Argulus Ambystoma, a New Species Parasitic on the Salamander Ambystoma Dumerilii from Mexico (Crustacea: Branchiura: Argulidae)

A new species of Argulus is described based on 18 specimens taken from the salamander ("achoque" or "ajolote") Ambystoma dumerilii Duges, collected in Lake Patzcuaro, Michoacan, Mexico. Diagnostic characters include the shape of the respiratory areas, number of sclerites in suction cup rods, and structures on the legs of males. Females are heavily stippled, whereas males have a very distinctive pigment pattern consisting of abundant melanophores covering the testes dors ally and two dark, inverted triangular patches on the carapace dorsally. The new species is similar to the North American species, A versicolor,A. americanus, A. maculosus, and A diversus. A single, dorsal pore was observed on each caudal ramus using scanning electron microscopy; these pores have not been reported previously in the Branchiura. OHIO J SCI 103 (3>52-6l, 2003 INTRODUCTION Ten species of Argulus Miiller have been collected in Mexico, Central America, and the West Indies. The widespread exotic, Argulus japonicus Thiele, was found on aquarium fishes, Carassius auratus (Linne) and Astronotus ocellatus (Agassiz), in Puerto Rico (Bunkley-Williams and Williams 1994), and Vargas and Fallas (1976) found A. japonicus on Carassius sp. in Costa Rica in 1972 and 1973Argulus dactylopteri Thorell was described from specimens collected from the gills of Dactylopterus volitans (Linne) in the West Indies (Thorell 1865, 1867); neither Cressey (1982) nor Pineda and others (1995) mentioned A. dactylopteri. Wilson (1902) gave the locality for A. dactylopteri as the East Indian Ocean from the branchial cavity of D. volitans, whereas Yamaguti (1963) cited Europe and Dactylopterus sp. If the type locality of A. dactylopteri is somewhere other than the Atlantic Ocean or Mediterranean Sea, the host would not be D. volitans but a species of Dactyloptena (see Eschmeyer 1997). Only six species of Argulus, A. chromidis Kr0yer; A. flavescens Wilson; A. megalops Smith; A. mexicanus Pineda, Paramo, and Del Rio; A. melanostictus Wilson; and A. rhamdiae Wilson have been found in Mexico (Wilson 1936; Causey I960; Olson 1972; Cressey 1982; Fucugauchi and others 1988; Pineda and others 1995; Suarez-Morales and Gasca 1997; Suarez-Morales and others 1998). In addition, an unidentified species of Argulus was reported from Potamarius nelsoni (Evermann and Goldsborough) by Pineda-Lopez and others (1985). The species described herein was collected from Lake Patzcuaro (Lago de Patzcuaro) in Central Mexico, State of Michoacan. Both the invertebrate and vertebrate faunas of the lake have been investigated; however, the genus Argulus has not been recorded as a component of the fauna (Ueno 1939; Ancona and others 1940; Brehm 1942; Tressler 1954; Brandon 1970; Barbour 1973; Rosas and others 1985; Chacon-Torres and others 'Manuscript received 20 September 2001 and in revised form 5 May 2002 (#01-24). Present Address: Department of Ichthyology, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118 1991; Osorio-Sarabia and others 1986; Perez-Ponce de Leon and others 1994; Peresbarbosa-Rojas and others 1994; Espinosa-Huerta and others 1996; PeresbarbosaRojas and others 1997). A number of endemic taxa exist in Lake Patzcuaro including several fishes (Cbirostoma patzcuaro Meek, C. e. estor Jordan, C. a. attenuatum Meek), a crayfish (Cambarellus patzcuarensis Villalobos), and the salamander host of the new argulid (Ambystoma dumerilii (Duges)) (Brandon 1970; Barbour 1973; Hobbs 1989). MATERIALS AND METHODS Three males and two females were examined using scanning electron microscopy (SEM). Preparation procedures were modified slightly from those of Rupp (1990). All specimens were initially preserved and stored in 70% ethanol (EtOH, J. A. Beatty pers. comm.). Specimens were dehydrated in an EtOH series consisting of 80% (5 min), 90% (5 min), 100% (1st, 5 min; 2nd, 10 min), then immediately critical point dried (CO2), mounted on metal stubs with carbon paint, allowed to dry overnight in an oven (60° C), and sputter coated with gold/ palladium. Specimens were stored in an oven at 60° C between uses. Some specimens were coated up to three times and were examined in a Hitachi S570 Scanning Electron Microscope at 15kV. Six females and seven males (including the holotype, all adult) were examined under dissecting and compound microscopes in a watchglass or temporary slide mount (with 70% EtOH/ glycerin). The holotype and allotype were photographed using a camera tube on a light microscope. In addition, some counts and measurements were taken from 3 male and 2 female specimens later used for SEM. All measurements were made using an ocular micrometer, and measurements reported in the description are arranged as follows: range (mean, holotype) with allotype values substituted in the case of females. Values for the right and left sides refer to the right and left of the specimen in dorsal view. Type specimens were deposited in the National Museum of Natural History, Washington, DC (USNM), and specimens of Argulus americanus Wilson (USNM 274331, 274332), A. diversusWihon (USNM OHIO JOURNAL OF SCIENCE W. J. POLY 53 32823,32824), A. maculosusWilsonOJSNM 12226,74329), and A. versicolor Wilson (USNM 74321; MCZ 19604, 19607) were examined by light microscopy for comparative purposes (MCZ, Museum of Comparative Zoology, Harvard University, Cambridge, MA). RESULTS Family Argulidae Rafinesque, 1815 Argulus Miiller, 1785 Argulus ambystoma, new species Material Examined Holotype: adult male, 3-48 mm total length, USNM 282782, Lake Patzcuaro, Michoacan, Mexico. Allotype: adult female, 4.18 mm total length, USNM 282783, collected with holotype. Paratypes: 5 adult males, 4 adult females, USNM 282784, collected with holotype; 4 adult males, 3 adult females (used for SEM or cleared, not retained), collected with holotype (host collected by J. Arnett; Argulus collected from hosts, Ambystoma dumerilii (Duges), byj. A. Beatty and R. A. Brandon on 27 May 1981). Diagnosis Respiratory areas with a smaller, oblong "area" situated anterior to and abutting or nearly contacting a larger posterior "area"; respiratory areas outlined with melanophores and very distinct; caudal rami near posterior end of abdomen in anal sinus; paired postantennal spines with reduced anterior spine and stout posterior spine; males with 43-56 support rods per suction cup; females with 45-54 support rods per suction cup; usually 3 to 4 sclerites per rod in males and 4 to 5 sclerites per rod in females; basal plate of second maxilla with three stout, digitate spines; first two pairs of legs each with a recurved flagellum on dorsal surface; coxae of second legs of males with bilobate structure posteroventrally, scales on lobes distally; third legs of males with paddle-like extension issuing anterodistally from the coxae, extending over bases. Description Total length (mm) 3.26-3.72 (3.53, 3.48) in males, 4.06-5.85 (4.92, 4.18) in females. Carapace ovoid to circular, narrower anteriorly. Carapace length (mean of both sides, mm) 2.16-2.47 (2.33, 2.26) in males, 3.203-97 (3.57, 3-22) in females. Maximum carapace width (mm) 2.13-2.57 (2.41, 2.38) in males, 3.21-4.15 (3-66, 3-30) in females. Carapace extending to middle of third legs or posterior edge of third legs/anterior margin of abdomen in males. Carapace extending to middle of third legs or posterior of fourth legs/anterior of abdomen in females, varies considerably in females. Females -without eggs in carapace alae. Carapace in males with inverted, dark, triangular patch of melanophores on dorsal surface of both left and right alae (much more apparent at lower magnifications) as well as scattered melanophores on alae and diffuse pigment scattered on cephalic region. Females with numerous scattered melanophores on carapace; inverted, triangular patches not as apparent in females (Fig. 1). Small pores and sensilla (Fig. 2) FIGURE 1. Argulus ambystoma, new species, dorsal view, male (holotype, left) and female (allotype, right) (scale bar = 1.0 mm). scattered on dorsal surface and margins of carapace. Smaller type of sensillum present on anterior margin (between sinuses) of cephalic region, abundant in interspaces between larger sensilla. Pair of compound eyes anteriorly with diameters (left and right eyes, Jim) 170210 (187, left: 170 and right: 180) (n = 18 eyes) in males, 200-240 (224, left: 230 and right: 210) (n = 14 eyes) in females. Transverse distance between eyes (|Xm) 390440 (419, 410) in males, 510-650 (604, 570) in females. Nauplius eye with one anterior and two posterior ocelli, pigment between ocelli forming a dark, prominent "Y" or "V." Sclerotized dorsal ridges forked anterior of eyes, inner branches longer and less developed than outer branches. Bifurcation of dorsal ridges usually more obvious in males and sometimes appears absent in females. Ventrally, carapace with small, posteriorly-projecting spines along outer margin, more numerous anterior of respiratory areas with a few rows extending along outer margin of carapace and respiratory areas to level of second legs. Respiratory areas consist of smaller, oblong "area" situated anterior to and abutting or nearly contacting larger posterior "area"; respiratory areas distinct, outlined with melanophores (Fig. 3). Thorax dorsoventrally compressed, indistinctly segmented with two pairs of posteriorly-projecting spines ventrally. Spines digitate, rounded distally, the anterior pair (accessory spines) larger than posterior pair (postmaxillary spines). Accessory spines situated between basal segments of second maxillae; postmaxillary spines positioned farther apart than accessory spines. Thorax with coarse-pectinate scales scattered on ventral surface. Four pairs of biramous swimming legs composed of a precoxa, coxa, basis, exopod, and endopod; with plumose setae on all exopods, endopods, coxae, and bases. First two pairs of legs each with a recurved flagellum on dorsal surface. Flagella laterally compressed, bearing plumose setae. Endopods of first pair of legs three-segmented, bearing three "setae" or "spines" at distal end. Endopods of second pair of legs unsegmented. Endopods of third and fourth pairs of swimming legs NEW SPECIES OF ARGULUS VOL. 103 FIGURES 2-3. Argulus ambystoma, new species. 2) Sensillum on dorsal surface of carapace (scale bar 5.0 |J.m); 3) Shape of respiratory areas (right ala of female) (scale bar =1.0 mm). two-segmented. Second, third, and fourth legs of males with accessory sexual structures. Coxae of male second legs with a bilobate structure posteroventrally, with scales on lobes distally (Fig. 4). Third legs each with paddle-like extension issuing anterodistally from the coxae, extending over bases (extending beyond distal margin of bases in larger males) (Fig. 4) and a hyaline, bubble-like area ("socket") on posterior of bases with opening of socket dorsally (Fig. 5). Dorsal surface of basis of third leg with conical pit ("pocket") ringed with papillae and minute pores; posterior wall of pocket split completely; opening of pocket leads to opening of socket below it. Paddle-like structure of coxa extends over pocket. Adjacent to pocket is a small, fleshy lobe tipped with papillae; lobe appears to be at base of exopod rather than on basis (Fig. 6). Bases of fourth legs each with large peg, sclerotized at its base; a smaller peg (dorsal to larger peg) and small sclerotized hump with minute spines ventral to larger peg (Fig. 5). Distal end of peg with notch on ventral surface and bearing blunt "tentacles" along its rim and numerous forked "tentacles" in concavity (Fig. 7). Precoxae + coxae of fourth legs of males and females with posterior bootshaped natatory lobes fringed with plumose setae and bearing scattered, coarse-pectinate scales; distal end of lobes extend beyond middle of bases but not beyond distal end of bases. Dorsal surface of all legs with finepectinate scales (Fig. 8), including dorsal surfaces of exopods and endopods. Ventral surfaces of all leg segments, including endopod, with coarse-pectinate scales (Fig. 9); ventral surface of exopod with fine-pectinate scales (Fig. 8). Sensilla and pores scattered on ventral surfaces of legs, pores also on dorsal surfaces of legs (Fig. 8). Abdomen bilobate, with pair of caudal rami near posterior end in anal sinus. Abdomen length (mm) 1.13-1.30 (1.21, 1.19) in males, 0.95-1.31 (1.14, 1.01) in females; maximum width (mm) 0.94-1.06 (1.00, 0.97) in males, 1.07-1.40 (1.26, 1.12) in females. Anal sinus length Qun) 180-220 (202, 180) in males, 350-430 (392, 350) in females. Male abdomen with spine-covered crests at each anterior corner (Fig. 10b), narrower than female abdomen, which is rounded at anterior corners (Fig. 10a). Small, lateral spines along most of edge of abdomen in both sexes. Each caudal ramus with five stout, naked "setae" (possibly not true setae) and dorsal pore just anterior of "setae" (Figs. 11, 12). Some of the "setae" extend beyond posterior margin of abdomen in males but not in females. Paired spermathecae of female brownish, round to oval, located anteriorly on abdomen. Pair of papillae on female abdomen anteroventrally, with small spines on ventral surface of papillae. Male abdomen with prominent black patches over testes dorsally (Fig. 1) and fewer, scattered melanophores ventrally over testes. Pores scattered on dorsal and ventral surfaces of abdomen, but only one short, sensillum observed dorsally OHIO JOURNAL OF SCIENCE W. J. POLY 55 FIGURES 4-7. Argulus ambystoma, new species. 4) Bilobed structure (B) on posteroventral surface of coxa of second leg and paddle-like structure (P) extending from the anterodistal margin of the coxa of the third leg of male (ventral view of left, second, and third legs) (scale bar = 100.0 |Xm); 5) Socket (S) and peg (P) on third and fourth legs, respectively, of male (dorsal view) (scale bar = 50.0 (im); 6) Conical pit or "pocket" (with papillae and pores) and fleshy lobe (with papillae, indicated by arrow) located anterodorsally on basis of the third leg of male (dorsal view) (scale bar = 50.0 ]im); 7) Tip of peg on basis of fourth leg of male (dorsal view) (scale bar = 5.0 |im). on a male's abdomen. First antennae composed of four segments: first segment sclerotized, large, with stout posteriorly-projecting posterior spine; second segment sclerotized with hump anteriorly, a smaller, posteriorly-projecting medial spine and large recurved terminal spine; third segment fleshy, cylindrical and smaller with large, stout seta distally that projects ventrally and several smaller setae; fourth segment fleshy, small, with few setae distally (Fig. 13). Second antennae with five fleshy segments; basal segment with posteriorly-projecting posterior spine. First two segments rounded, bulbous; remaining three thin, cylindrical. All segments of second antennae with several setae that project distally (Fig. 13). Paired postantennal spines consist of reduced anterior spine and stout posterior spine (Fig. 13). Posterior first antennal spines and 56 NEW SPECIES OF ARGULUS VOL. 103 postantennal spines more robust in females than in males. Reduced anterior postantennal spines vary in degree of development, sometimes partially hidden by posterior first antennal spines. First maxillae modified into suction cups in adults. First maxillae inner diameter (u.m) 290-320 (303, left: 300 and right: 290) (n = 15, left and right) and outer diameter (|im) 430-490 (460, left: 450 and right: 450) (« = 15) in males, inner diameter (|lm) 500-620 (564, left: 500 and right: 500) (n = 11, left and right) and outer FIGURES 8-10. Argulus ambystoma, new species. 8) Fine-pectinate scales on dorsal surface of exopod base; two pores are visible also (scale bar = 10.0 urn); 9) Example of a coarse-pectinate scale (on basal plate) (scale bar = 5.0 um); 10A) abdomen of female, 10B) abdomen of male, showing sexual dimorphism (dorsal view). The male abdomen has anterolateral crests with small spines, whereas the female abdomen is rounded anterolaterally and lacks the spines (scale bar 0.25 mm). OHIO JOURNAL OF SCIENCE W. J. POLY diameter (jim) 700-850 (787, left: 700 and right: 720) (w = 9) in females. Number of support rods (left and right suction cups) in males 43-56 (49.9, left: 50 and right: 52) and in females 45-54 (49.5, left: 47 and right: 52) (see Table 1). Number of sclerites per support rod in males 1-5 (3-3, holotype: mean: 3.4, range: 3-4) (n = 589 rods) and in females 1-7 (4.6, allotype: mean: 4.9, range: 3-7) (w 445 rods). Number of sclerites variable with position 58 NEW SPECIES OF AKGULUS VOL. 103 on suction cup, shape of sclerites variable. Usually 3 to 4 sclerites per rod in males, 4 to 5 sclerites per rod in females (Figs. 13, 14); lower counts, such as one, usually due to missing sclerite(s), uncommon. Proximal sclerite usually rodor vase-shaped, longer than other sclerites, which may be barrel-shaped to teardrop-shaped, slightly imbricated to offset, barely connecting with adjacent sclerites, and diminishing in size distally. Second maxillae five-segmented with broad basal plate bearing three digitate spines; spines narrower, rounded distally. Outer two spines closer together than median spine and innermost spine; outermost spine slightly shorter than remaining two spines. Basal plate with elevated pad bearing coarse-pectinate scales and about three stout, simple setae that extend over base of median posterior spine (Figs. 9, 15). Bidentate to multidentate spines and coarse-pectinate scales clustered in distinct patches on ventral surfaces of last four segments. Two stout, simple setae on second segment, one on third and fourth segments posterodistally. Distal segment with two sharp claws (usually appearing offset and not side-by-side) and blunt, elongate lobe positioned above claws, short sensillum at tip of lobe. Mouth tube short, lacking armature, not reaching thoracic accessory spines. Labium with three to four rows of small, embedded scales below mouth; short sensilla scattered on remainder of labium. Pair of serrated mandibles inside mouth tube. Preoral stylet present. Etymology The specific name is derived from the name of the host genus, Ambystoma (gender neuter), as a noun in apposition to the generic name, Argulus (gender masculine). Remarks Argulus ambystoma is most similar to A. versicolor, A. americanus, A. maculosus, and A. diversus, but can be distinguished from them by the shape and position of the respiratory areas and the secondary sexual modifications on the legs of males. The paddle-like structure on the third leg of male Argulus ambystoma resembles those of males of A. americanus and A. diversus. The number of suction cup rod sclerites also distinguishes A. ambystoma from both A. maculosus and A. americanus (usually 2-3 and 2 sclerites per rod in the latter two species, respectively). Argulus ambystoma is similar to A. versicolor in number and shape of sclerites in suction cup rods, but can be distinguished readily by the shape and position of the respiratory areas and pigmentation of the carapace (refer to Wilson 1902, 1904; Yeatman 1965 for figures of other species; except, note that at least some of the illustrations of A versicolor in Yeatman do not apply to that species). DISCUSSION Only rarely have Argulus been recorded as parasites of amphibians (for example, Goin and Ogren 1956; Bower-Shore 1940; Sauer 1977; Cuvier 1798 in Wilson 1902; Lemos de Castro and Gomes-Correa 1985; Clark 2001). The first record of Argulus from Ambystoma dumerilii was obtained when salamanders were purchased from local fishermen at Lake Patzcuaro to study their life history (R. Brandon pers. comm.; Brandon 1970). Salamanders harboring Argulus were captured in August 1968 (1 female) and December 1968 (1 female and 1 male), but the parasites were not noticed until April and May 1969, indicating that they were too small to observe easily when the salamanders were initially brought into the lab or that the species is cryptic on its host. Either may be the case, but the parasites were hidden among the gills of its host and were observed on the heads of salamanders in the evening only after the lights had been off for some time (R. Brandon pers. comm.). Also, when the lights were turned on, the parasites quickly moved toward the gills (R. Brandon pers. comm.). These observations support the cryptic nature of this argulid on Ambystoma dumerilii and may be the reason the species was not noticed until recently. None of the specimens collected by R. Brandon in 1969 could be located. The second record of Argulus on Ambystoma dumerilii resulted in the collection of the material used for the description; R. Brandon received some A. dumerilii from the Cincinnati Zoo and collected the Argulus from these animals. An additional observation of Argulus around the gills and on the head of wild caught A. dumerilii was supplied by S. Randal Voss (pers. comm.) in 1996.