Stilestrongylus azarai

ARGENTINA • ♂, holotype; Buenos Aires Province, Balcarce; 1977–1978; Sutton leg.; Akodon azarae; MLP-He 0687-1• ♀, allotype; same data as for holotype; MLP-He 0687-2 • 6 ♂♂, 6 ♀♀, paratypes; same collection data as for preceding; MLP-He 0687-3.

Stilestrongylus oryzomysi

ARGENTINA • 2 ♂♂, 10 ♀♀, 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 • ♂, holotype; Artigas Department, Bella Unión, Colonia España; [30°21'52"S, 57°38'34"W]; Jul. 1989; Sutton leg.; “Oryzomys flavescens”; MLP-He 2046-1 • ♀, allotype; same data as for holotype; MLP-He 2046-2 • 2 ♂♂ (distal fragments), 3 ♀♀, paratypes; same collection data as for preceding; MLP-He 2046-3.

Type material of G. zeta 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 (Simões et al. 2012a) and of voucher specimens from Brazil and Argentina (Misiones province) (Digiani et al. 2012).

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. azarai, 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 unidentified. Therefore, each set examined corresponded to an 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 II (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 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 División Zoología 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. III 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).

Worms were studied in toto under a light microscope (Leica DM2500 equipped with a drawing attachment). Measurements 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 synlophe examination. The sections are oriented with the dorsal side of the worm towards the top of the page and the left side of the worm towards the left of the page.

All attempts to obtain DNA from the aged material deposited in the MLP-He were unsuccessful, and DNA had to be extracted from specimens collected in localities other than the type localities (Table 6). These were recovered from A. azarae and Oligoryzomys nigripes (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’-terminus (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-Lógicos, Argentina), and 6 µl of DNA for a final volume of 50 µl under the following PCR conditions: an initial denaturation 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, and a 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 transilluminator. 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 phylogenetic position. Alignments were made with MUSCLE through MEGA v10.2.6 (Kumar et al. 2018) and 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 selected using jModelTest v2.1 based on the best-fitting model indicated by the Bayesian Information Criterion (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 implemented in MrBayes v3.2, and analyses were run for 20,000,000 generations (Ronquist et al. 2012). Uncinaria 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 COIintF (5´-TGATTGGTGGTTTTGGTAA-3´) and COIintR (5´-ATAAGTACGAGTATCAATATC-3´) (Casiraghi et al. 2001), LCOI1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’), and HCOI2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994).

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 sampling event (14 males, 13 females).

General. Worms small to medium-sized, varying from loosely to tightly coiled, usually with 2–3 spires in anterior portion of body.

Head. Observed in two voucher specimens. Rounded buccal opening surrounded by thin ring; 2 amphids; 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).

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. 2l-6l - left bursal rays 2 to 6; 8l - 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 µm (A, B, E); 50 µm (C, D, F, G). C, G from paratypes. A, B, and D–F from other specimens in A. azarae from the type locality (Balcarce).

Males. Measurements in Table 4. Bursa large, bell-shaped, and dissymmetrical, with dorsal lobe well developed and right lobe markedly larger than left one. Prebursal papillae not observed. Bursa characterised by papillae 2 (ventral) very close (45–50 μm) to each other (Fig. 1C), implying that lobes were studied separately 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 arising at mid-length of common trunk, but distance between papillae 2 and 3 larger than that between papillae 3 and 4. Rays 4–6 diverging nearly at same level at distal quarter of common trunk, forming the characteristic “lateral trident”. Ray 6 curved posteriorly, may be slightly longer than rays 4 and 5. Rays 3, 4, 5, and 6 reaching bursal margin. Left lobe (Fig. 1E): pattern of type 2-2-1. Rays 2 and 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 approximately 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 arising 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, running between right trunk and dorsal ray, then heading distally towards right ray 6 but ending far from it. Genital 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 conspicuous papillae 7; ventral lip with single lappet-shaped papilla 0 (Fig. 1G). Gubernaculum conspicuous. Spicules thin and alate, ending in single sharp tip.

Table 4.

Download as

CSV

XLSX

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)	♀ (n = 19)	♂ (n = 16)	♀ (n = 20)	♂ (n = 10)	♀ (n = 10)
	2.12–2.75 (2.35) 6%	2.3–3.8 (3.1) 14.2%	2.03–3.39 (2.67) 19.4%	1.88–5.63 (3.33) 40.9%	3.00–4.00 (3.33)	4.5–5.7 (5.1)
Body width	50–90 (78) 15%	70–100 (82) 11.4%	50–120 (72) 34.4%	75–130 (106) 19.1%	120–150 (132)	100–140 (120)
Cephalic vesicle length	45–60 (53) 8.4%	55–60 (57) 4.6% (n = 6)	42–60 (54) 10.6%	40–65 (52) 13.5%	55–65 (61)	50–75 (65)
Cephalic vesicle width	18–30 (24) 15.1%	20–28 (25) 11.6% (n = 6)	20–28 (23) 14.4%	14–45 (24) 28.8%	30–40 (33)	35–40 (38)
Oesophagus length (OeL)	230–310 (277) 6.8%	280–330 (298) 5.8%	245–295 (273) 6.7%	210–340 (276) 13.6%	290–310 (300)	320–360 (344)
Nerve ring†	100–135 (113) 9.7% (n = 8)	110–135 (118) 9.8% (n = 5)	110–150 (130) 10.4%	90–170 (124) 14.4%	140–175 (155)	120–170 (150)
Excretory pore† (EP)	170–195 (176) 11% (n = 5)	195–220 (203) 7.1% (n = 3)	185–245 (214) 9.7% (n = 6)	157–250 (197) 16%	220–260 (239)	190–240 (220)
EP/OeL (%)	59.6–70.9 (64) 10.3% (n = 5)	59.1–73.3 (66.6) 10.7% (n = 3)	72.5–83.1 (77.8) 5.1% (n = 6)	61.3–76.9 (69.7) 8.3%	71.0–88.1 (79.2)	54.3–66.7 (61.8)
Deirids†	140–205 (177) 12.9% (n = 10)	195–220 (206) 6.4% (n = 4)	185–245 (219) 9.3% (n = 7)	160–245 (200) 16.2%	220–260 (239)	190–240 (220)
Spicule length (SpL)	310–460 (380) 10.2%	–	290–445 (366) 13.2%	–	440–480 (456)	–
SpL/BL (%)	14.4–18.9 (16.5) 8.7%	–	11.7–16.5 (13.8) 10%	–	11.3–16.0 (13.8)	–
Genital cone length	70–125 (88) 16.3%	–	60–135 (109) 20.7%	–	–	–
Genital cone width	27–50 (40) 16.7%	–	32–50 (42) 14.9%	–	–	–
Vulva‡	–	55–80 (68) 11.6% (n = 9)	–	32–88 (62) 27.5%	–	70–100 (82)
Vestibule length	–	30–65 (48) 22%	–	35–65 (54) 16.3% (n = 9)	–	55–75 (62)
Sphincter length	–	25–30 (27) 7.9%	–	20–30 (28) 12.9%	–	30–40 (38)
Infundibulum length	–	65–85 (77) 8.7% (n = 10)	–	70–80 (78) 6.5% (n = 4)	–	90–110 (104)
Uterus length (UtL)	–	270–880 (460) 31.2%	–	270–1000 (575) 45.4%	–	800–980 (874)
UtL/BL (%)	–	11.2–23.2 (15.5) 22.7%	–	11.3–21.1 (14.8) 22.2%	–	15–19 (17)
Tail length	–	20–35 (28) 16.7% (n = 7)	–	22–45 (34) 25.4% (n = 9)	–	20–25 (23)
Egg number	–	3–24 (9) 61.1%	–	3–12 (7) 42.4%	–	24–43

†distance from apex. ‡distance from posterior extremity.

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 cuticular inflation from level of distal uterus up to vulvar aperture. Tail short and stout, ending in mucron (Figs 2A, B). Posterior end moderately retractile.

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 midbody; 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; p0 - single papilla 0; p7 - paired papillae 7. Scale bars: 50 µm (A–D, G–I); 20 µm (E, F). A–C from paratypes of S. azarai. D from another specimen in A. azarae from the type locality (Balcarce). E, H, and I from a paratype of S. oryzomysi from Campana. F and G from other specimens in A. azarae from Campana.

Voucher material examined

Argentina • 2 ♂♂, 2 ♀♀; Buenos Aires Province, Balcarce; 1977–1978; Sutton leg.; Akodon azarae; MLP-He 0674-1 • 3 ♂♂; same collection data as for preceding; MLP-He 0675-1 • 1 ♂, 1 ♀; same collection data as for preceding; MLP-He 0676-1 • 5 ♂♂, 6 ♀♀; same collection data as for preceding; MLP-He 0683-1 • 3 ♂♂, 4 ♀♀; 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 posterior 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.

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-Desset (1991), was originally attributed to “Oryzomys flavescens” (current name= Oligoryzomys flavescens). After re-examination of the voucher (now designated as symbiotype) 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.

Figure 3. 

Akodon azarae MLP-Mz 3076 (field number 1914), symbiotype of Stilestrongylus oryzomysi and Trichofreitasia lenti.

Examination of the type series. Head. Observed in one female paratype. Identical to that illustrated by Sutton 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 orientation inclined at about 60° to sagittal axis. Right-ventral ridges smallest (Fig. 2E).

Males. Proximal part of body loosely coiled. Genital 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 0 not observed. Gubernaculum conspicuous. Spicules thin and alate, ending in single sharp tip (Fig. 2F).

Females. Body varying from loosely coiled in 2–3 irregular spirals to tightly coiled in up to 5 spirals. Monodelphic. Uterus less than 20% of body length; eggs few (3–9). Infundibulum longer than vestibule. Posterior extremity mostly straight, sometimes slightly curved ventrally. Cuticular inflation present from level of ovejector up to vulvar aperture. Posterior end retractile into inflation, never completely invaginated. Tail short and stout, ending in mucron (Fig. 2H, I).

Remarks. The worms in the type series of S. oryzomysi were relaxed and stretched, and diaphonisation was unnecessary for taking measurements. However, the males showed excessive flattening of the body, hindering the study of the bursa. Additional worms had to be included from the same sampling event due to material 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, Simões, 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 nippostrongylines of other genera: two A. azarae specimens harboured T. lenti, and all Oligoryzomys spp. harboured G. zeta (P=100%, MI=3.7) (Table 2).

Voucher material examined. Argentina • 11 ♂♂, 10 ♀♀; 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 ♂♂; same collection data as for preceding; MLP-Mz 3079; MLP-He 1940-1.

Fig. 4, Table 5

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 “Oryzomys flavescens” (current name= Oligoryzomys flavescens). 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 internal 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 examined. All of them harboured several specimens of Guerrerostrongylus sp., which could be studied and measured.

General. Worms large to very large, uncoiled.

Head. Cephalic vesicle short and stout. Apical structures hardly visible. Oral aperture large, triangular, with rounded corners, and surrounded by thick ring. Four externo-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. Ridges continuous, subequal, oriented from right to left (right, dorsal, and ventral ridges), or perpendicular to body surface (left ridges). Two prominent lateral cuticular dilatations, left larger than right one. Synlophe identical to those described by Digiani et al. (2012, figs 3, 4) and (Simões et al. 2012a, figs 4, 5) for G. zeta.

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. 8l - left ray 8; 2r-6r - right bursal rays 2 to 6; 8r - right ray 8. Scale bars: 50 μm (A, D); 100 μm (B, C, E). A, B, D, E from specimens of Oligoryzomys nigripes from Bella Unión. C from a paratype of G. uruguayensis.

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.

Males. Large to very large, uncoiled or coiled irregularly. 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 margins of rays 4 or 4–5 and/or thickened bases of dorsal ray and rays 8 (Fig. 4B). Spicules long and thin with undulating 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 numerous. Posterior extremity straight; tail conical, not retractile. Tail length about 1/3 of distance from vulva to posterior extremity (Fig. 4E).

Voucher material examined. Uruguay • 1 ♂, 2 ♀♀; Artigas Department, Bella Unión, Colonia España; [30°21'52"S, 57°38'34"W]; Jul. 1989; Sutton leg.; Oligoryzomys nigripes, MLP-Mz 3080; MLP He-2047-1 • 3 ♂♂, 2 ♀♀; same data as for preceding; MLP-Mz 3084; MLP-He 2058-1 • 2 ♂♂, 2 ♀ ♀; same collection data as for preceding; “Oryzomys”; MLP-He 2056-1 • 4 ♂♂, 5 ♀♀; same data as for preceding; MLP-He 2057-1 • 6 ♂♂, 5 ♀♀; same data as for preceding; “colilargo”; MLP-He 2059-1 • 3 ♂♂, 1 ♀; same data as for preceding; O. flavescens, 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 the helminth sets MLP-He 2047, 2049, 2052, 2058, and 2063 (O. nigripes); MLP-He 2048, 2064, and 2068 (O. flavescens) (Table 3). The hosts of the remaining helminth sets were a priori identified in the field and subsequently labelled as “Oryzomys sp.” or “colilargo” (Table 3). Whether the identity of these hosts was further confirmed or not, they had helminth assemblages composed of Guerrerostrongylus spp. showing the features described above and of S. flavescens (P=94.1%, MI=21.2). One specimen identified as O. flavescens also harboured Hassastrongylus hoineffae (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).

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 Nippostrongylus brasiliensis (Travassos, 1914).

No genetic divergence is found between the sequences 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.

Figure 6. 

Maximum likelihood bootstrap consensus tree. Values next to the nodes are bootstrap proportions (%).

S. azarai / S. oryzomysi

We found no morphological differences among the type series of S. oryzomysi, the specimens of S. azarai harboured 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. IIc 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 papillae 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 Durette-Desset (1991) in worms from the locality of Campana were also observed in voucher specimens of S. azarai.

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) (Table 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 × 36 μm vs. 80 × 40 μm) (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 reported for both S. azarai and S. oryzomysi were taken from their holotypes and allotypes, probably accounting for their metric discrepancies. Moreover, the measurements 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 (Waterhouse) 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 discrepancies, 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).

In conclusion, both type series appear to be morphologically identical, and after reassigning the symbiotype of S. oryzomysi (MLP-Mz 3076) to A. azarae, here we propose S. oryzomysi as a junior synonym of Stilestrongylus azarai.

Stilestrongylus oryzomysi was possibly regarded as a new species because it was described from a host mislabelled as Oligoryzomys spp. This assumption is based on the fact that Oligoryzomys spp. can be easily distinguished from A. azarae, even by non-experts. We believe that this labelling mistake would have been noticed if more hosts from the same sampling were examined at the time of describing the species.

Additionally, since the rodent MLP-Mz 3076 was also the symbiotype of Trichofreitasia lenti (Sutton and Durette-Desset 1991), it should be reassigned from O. flavescens to A. azarae.

G. zeta / G. uruguayensis

We found no morphological or metric differences among the type specimens of G. uruguayensis, the specimens of Guerrerostrongylus harboured by the remaining colilargos from the same sampling, and the specimens of G. zeta redescribed by Digiani et al. (2012) and Simões et al. (2012a). Diagnostic characters of G. zeta, such as the proportions of the cephalic vesicle, ray pattern of the bursa, details of the genital cone, telamon, and spicules, were observed in all the worms examined from the colilargos from Uruguay. In regard to morphometry, G. uruguayensis was differentiated from G. zeta by its larger body size. However, the description of G. uruguayensis was based only on the holotype and allotype, and it did not account for morphological or morphometric variability. On the other hand, our body length values obtained from the holotype and allotype were lower than those reported by Sutton and Durette-Desset (1991). Additionally, G. uruguayensis was differentiated from G. zeta by a widening of the body just anterior to the tail in the female; by bursal rays 6 longer than rays 8; by rays 8 arising from the base of the dorsal ray; and by the presence of cuticular ornamentation on rays 4 and 5 in the male (Sutton and Durette-Desset 1991). In females, body size is often related to age and the posterior widening to reproductive status, as gravid specimens bearing numerous eggs in the uterus usually have a widened pre-vulvar region. On the other hand, the bursal characters argued as diagnostic for G. uruguayensis are ambiguous and dependent on the degree of spreading of the bursa, or unspecific and subjected to individual variation. The following characters should be dismissed as diagnostic for G. uruguayensis: 1) rays 8 arising from the base of the dorsal ray, because all the specimens examined clearly showed a proximal common trunk to rays 8 and the dorsal ray; 2) rays 6 longer than rays 8, because in the original description of L. zeta Travassos (1937) pointed out that rays 6 were the longest; 3) cuticular ornamentation on rays 4 and 5, because it was mostly absent and could be seen on the margins of rays 4 and 5 only in a few males. In addition, males with and without ornamentation coexisted in the same infrapopulation, suggesting that it may be a polymorphic trait or an artefact of fixation.

In conclusion, the comparison of the type series of G. uruguayensis against other specimens of Guerrerostrongylus from the same sampling event and against all available descriptions of G. zeta reported so far confirms that the worms examined herein parasitising Oligoryzomys spp. from Uruguay belong to G. zeta. This species was originally described as Longistriata zeta by Travassos (1937), then transferred to Hassalstrongylus by Durette-Desset (1971), and finally to Guerrerostrongylus by Sutton and Durette-Desset (1991). The identification of G. uruguayensis as a distinct species from that described by Travassos was likely due to the fact that both original descriptions were based on very few specimens, leading to the overestimation of metric differences and morphological variations without specific value. In addition, the description of G. uruguayensis was published in 1991, while the synlophe of G. zeta remained undescribed until many years later. Further redescriptions of G. zeta (Digiani et al. 2012; Simões et al. 2012a) based on paratypes and, especially, on larger sets of voucher specimens provided the description of the synlophe and a range of metric variability broad enough to include Travassos’ specimens and the type material of G. uruguayensis. Consequently, G. uruguayensis is proposed as a junior synonym of G. zeta.

The rodent with field number 2046, consigned “Oryzomys flavescens” by Sutton and Durette-Desset (1991), is also the type host of Stilestrongylus flavescens. Due to the absence of a voucher, the identity of this rodent could not be ratified or rectified (see Table 3), and therefore it must follow the one designated in the original description. Then, the type host species of S. flavescens is, according to the current nomenclature, Oligoryzomys flavescens (= Oryzomys flavescens).

The examination of the material has afforded an opportunity to clarify the taxonomic status of two of the examined nippostrongyline species as well as to shed light on the host spectrum and geographic distribution of some others. According to Sutton and Durette-Desset (1991), O. flavescens harboured S. oryzomysi and T. lenti in Argentina, and S. flavescens and G. uruguayensis in Uruguay. After examining numerous helminth sets, we found not only that this rodent harboured the same nippostrongyline assemblage on both sides of the Uruguay River but also that it was shared with O. nigripes. On the other hand, A. azarae, which is present in the same sampling localities as above, was strongly parasitised by S. azarai and, at least on the Argentinean side and to a lesser degree, by T. lenti (see Results and Tables 1, 2). Thus, this study extends the distribution range of S. azarai to include Uruguay and reports a new host record for T. lenti (A. azarae, type host species), a new host record for G. zeta in Argentina (O. flavescens), and the first record of helminths for O. nigripes in Uruguay.

Stilestrongylus azarai. This species seems to be the main component of the helminth community of A. azarae. It has been found in most of the rodent’s geographic distribution, which ranges from southern Brazil and eastern Paraguay south through Uruguay and central Argentina (Pardiñas et al. 2015). Up to the present, S. azarai has been recorded in the Argentinean provinces of Formosa, Chaco, Santa Fe, Entre Ríos, Corrientes, and Buenos Aires, and in Uruguay (Durette-Desset and Sutton 1985; Navone et al. 2009; Miño et al. 2012, 2019; Serrano 2024; this work). It has been rarely found in other hosts, always with relatively low prevalences and intensities of infection, e.g., in Oligoryzomys fornesi (Massoia) (P=14%, IM=4, n=7) and Calomys callosus (Rengger) (P=33%, IM=1, n=3) both from Chaco province (Serrano 2024). Four out of five Reithrodon auritus (Desmarest) from Balcarce (helminth sets housed in the MLP-He) harboured a few S. azarai specimens coparasitic with Stilestrongylus aureus Durette-Desset & Sutton, 1985 (Digiani pers. obs.). The three latter records were obtained from localities where the respective hosts live in sympatry with A. azarae, which is clearly the main host. Stilestrongylus azarai was also reported in Graomys griseoflavus from San Luis province (P=27.8%, and IM= 16.2, n=18), mainly coparasitic with a species of Hassalstrongylus (Digiani and Durette-Desset 2003c). To our knowledge, no other species of Akodon have been reported to harbour S. azarai. It would be interesting to investigate the role of Akodon dolores Thomas as the main host of this parasite in San Luis Province, where it is the only Akodon species present (Pardiñas et al. 2015).

On the other hand, A. azarae specimens have been rarely found harbouring heligmonellid species other than S. azarai, with the exception of T. lenti. In Corrientes province, this rodent was reported to be parasitised by Stilestrongylus stilesi Freitas, Lent & Almeida, 1937 (P=57%, Gómez-Muñoz et al. 2020) and Hassalstrongylus dollfusi (Díaz Ungría, 1963) (P=3.6%, Serrano et al. 2021), neither of which were coparasitic with S. azarai.

Trichofreitasia lenti. Although it was not found in the Uruguayan sample, several helminth sets still remain unexamined. Moreover, its presence in Uruguay is expected given the fact that this species has already been reported from different Akodon species in Argentina and Brazil (Digiani et al. 2007; Simões et al. 2011, 2012b; Panisse et al. 2017; Cardoso et al. 2018; this work). In Brazil, T. lenti has been recorded in other host species, particularly O. nigripes (Simões et al. 2011, 2012b; Cardoso et al. 2019; Benatti et al. 2021), but these reports do not include either descriptions or illustrations and may deserve revision. In any case, none of the 22 samples of Oligoryzomys spp. examined in the present survey contained T. lenti. Moreover, in another, more extensive survey, Serrano (2024) examined 179 Oligoryzomys spp. that were not parasitised by T. lenti.

Guerrerostrongylus zeta. Numerous publications have confirmed that the main host of G. zeta is O. nigripes rather than its type host species, Nectomys squamipes (Brants). It was repeatedly reported parasitising O. nigripes from Argentina and Brazil (Pinto et al. 1982; Gomes et al. 2003; Simões et al. 2011, 2012a, 2012b; Digiani et al. 2012; Werk et al. 2016; Panisse et al. 2017; Cardoso et al. 2018, 2019; Boullosa et al. 2020; Kersul et al. 2020; Gentile et al. 2022). In Brazil, this species was also recorded in O. flavescens (Cardoso et al. 2018), N. squamipes, Cerradomys subflavus (Wagner), Galea spixii (Wagler) (Pinto et al. 1982), Euryoryzomys russatus (Wagner), Akodon cursor Winge, and A. montensis Thomas (Gomes et al. 2003; Simões et al. 2011; Boullosa et al. 2020; Kersul et al. 2020). However, these reports lack descriptions or illustrations. It is possible that at least the worm from E. russatus could belong to Guerrerostrongylus ulysi Digiani, Notarnicola & Navone, 2012, a species described from Sooretamys angouya (Fischer) in Misiones province, Argentina (Digiani et al. 2012) and later found in E. russatus in the same province by Panisse et al. (2017).

A special case involves females identified as Guerrerostrongylus sp. in Akodon simulator Thomas from Tucumán province (northwestern Argentina) (Digiani et al. 2007). These females showed the typical synlophe of Guerrerostrongylus, but their opacity precluded the observation of any diagnostic character and thus the identification at the species level. In turn, the males recovered from the same host were identified as G. uruguayensis before redescriptions and further reports of G. zeta from Argentina were available (Digiani et al. 2007). Although our results suggest that these males would belong to G. zeta, the information on their hosts is incomplete and uncertain. Therefore, newly collected material from a larger number of individually identifiable hosts is required for a more accurate identification of Guerrerostrongylus spp. associated with A. simulator.

Despite the wide distribution of South American Nippostrongylinae (SAN) as parasites of sigmodontines, they remain poorly studied from a molecular approach, with only two studies involving molecular phylogenetic reconstructions. Scheibel et al. (2014) mainly focused on the nematode family Viannaiidae and solely included Hassalstrongylus sp. as a representative of SAN. Later, Weirich et al. (2016) used a fragment of the mitochondrial gene coding for the large ribosomal subunit RNA (rrnL) to analyse the phylogenetic position of Guerrerostrongylus marginalis (Weirich, Catzeflis & Jiménez, 2016), Hassalstrongylus sp., and Stilestrongylus sp., among other Trichostrongylina. In their phylogenetic tree, these species were clustered in a strongly supported monophyletic clade, though with greater genetic similarity between Hassalstrongylus sp. and Stilestrongylus sp. In the present study, we confirm, using a nuclear gene, the monophyly of the clade formed by Stilestrongylus, Hassalstrongylus, and Guerrerostrongylus, which are the three most speciose and widely distributed genera of SAN.

In the present study, we provide a significant amount of information on the taxonomy and geographic distribution of three nippostrongyline species obtained from the examination of several lots of material deposited in the Helminthological and Mammal Collections of the Museo de La Plata. Our findings confirm the importance of examining the largest possible number of specimens to describe new taxa and highlight the role of public biological collections as biodiversity repositories.