 2012a, figs 4, 5) for G. zeta. Males. Large to very large, uncoiled or coiled irregu- larly. Bursa subsymmetrical, elliptical, with pattern 2-2-1 tending to 1-3-1. Rays 6 longest, arising at same level as rays 2; rays 8 arising at proximal 1/4 of dorsal trunk. Some specimens show serrated ornamentation on mar- gins of rays 4 or 4-5 and/or thickened bases of dorsal ray and rays 8 (Fig. 4B). Spicules long and thin with undulat- ing pattern (Fig. 4C). Genital cone moderately developed, telamon V-shaped (Fig. 4D). Females. Large to very large; vestibule as long as infundibulum; uterus ca. 20% of body length; eggs nu- merous. Posterior extremity straight; tail conical, not re- tractile. Tail length about 1/3 of distance from vulva to posterior extremity (Fig. 4E). Voucher material examined. UruGuay ° 1 <4, 2 99; Artigas Department, Bella Union, Colonia Espafia; [30°21'52"S, 57°38'34"W]; Jul. 1989; Sutton leg.; Oli- goryzomys nigripes, MLP-Mz 3080; MLP He-2047-1 «3 Soh, 2 2: same data as for preceding; MLP-Mz 3084: MLP-He 2058-1 * 2 do, 2 2 9; same collection data as for preceding; “Oryzomys”, MLP-He 2056-1 * 4 ¢¢, 5 © ©; same data as for preceding; MLP-He 2057-1 °6 2, 5 29; same data as for preceding; “colilargo”; MLP-He 2059-1 + 3 do, 1 Y; same data as for preceding; O. fla- vescens, MLP-Mz 3086; MLP-He 2064-1. Prevalence and mean intensity. P=100% (n=17), MI=19.8 (range 5-48) (Table 3). Remarks. Some helminth sets from this sampling lacked the voucher host in the mammal collection. Only eight of them could be located, which corresponded to 1325 the helminth sets MLP-He 2047, 2049, 2052, 2058, and 2063 (O. nigripes); MLP-He 2048, 2064, and 2068 (O. fla- vescens) (Table 3). The hosts of the remaining helminth sets were a priori identified in the field and subsequently la- belled as “Oryzomys sp.” or “colilargo” (Table 3). Whether the identity of these hosts was further confirmed or not, they had helminth assemblages composed of Guerreros- trongylus spp. showing the features described above and of S. flavescens (P=94.1%, MI=21.2). One specimen iden- tified as O. flavescens also harboured Hassastrongylus ho- ineffae (Durette-Desset, 1969). In addition, we examined six A. azarae from the same sampling event and found that they were parasitised by S. azarai (P=100%, MI=81.7), but not by any species of Guerrerostrongylus (Table 3). Molecular study Sequences of ITS+ were obtained from three specimens of G. zeta (970-973 bp) and one of S. azarai (950 bp) (Table 6). After alignment with MUSCLE, we obtained 1056 positions, which were reduced to 910 using GBLOCKS. GTR+G was selected as the best-fit substitution model using MEGA and MRBAYES. The resulting trees show similar topology except for the position of Nippostrongy- lus brasiliensis (Travassos, 1914). No genetic divergence is found between the sequenc- es of G. zeta specimens (Table 7), which cluster together with Hassalstrongylus sp., forming a strongly supported monophyletic clade in both phylogenetic reconstructions (Figs 5, 6). In turn, a clade including these two species and S. azarai (South American nippostrongylines) was recovered in both phylogenetic analyses, though with lower support. Discussion and conclusions Taxonomic aspects S. azarai /S. oryzomysi We found no morphological differences among the type series of S. oryzomysi, the specimens of S. azarai har- boured by A. azarae from the same sampling event, and the type series of S. azarai. The synlophe of a paratype of S. oryzomysi was identical to that of S. azarai in fig. Ic of Durette-Desset and Sutton (1985). Details of the ovejector and tail in the female and the bursa and genital cone in the male were identical in both type series. Therefore, we propose to consider the relative distances between papil- lae 2, 3, and 4 of the right lobe, the pathway of rays 8, and the appearance of the papillae on the genital cone as diagnostic characters of S. azarai. Moreover, the cephalic structures and arrangements observed by Sutton and Du- rette-Desset (1991) in worms from the locality of Campa- na were also observed in voucher specimens of S. azarai. zse.pensoft.net 1326 0.05 Uneinaria lucasi HO262141 Digiani, M.C. & Serrano, P.C.: Rodent parasitic nematodes from La Plata Museum Collection Guerrerostrongylus zeta PP447917 100 Guerrerostrangylus zeta PP447916 Guerrerostrongylus zeta PP447919 Hassalstrongylus sp, JX877694 Stilestrongylus azarai PP447916 Vexillata convoluta JXS7TT692 Vexillata liomyos MN366464 Carolinensis perezponcedeleoni JX877666 Paraheligmonella lamothei MN366457 Nippostrongylus brasiliensis AY332646 Figure 5. Phylogram resulting from Bayesian inference. The values next to the nodes are posterior probabilities (%). The scale bar represents the number of substitutions per site. 78 af Guerrerostrongylus zeta PP447917 Guerrerostrongylus zeta PP447919 Guerrerostrongylus zeta PP447918 Hassalstrongylus sp. JX877694 Stilestrongylus azarai PP447916 Paraheligmonella Jamothei MN366457 Vexillata convoluta JX877692 Vexilfata liomyos MN366464 Carolinensis perezponcedeleoni JX877686 Nippostrongylus brasiliensis AY332646 Uncinaria lucasi HQ262141 Figure 6. Maximum likelihood bootstrap consensus tree. Values next to the nodes are bootstrap proportions (%). In regard to morphometry, most of the body length values obtained by us for S. oryzomysi were lower than those reported by Sutton and Durette-Desset (1991) (Ta- ble 4). Stilestrongylus oryzomysi was differentiated from S. azarai by the more distal position of the excretory pore (72-74% of the oesophagus length vs. 41-51%) and by the more developed genital cone (90 x 36 um vs. 80 x 40 um) (Sutton and Durette-Desset 1991). These characters are of poor diagnostic value in the Trichostrongylina, in which morphometric characters of soft organs may vary even within individual hosts (Durette-Desset et al. 2017). It should be kept in mind that the only measurements re- ported for both S. azarai and S. oryzomysi were taken from their holotypes and allotypes, probably accounting for their metric discrepancies. Moreover, the measurements zse.pensoft.net of the anterior organs provided in the original description of S. azarai were most likely underestimated because the specimens of the type series were wrinkled. Digiani & Durette-Desset (2003c) also found differences between S. azarai specimens parasitising Graomys griseoflavus (Wa- terhouse) from San Luis province, Argentina, and those described in the original description (i.e., larger body measurements, a more distal position of the nerve ring and excretory pore, a larger number of eggs in the uterus) (see Table 4). Notwithstanding these metric discrepan- cies, the worms from San Luis unequivocally exhibited the diagnostic characters of S. azarai. Arc-like structures connecting the cephalic papillae were also present in the helminths of G. griseoflavus (figs 26, 33-35 of Digiani and Durette-Desset 2003c). Zoosyst. Evol. 100 (4) 2024, 1315-1331 1327 Table 6. Nematode family and species, host species, collection locality, and GenBank accession number for sequences of ITS+ rDNA used for phylogenetic analysis. In bold, the sequences obtained in this work. Family Species Host Locality Accession number Heligmonellidae S. azarai A. azarae Berisso, Buenos Aires province, Argentina PP447916 G. zeta (1) O. nigripes Campo San Juan, Misiones province, Argentina PP447917 G. zeta (2) O. nigripes Campo San Juan, Misiones province, Argentina PP447918 G. zeta (3) O. nigripes Campo San Juan, Misiones province, Argentina PP447919 Hassalstrongylus sp. Calomys sp. Santa Barbara, Jujuy province, Argentina JX877694. Nippostrongylus brasiliensis Rodents Brest, France AY332646 Carolinensis perezponcedeleoni Nyctomys sumichrasti Catemaco, Veracruz, Mexico JX877686 Paraheligmonella lamothei Sylvilagus floridanus Chiapas, Mexico MN366457 Ornithostrongylidae Vexillata liomyos Liomys pictus Jalisco, Mexico MN366464 Vexillata convoluta Cratogeomys merriami Morelos, Mexico JX877692 Ancylostomatidae Uncinaria lucasi Eumetopias jubatus Hazy Island, Southeast AK, USA HQ262141 Table 7. Genetic divergence among species of Heligmonellidae, estimated through the uncorrected p-distance of the ITS+ rDNA region. 1 2 3 1 ~—_~Uncinaria lucasi (outgroup) 2 S. azaral 0.30 3 G. zeta (specimen 1) 0:28" "2OLKS8 4 G. zeta (specimen 2) 0.28" 20:138~