Fugassa Et Al. 2007 J Parasitology

J. Parasitol., 93(2), 2007, pp. 418–421 䉷 American Society of Parasitologists 2007

Infection of Myxobolus galaxii (Myxozoa) in Galaxias maculatus (Osmeriformes: Galaxiidae) From Northwestern Patagonian Andean Lakes (Argentina) Vero´nica Flores and Gustavo Viozzi, CONICET-UNC Laboratorio de Parasitologı´a, Centro Regional Universitario Bariloche, Quintral 1250 (8400), San Carlos de Bariloche, Argentina. e-mail: [email protected] ABSTRACT: The infection of Myxobolus galaxii Szidat, 1953, from the musculature and abdominal organs of northwestern Patagonian Galaxias maculatus is described. Plasmodia are histozoic and intercellular. Spores are pyriform in valvar view and biconvex in sutural view, with 4–9 edge notches in the sutural line, varying in shape within the same plasmodium. Myxobolus galaxii was detected in fish from 7 of 17 Andean Patagonian lakes, with prevalences ranging between 2 and 17%. A repeating pattern of summer increment in prevalence was observed, which could be explained by the ontogenetic migratory movements of the fish in Lake Gutie´rrez. Also, accumulation of plasmodia through the life span of fish was detected.

TABLE II. Seasonal sample size and mean length (⫾SD) of Galaxias maculatus from Lake Gutie´rrez.

Galaxias maculatus (puyen) is a circum-Antarctic fish species distributed in Chile, Argentina, Malvinas Islands, Australia, and New Zealand (Berra et al., 1996). Four species of Myxobolus (Bu¨tschli, 1882) parasitizing G. maculatus (Jenyns) have been recorded: Myxobolus galaxii Szidat, 1953, is from the musculature and other abdominal organs and Myxobolus magellanicus Szidat, 1953, is reported from the gills of fish collected in Tierra del Fuego (southern Patagonia, Argentina); Myxobolus iucundus Hine, 1976, was found to parasitize muscles of fish in New Zealand; and Myxobolus bartoni Kalavati, Brickle, et MacKenzie, 2000, was reported in the trunk musculature of specimens from the Malvinas Islands (Szidat, 1953; Hine, 1976; Kalavati et al., 2000). During a survey of parasites of G. maculatus from northwestern Patagonian lakes, plasmodia containing spores like those of M. galaxii described by Szidat (1953) were found in the musculature and abdominal organs of fishes. The original description of M. galaxii did not report the distribution in tissues, the type of sporogenesis, the number and the position of polar filaments, or the histological and ecological features of infection. The aim of this study was to characterize the infection of M. galaxii from muscles and other organs of G. maculatus from northwestern Andean Patagonian lakes. Fish were captured with baited traps in 17 oligotrophic lakes located in northwestern Argentinean Patagonia between 40⬚10⬘S and 41⬚30⬘S (Table I). All of these lakes have only landlocked populations of G.

TABLE I. Coordinates of lakes, sample size, and prevalence of infection of Myxobolus galaxii from Galaxias maculatus.

Lake

Coordinates

Sample size

Prevalence (%)

La´car Villarino Filo Hua Hum Espejo Traful Correntoso Pire´ Nahuel Huapı´ Moreno Escondido Gutie´rrez Mallı´n Ingeniero Mascardi Fonck Hess Guillelmo Steffen

40⬚10⬘S–71⬚30⬘W 40⬚28⬘S–71⬚35⬘W 40⬚32⬘S–71⬚20⬘W 40⬚41⬘S–71⬚42⬘W 40⬚41⬘S–71⬚35⬘W 40⬚44⬘S–71⬚39⬘W 40⬚44⬘S–71⬚39⬘W 40⬚48⬘S–71⬚39⬘W 41⬚04⬘S–71⬚33⬘W 41⬚04⬘S–71⬚35⬘W 41⬚11⬘S–71⬚25⬘W 41⬚15⬘S–71⬚40⬘W 41⬚17⬘S–71⬚34⬘W 41⬚19⬘S–71⬚45⬘W 41⬚22⬘S–71⬚43⬘W 41⬚23⬘S–71⬚27⬘W 41⬚30⬘S–71⬚40⬘W

31 20 20 24 24 35 35 24 64 41 52 24 73 12 17 37 27

16 — — — — — 14 8 2 — 17 4 15 — — — —

Season

n

Mean length (mm)

SD

1995 Summer Autumn Winter Spring

52 141 96 61

47.4 49.2 40.0 44.5

3.9 5.8 3.1 5.1

1996 Summer Autumn Winter Spring

135 166 105 134

45.4 46.4 42.5 42.5

4.3 4.9 4.9 5.4

1997 Summer

154

44.2

5.0

maculatus. In Lake Gutie´rrez. The fish mature at 1 yr of age and spawn in the littoral zones of the lakes from early spring to early summer (Barriga et al., 2002). Lake Gutie´rrez has a surface area of 16.4 km2, and a maximum depth of 111.2 m. After capture, the fish were transported to the laboratory and kept alive at 6 C until killed and necropsied between 24 and 48 hr after capture. Samples of 12–73 fish from 17 Andean Patagonian lakes were collected in the summer–autumn (Table I). Specimens from Lake Gutie´rrez were collected monthly from February 1995 to February 1997. Sex and total length of fish were recorded. Musculature and abdominal organs were removed. Plasmodia were dissected to obtain fresh spores or fixed in 10% buffered formalin. Musculature was fixed in 10% buffered formalin, dehydrated in graded ethanol series, cleared in xylene, and embedded in paraffin. Histological sections (7 ␮m thick) were stained with hematoxilin and eosin. Morphometric measurements were based on 141 fresh and 80 fixed spores obtained randomly from plasmodia of different fish. Measurements are

FIGURE 1. Seasonal fluctuation of water temperature and prevalence of Myxobolus galaxii in Galaxias maculatus from Lake Gutie´rrez. 418

RESEARCH NOTE

419

FIGURE 2. Prevalence of Myxobolus galaxii in relation to length of Galaxias maculatus from Lake Gutie´rrez.

given in micrometers, with mean and standard deviation followed by range in parenthesis. The data for monthly samples from Lake Gutie´rrez were grouped into seasons according to water temperature. Sample size and mean length of fish are summarized in Table II. Prevalence of infection was calculated according to Bush et al. (1997). The correlation between prevalence and water temperature and length of fish was analyzed with a Spearman rank correlation test (Conover, 1980). A test of independence (Conover, 1980) was carried out to establish the relationship between prevalence and the host’s sex. A 1-way analysis of variance test FIGURE 4. Histological section of Myxobolus galaxii plasmodium in musculature of Galaxias maculatus. Scale bar ⫽ 100 ␮m (A, B).

FIGURE 3. Plasmodia of Myxobolus galaxii in Galaxias maculatus. (A) Plasmodia in musculature of caudal peduncle, visible through the integument. (B) Plasmodia dissected from musculature. (C) Plasmodium on gut surface surrounded by mesentery. Scale bar ⫽ 1,000 ␮m.

was employed to evaluate differences in the size of the fish among seasons and a multiple comparison Sheffe´ test was used to detect the differences in the length of fish between seasons (Sokal and Rohlf, 1981). All P ⬍ 0.05 were considered significant. In total, 1,044 G. maculatus from Lake Gutie´rrez were analyzed (Table II). Significant differences in fish length were detected between seasons (F ⫽ 81.328; P ⫽ 0.0001); a Scheffe´ test showed that the specimens in the winter samples were significantly smaller than the fish of the other seasons. Maximum values of prevalence were detected in the summers of 1995 and 1996 (17 and 13%, respectively) and minimum values in spring 1995 and 1996 (3 and 5%, respectively) (Fig. 1). No significant correlation was found between the water temperature and prevalence (rS ⫽ 0.515; P ⫽ 0.156). The prevalence of M. galaxii increased with total length of G. maculatus (Fig. 2), and there was a significant positive correlation between these 2 variables (rS ⫽ 0.786; P ⫽ 0.021). The sex of 587 fish specimens was determined; 372 were female and 215 were male. Prevalence was not significantly different (␹2 ⫽ 0.56; P ⬎ 0.05) between males (16%) and females (14%). The plasmodia located in musculature (14.4%) were visible through the integument as elongated spots and were distributed throughout striated muscle of the flank and caudal peduncle (Fig. 3A). In abdominal organs, plasmodia were located in the gut (58.9%), spleen (11.1%), kidney (8.9%), gonads (4.4%), and liver (2.2%). They were white and usually subspherical in shape (Fig 3B). In spleen, liver, and kidney, the plasmodia were found in the parenchyma. In the gonads, they were embedded in the tissues, and in the digestive tract, they were found in the tissues or protruding from the surfaces into the abdominal cavity surrounded by mesenteries (Fig. 3C). The plasmodia, 585.1 ⫾ 120.2 (426–850) ␮m long and 151.9 ⫾ 43.8 (85–227) ␮m wide (n ⫽ 13), have a thin wall (Fig. 4A). They are histozoic, intercellular, and polysporous. Sporogenesis is synchronous (Fig. 4B). No gross or tissue le-

420

THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 2, APRIL 2007

FIGURE 5. Photomicrographs of formalin-fixed spores of Myxobolus galaxii from muscles of Galaxias maculatus showing morphological variability. Scale bar ⫽ 15 ␮m (A, B).

sions were observed by light microscopy. The only host reaction detected was the formation of a connective tissue–like capsule around the plasmodia (Fig. 4B). Mature spores were smooth, pyriform to ovate in frontal view, and biconvex in sutural view. They have a straight sutural line, with 4–9 small edge notches (Fig. 5A). A mucous envelope and triangular intercapsular processes were absent. An iodophilous vacuole was not observed. Measurements of fresh and fixed spores are given in Table III. The polar filaments exhibited 9–12 coils perpendicular to the longitudinal axis of the polar capsules. The polar capsule dimensions and the relation of the polar capsule to spore length are shown in Table III. Variation in morphology of spores can be found even in the same plasmodium. A few spores with an elongated caudal zone of valves in plasmodia from musculature were observed (Fig. 5A–B). Seasonal fluctuation in the prevalence of myxosporeans appears to be common in those species having cysts on exposed host surfaces (Cone, 1994), like M. magellanicus, which are released from the fish when spores are mature (Flores and Viozzi, 2001). Histozoic species found deep in host tissues, like M. galaxii, have a dispersal strategy requiring host death (Marcogliese and Cone, 2001). These diverse strategies lead to differences in the annual infection cycle of M. magellanicus and M. galaxii in the same G. maculatus population. The infection pattern of M. galaxii in Lake Gutie´rrez showed that the young G. maculatus had the lowest values of prevalence and increased with fish length. Because plasmodia are accumulated through the life span of fish and spores are not released until death, the probability of becoming infected seems to increase with the size of the fish because of the length of time they are exposed to infection. During the study period, there was a repeating pattern of a summer increase of prevalence and a decline from summer to spring, which could be explained by the significantly smaller size of the specimens captured in winter (Table II). This infection pattern could be related to the life history of G. maculatus in Lake Gutie´rrez (i.e., the spawning period ranges from spring to summer so that larvae are found in autumn and juveniles are mostly found in winter) (Barriga et al., 2002). In contrast, M. magellanicus show the highest values of prevalence in the smallest fish, with a peak prevalence in winter (Flores and Viozzi, 2001). In Patagonia, Myxidium biliare Viozzi et Flores, 2003 (a coelozoic species with prevalences between 4.2 and 70%), and M. magellanicus (a surface species with prevalences between 3.2 and 50%) are widely distributed in lakes situated between 39⬚25⬘S and 41⬚30⬘S (Flores and Viozzi, 2001; Viozzi and Flores, 2003). In contrast, the histozoic M. galaxii was found only in 7 lakes with prevalences ranging between 2 and 17%. This prevalence and distribution pattern is similar to that suggested by Cone et al. (2004), in which coelozoic species shedding spores from the live host would have higher local prevalence and a wider geographical range than histozoic species in which transmission occurs only after host death. All G. maculatus populations parasitized by M. galaxii also harbored M. magellanicus (Flores and Viozzi, 2001). Although other native and introduced fishes have been examined for myxozoan infections in Patagonia (data not shown), M. galaxii is limited only to G. maculatus. Szidat (1953) reported the presence of white plasmodia mainly located in musculature, kidney, and ‘‘other organs’’ in G. maculatus. In this study, a multiorgan infection, mainly in the abdominal cavity (85.6%), was observed. The organ most frequently affected was the gut (58.9%) and the least frequently affected was the

TABLE III. Measures of Myxobolus galaxii spores from musculature and internal organs of Galaxias maculatus.

Spore source Musculature Gonads Stomach Intestine Szidat (1953)

Spore Condition n Fixed Fresh Fixed Fresh Fresh Fresh Fixed

60 30 20 11 80 20 —

Length 14.1 13.8 13.8 14.9 14.7 13.6

⫾ 1.0 (12–17) ⫾ 0.7 (13–15) ⫾ 0.8 (12–15) ⫾ 0.8 (13–16) ⫾ 0.8 (13–17) ⫾ 0.7 (13–16) (13.7–15)

Width 9.7 9.5 9.9 9.5 8.6 9.4

⫾ 0.7 (8–11) ⫾ 0.7 (8–11) ⫾ 0.5 (9–11) ⫾ 0.5 (9–10) ⫾ 1.0 (6–10) ⫾ 0.7 (8–10) (8.8–10)

Polar capsule Thickness

Length

8.0 ⫾ 0.8 (7–9) 6.9 ⫾ 0.7 (5–8) 3.4 — 6.3 ⫾ 0.6 (5–7) 3.0 9 6.8 ⫾ 0.7 (6–8) 8 7 7.9 ⫾ 0.5 (7–9) 8.1 ⫾ 0.7 (7–9) 3.1 8 7.6 ⫾ 0.5 (7–8) 3.2 — —

Width ⫾ 0.5 (3–4) ⫾ 0.2 (2.5–3) 3 3 ⫾ 0.3 (3–4) ⫾ 0.3 (3–4) —

Spore length/polar capsule length ratio 2.1 2.2 2.0 2.1 1.8 1.8

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.2 (1.6–2.5) 0.3 (1.9–2.8) 0.2 (1.6–2.5) 0.1 (1.8–2.3) 0.2 (1.4–2.3) 0.1 (1.6–2.0) —

RESEARCH NOTE

liver (2.2%). The spore length and width ranges of M. galaxii given by Szidat (1953) are within the range of our fixed and fresh spores, but variation in shape was not mentioned. In our material, the spores varied in length and width between those from the musculature and those from other organs. Such variations in spore sizes in multiorgan infections were also observed in Myxidium zealandicum Hine 1975 from the freshwater eel Anguilla australis (Hine, 1978). Mitchell (1989) demonstrated great intraspecific morphometric variation among spores of Myxobolus muelleri Bu¨tschli, 1881, within individual plasmodia and among spores from different tissues of an individual squawfish Ptychocheilus oregonensis. Financial support for this study was provided by UNC-B-115; and CONICET PIP 02752); V.F. had a fellowship funded by CONICET (Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Argentina). We thank Richard Sage for English revision of the manuscript. LITERATURE CITED BARRIGA, J. P., M. A. BATTINI, P. J. MACCHI, D. MILANO, AND V. E. CUSSAC. 2002. Spatial and temporal distribution of landlocked Galaxias maculatus and Galaxias platei (Pisces, Galaxiidae) in a lake in the South American Andes. New Zealand Journal of Marine and Freshwater Research 36: 349–363. BERRA, T. M., I. E. CROWLEY, W. IVANTSOFF, AND P. A. FUERST. 1996. Galaxias maculatus: An explanation of its biogeography. Marine and Freshwater Research 47: 845–849. BUSH, A. O., K. LAFFERTY, J. LOTZ, AND A. SHOSTAK. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology 83: 575–583. CONE, D. K. 1994. Annual cycle of Henneguya doori (Myxosporea) parasitizing yellow perch (Perca flavescens). Journal of Parasitology 80: 900–904. ———, D. J. MARCOGLIESE, AND R. RUSSEL. 2004. The myxozoan fauna of spottail shiner in the Great Lakes basin: Membership, richness, and geographical distribution. Journal of Parasitology 90: 921–932.

421

CONOVER, W. J. 1980. Practical nonparametric statistics, 2nd ed. John Wiley & Sons, New York, New York, 493 p. FLORES, V., AND G. VIOZZI. 2001. Redescription, seasonality and distribution of Myxobolus magellanicus (Myxosporea) in Galaxias maculatus (Osmeriformes: Galaxiidae) from Patagonian Andean lakes (Argentina). Acta Parasitologica 43: 159–163. HINE, P. M. 1976. Myxobolus iucundus n. sp. (Sporozoa, Myxosporidea) parasitic in Galaxias maculatus (Jenyns, 1842) in New Zealand. Journal of Royal Society of New Zealand 2: 157–161. ———. 1978. Variations in the spores of Myxidium zealandicum Hine, 1975 (Protozoa: Myxosporidea). New Zealand Journal of Marine and Freshwater Research 12: 189–195. KALAVATI, C., P. BRICKLE, AND K. MACKENZIE. 2000. Two new species of myxozoan parasites (Myxosporea, Multivalvulida, Bivalvulida) from fishes of the Falkland Islands. Acta Parasitologica 45: 285– 288. MARCOGLIESE, D. J., AND D. K. CONE. 2001. Myxozoan communities parasitizing Notropis hudsonius (Cyprinidae) at selected localities on the St. Lawrence River, Quebec: Possible effects of urban effluents. Journal of Parasitology 87: 951–956. MITCHELL, L. G. 1989. Myxobolid parasites (Myxozoa: Myxobolidae) infecting fishes of western Montana with notes on histopathology, seasonality, and intraspecific variation. Canadian Journal of Zoology 67: 1915–1992. SOKAL, R., AND F. ROHLF. 1981. Biometry, 2nd ed. W. H. Freeman and Co., San Fransisco, California, 859 p. SZIDAT, L. 1953. Einige neue Arten der Familie Myxobolidae The´lohan (Protozoa, Klase Sporozoa) aus Su¨ßwasserfischen Argentiniens. Gewa¨sser und Abwa¨sser 5: 7–16. VIOZZI, G. P., AND V. R. FLORES. 2003. Myxidium biliare sp. n. (Myxozoa) from gall bladder of Galaxias maculatus (Osmeriformes: Galaxiidae) in Patagonia (Argentina). Folia Parasitologica 50: 190– 194.

J. Parasitol., 93(2), 2007, pp. 421–422 䉷 American Society of Parasitologists 2007

Paleoparasitological Analysis of a Raptor Pellet From Southern Patagonia M. H. Fugassa, N. H. Sardella, and G. M. Denegri, Laboratorio de Zoonosis Parasitarias, Deptamento de Biologı´a, Universidad Nacional de Mar del Plata—CONICET, Argentina. e-mail: [email protected] ABSTRACT: Organic remains attributable to one regurgitated pellet were examined. The pellet, belonging to a bird of prey and collected from a cave of Southern Patagonia, was dated at 6,540 ⫾ 110 yr. With standard paleoparasitological procedures, eggs of Capillaria sp. and a mite, Demodex sp., were found. The parasites found in the pellet belong to a rodent ingested by the bird. The present report constitutes the first paleoparasitological study of a regurgitated pellet.

Paleoparasitology includes the study of parasites isolated at archaeological and paleontological sites. Coprolites are the main sources of parasite findings, together with sediments of latrines, middens, and mummified tissues (Bouchet et al., 1999, 2003). The magnitude of the contributions that paleoparasitology offers to archaeology depends on the number of samples examined, the availability of different methods, i.e., the application of immunological techniques (Gonc¸alves et al., 2003), DNA amplification of ancient material (Arau´jo et al., 1998), and the source of evidence. The improvement of methods applied to free sediments of burials, shell middens, and deposits of refuse have extended the scope of paleoparasitology in Patagonia (Fugassa and Guicho´n, 2005; Fugassa et al., 2006). The aim of the present study was to examine new archaeological material for paleoparasitological purposes. The sample was collected from the archaeological site, Cerro Casa de Piedra 5 (CCP5), located in the Perito Moreno National Park, Santa

Cruz Province, Argentina, belonging to a deposit dated 6,540 ⫾ 110 yr B.P. (before present) (Aschero, 1996). CCP5 is a hill in the Nothofagus sp. forest Patagonian steppe ecotone, associated with human occupation of hunter-gatherer groups (Aschero, 1982; Aschero et al., 1992). The sample examined consisted of a light concretion, externally covered by hair, suggestive of a pellet regurgitated by a bird of prey (Marti, 1987). The pellet was described, measured, and weighed according to the method of Jouy-Avantin (2003). Two samples of approximately 0.5 g each, one from the surface and the other from the interior, of the concretion were rehydrated with a 0.5% trisodium phosphate aqueous solution (Callen and Cameron, 1960) and concentrated by spontaneous sedimentation according to Lutz (1919). The macroscopic remains were separated and dried to room temperature for diet analysis. Weight and volume of the sample were 7.78 g and approximately 38 cm3, respectively (Fig. 1). Externally, it consisted of rodent hairs and quills. The partial dissection showed numerous bones of a small rodent. The sediment obtained was limited. Microscopic examination of the sample revealed eggs of Capillaria sp. with ornamented walls possessing radial perforations (Fig. 2a), 37.5–42.5 ␮m (36.25 ⫾ 6.29; n ⫽ 4) ⫻ 63.75–68.75 ␮m (65.93 ⫾ 2.13; n ⫽ 4) in diameter. A mite, identified as Demodex sp. (112.5 ⫻ 32.5 ␮m in diameter), was also identified (Fig. 2b). Microscopic examination of the concretion revealed that the hair fragments of the rodent were more predominant (Chehebar and Martı´n,

422

THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 2, APRIL 2007

hepatica, mostly parasites of rodents (Thienpont et al., 1979). The presence of Demodex sp. is the first documentation from an ancient sample. The present report is the first paleoparasitological study of a regurgitated pellet. The pellet provides evidence of nondigestible scales, feathers, hair, and bones, most likely of rodent origin (Marti, 1987). Although pellets do not reflect the parasitic fauna of an individual host, they may provide a parasitological record of prey. We thank Marı´a Teresa Civalero (INAPL-CONICET) and Carlos Aschero (UNT-CONICET) for providing the samples. Ricardo A. Guicho´n, Susana L. Burry, and Pablo A. Martinez contributed to the laboratory work. Editorial comments were valuable in an early version of the manuscript. This work is supported by PICT 03-13889, PICTO 04-849, and CAPES/SECyT. LITERATURE CITED

FIGURE 1. Pellet (CCP5, Santa Cruz province, Argentina, 6,540 ⫾ 110 yr B.P.). Scale bar ⫽ 2 cm.

FIGURE 2. (a) Adult of Demodex sp. (b) Capillaria sp. egg. Scale bar ⫽ 40 ␮m. 1989) than the large charcoal fragments and scanty segments of plant tissue and pollen grains. The scanty presence of sediment and the high proportion of hairs and bones support the idea that the concretion is a regurgitated pellet. Its size and the zoological origin suggest it originated from Tyto alba or Bubo virginianus (Narosky and Yzurieta, 1988). Today, the site is a refuge of barn owls, T. alba (M. T. Civalero, pers. comm.). The eggs of the nematode are compatible with Capillaria

ARAU´JO, A., K. REINHARD, O. M. BASTOS, L. C. COSTA, C. PRIMES, A. IN˜IGUEZ, A. C. VICENTE, C. M. MOREL, AND L. F. FERREIRA. 1998. Paleoparasitology: Perspectives with new techniques. Revista do Instituto de Medicina Tropical de Sao Paulo 40: http://www.scielo.br/ scielo.php?script⫽sci㛮arttext&pid⫽S0036-46651998000600006&lng ⫽es&nrm⫽iso (last update: 6 January 2006). ASCHERO, C. A. 1982. Nuevos datos sobre arqueologı´a del Cerro Casa de Piedra, sitio CCP5. Relaciones 14: 267–284. ———. 1996. El a´rea Rı´o Belgrano-Lago Posadas (Santa Cruz): problemas y estado de problemas. In Arqueologı´a so´lo Patagonia, J. G. Otero (ed.). CENPAT, CONICET, Buenos Aires, Argentina, p. 17– 26. ———, C. BELLELI, M. T. CIVALERO, R. A. GON˜I, G. GURAIEB, AND R. MOLINARI. 1992. Cronologı´a y tecnologı´a en el Parque Nacional Perito Moreno (PNPM): ¿Continuidad o reemplazos? Arqueologı´a 2: 89–106. BOUCHET F., N. GUIDON, K. DITTMAR, S. HARTER, L. F. FERREIRA, S. M. CHAVES, K. J. REINHARD, AND A. ARAU´JO. 2003. Parasite remains in archaeological sites. Memorias do Instituto Oswaldo Cruz 98 (Suppl. I): 47–52. ———, C. LEFEVRE, D. WEST, AND D. CORBETT. 1999. First paleoparasitological analysis of the midden in the Aleutian islands (Alaska): Results and limits. Journal of Parasitology 85: 369–372. CALLEN, E. O., AND T. W. M. CAMERON. 1960. A prehistoric diet revealed in coprolites. New Scientist 8: 35–40. CHEHEBAR, C., AND S. MARTI´N. 1989. Guı´a para el reconocimiento microsco´pico de los pelos de los mamı´feros de la Patagonia. Don˜ana, Acta Vertebrata 16: 247–291. FUGASSA, M. H., G. M. DENEGRI, N. H. SARDELLA, A. ARAU´JO, R. A. GUICHO´N, P. A. MARTINEZ, M. T. CIVALERO, AND C. ASCHERO. 2006. Paleoparasitological records in canid coprolite from Patagonia, Argentina. Journal of Parasitology 92: 1110–1111. ———, AND R. A. GUICHO´N. 2005. Ana´lisis paleoparasitolo´gico de coprolitos hallados en sitios arqueolo´gicos de Patagonia Austral: Definiciones y perspectivas. Magallania 33: 13–19. GONC¸ALVES, M. L., A. ARAU´JO, AND L. F. FERREIRA. 2003. Human intestinal parasites in the past: New finding and a review. Memorias do Instituto Oswaldo Cruz 98 (Suppl. I): 103–118. JOUY-AVANTIN, F. 2003. A standardized method for the description and study of coprolites. Journal of Archaeological Science 30: 367– 372. LUTZ, A. 1919. Schistosoma mansoni e a schistosomatose segundo observacoes feitas no Brasil. Memorias do Instituto Oswaldo Cruz 11: 121–155. MARTI, C. D. 1987. Raptor food habits studies. In Raptor management techniques manual, Scientific Technique Series No. 10, B. A. Pendleton, B. A. Millsap, K. W. Cline, and D. M. Bird (eds.). National Wildlife Federation, Washington, D.C., p. 67–80. NAROSKY, T., AND D. YZURIETA. 1988. Guı´a para la identificacio´n de las aves de Argentina y Uruguay. Asoc. Ornitolo´gica del Plata, Buenos Aires, Argentina, 345 p. THIENPONT, D., F. ROCHETTE, AND O. F. J. VANPARIJS. 1979. Diagno´stico de las helmintiosis por medio del examen coproparasitolo´gico. Janssen Research Foundation, Beerse, Be´lgica, 187 p.