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Protistology 4 (4), 339-345 (2006/7)
Protistology
Lagenophrys lenticula and L. patina (Peritricha), epibionts of Hyalella azteca (Amphipoda). A study using scanning electron microscopy to reveal details of the lorica aperture
R. Mayén-Estrada and M. A. Aladro-Lubel
Laboratorio de Protozoología, Depto. Biología Comparada, Fac. Ciencias, Universidad Nacional Autónoma de México
Summary
The peritrich ciliates Lagenophrys lenticula and L. patina are epibionts of the amphipod Hyalella azteca, from Patzcuaro Lake, Michoacan, Mexico. The two species were studied with scanning electron microscopy, enabling fine characterization of the lips of the lorica aperture and complete description of each species. Prevalence and distribution of each lagenophryid species on the basibiont body and their biogeographical record are updated.
Key words: lorica aperture, lagenophryids, ciliated epibionts, amphipods
Introduction
Members of the genus Lagenophrys are ciliated protozoa adapted to living as epibionts on crustaceans. Species of Lagenophrys spend most of their life cycle attached to their host's exoskeleton by means of the ventral surface of their distinctive lorica or, in some cases, by means of a pseudostalk formed from a narrowed region of the lorica. The free-swimming phase of the life cycle of Lagenophrys is the migratory larva, or telotroch, that is characteristic of all sessiline peritrichs. It is ephemeral, having the function of seeking out and settling on a new host once it is released.
Extra telotrochs are produced when the host undergoes ecdysis, as part of the process of abandoning the loricae on the host's old exoskeleton. Some characters that define species of Lagenophrys are the structure of the lorica and the lips of the lorica aperture.
To date, 62 species of Lagenophrys have been described (Clamp and Kane, 2003), of which 26 use marine and freshwater amphipods as substrates. Only 10 species of this genus have been studied with scanning or transmission electron microscopy (Couch, 1973; Felgenhauer, 1979, 1982; Schodel, 1983, 1985, 1986; Walker et al., 1986; Clamp, 1988; Claps and Sampons, 1994; Veltkamp et al., 1994; Roberts and Chubb, 1998;
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Mayé n-Estrada and Aladro-Lubel, 2000). Of these studies, only four have focused on species of Lageno-phrys which attach to amphipods. Two species, L. lenticula (Kellicott, 1885) and L. patina Stokes, 1887, have been reported as associates of freshwater amphipods. Only optical microscopy has been used to describe the morphology of L. lenticula (Felgenhauer, 1979). The structure of the lips of the lorica aperture is a key diagnostic feature used to identify species of Lageno-phrys. The purpose of this paper is to add new information that will improve diagnostic features of these species with emphasis on the details of the lips as revealed by scanning electron microscopy. In addition, morphometric data, biogeographical record and distribution on the host's body are provided.
Material and Methods
The crustacean amphipod Hyalella azteca was collected from Pátzcuaro Lake, Michoacán, Mexico with the aid of a 5 mm-mesh net over a five month period through the years 2000-2002 (January and November, 2000; February and June, 2001, and February 2002). In order to observe living ciliates attached to the exoskeleton, amphipods were maintained in laboratory aquaria at room temperature, using unfiltered water from the lake with submerged plants added. To record distribution on the host's body, preferential habitat, and prevalence, each amphipod was dissected and separated into 12 units: head (H), antennae (A), mouth parts (Mp), gnathopods (G), coxae (C), pereionites (Pe), pleonites (Pn), uronites (Un), pereiopods (P), pleopods (Pl), uropods (U) and telson (T). Each part was fixed with 5% formaldehyde and stained or impregnated with Harris hematoxylin and protargol in order to reveal the cytological characters of epibionts. For scanning electron microscopy, material was fixed in 1% glutaraldehyde, transferred to 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), critical point dried, and coated with carbon and gold. Morphometric data of
lagenophryid peritrich cilates were recorded, and the mean, maximum, minimum and standard deviation calculated.
Results
Morphological aspects
Morphometric data ofboth species of Lagenophrys are shown in Table 1. Details and disposition of both lips bordering the lorica aperture in L. lenticula are shown in figures 1-4. The lips were observed only in the closed position. Viewed laterally, the posterior lip showed only fine projections (Fig. 2), but seen from above (Fig. 3), projections were irregularly disposed and up to eight in number, with a folded appearance. These folds were not visible by optical microscopy (Fig. 4).
Both lips of L. patina are shown in figures 5-10, with the loricastome open to different degrees. The anterior lip carried from 12 to 17 indentations (Fig. 7), but specimens with few (Fig. 6) or no teeth (Figs. 5 and 10) were also observed. Teeth were observed across the entire edge. The posterior lip always had 7 to 18 teeth (Figs 5-10) distributed along its edge. In some specimens, these teeth were different in size, but in others, all were of the same size. Teeth ofboth lips were separated from one another by grooves or folds that penetrated the lips to different depths. In the case of the posterior lip, this can be seen easily with optical microscopy when the loricastome is open (Fig. 10). Observation of teeth on the anterior lip is difficult with optical microscopy, either because of the shallow depth of the grooves that delimits each one or because the teeth are disposed on the inner face of the loricastome.
Distribution and prevalence
Altogether, 584 individuals of Hyalella azteca were collected, ofwhich 67.6% had Lagenophrys patina and 10.9% had L. lenticula attached to the exoskeleton (Table 2). L. patina was recorded on amphipods during
Table 1. Morphometric data of Lagenophrys lenticula and L. patina epibionts of Hyalella azteca.
Characters
Attributes Lorica Lorica width Lips of Pseudostalk Pseudostalk Zooid Zooid Macronucleus Macronucleus
lenqth lorica lenqth width lenqth width lenqth width
L. lenticula
Average 47.7 31.2 14.3 14.3 4.2 31.4 24.5 22.9 4.3
Minimum 39.2 22.4 11.2 8.4 2.8 22.4 19.6 16.8 2.8
Maximum 66.6 44.8 16.8 29.6 7.4 51.8 37.0 30.8 5.6
SD 4.6 4.4 1.6 3.7 1.4 5.0 3.6 3.1 1.3
N 90 90 53 43 43 55 55 31 31
L . patina
Average 57.9 53.9 14.4 46.6 37.9 29.3 5.5
Minimum 42.0 36.4 14.0 33.6 28.0 19.6 2.8
Maximum 81.4 74.0 16.8 66.6 52.2 36.5 11.2
SD 5.2 5.3 1.0 13.4 5.3 3.3 1.3
N 150 150 105 104 104 101 101
SD = Standard deviation; N= number of individuals. Measurements in pm.
Figs 1-4. Micrographs of Lagenophrys lenticula attached to amphipod Hyalella azteca. 1-3 - Scanning electron micrographs; 4 - live individual. Abbreviations: al- anterior lip, l- lorica, lal- lorica aperture lips, pl- posterior lip, ps- pseudostalk, s- setae of basibiont. Scale bars: 1- 10 ^m; 2, 3 - 5 ^m; 4 - 13 ^m.
all five months in which collections were made, but L. lenticula was recorded during only three months (January 2000, February 2001 and 2002).
Lagenophrys patina attached to all 12 appendages of H. azteca but with differences in prevalence. Ciliates were recorded on the coxal plates with the highest frequency (70%) and on the head with the least frequency (0.6%). L. lenticula showed the greatest prevalence on setae of pereiopods (63%) and was found attached to only five appendages (antennae, pereiopods, pleopods, uropods and telson).
Discussion
According to Clamp and Kane (2003), species included in the genus Lagenophrys have a lorica aperture bounded and closable by two opposing lips, which are formed from folds of lorica material. Edges of one or both lips may be tuberculate or serrate, but spines are
never present on the anterior lip. The form, disposition, number of elements and presence of processes in both lorica aperture lips are several characters confirming that the specimens we collected should be placed within the genus Lagenophrys and identified as L. lenticula and L. patina.
We confirmed, as Clamp (1991) stated in the emended description of L. lenticula, that the lorica aperture lips are moderately arched and smooth, without indentations. However, we also observed several folds in the edge of posterior lip, not easily distinguishable under optical microscopy. With respect to morphometric data, we found that some features differ from previously reported data (Table 3). According to Clamp (1991), L. lenticula attaches only to setae at the joints of the sixth and seventh pereiopods. Additionally, we found that L. lenticula also attaches to setae of four additional regions of the body (Tables 2 and 3). This data allows for an emended description as follows:
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anterior lip smooth; posterior lip with slight folds on its edge. The epibiont attaches to setae ofseveral appendages.
For L. patina, Clamp (1990) reported morphological variability of the lips. Clamp (1990) observed that the edge of anterior lip could be both smooth and with many shallow evenly spaced indentations, and that only the medial three-fifths in the edge of posterior lip had several large blunt tooth-like projections, separated by shallow notches, which were absent in some individuals. In describing L. patina (misidentified as L. labiata), Felgenhauer (1979) documented that the borders of both lips were crenated and irregular. We assume that these observations correspond to the teeth. We confirm variation of the two aperture lips of L. patina, exemplified in this case among individuals that were attached to hosts from a population of H. azteca in Michoacán, Mexico. On both lips we observed the presence of variable number of teeth. Lips of L. patina vary as follows: the anterior lip was either smooth or with indentations (number of teeth ranged from 12 to 17), and each one was delimited by grooves on the inner face of the loricastome. The posterior lip showed 9-18 teeth, whose size increased from the lateral edge to the medial edge. We also found some morphometric difference between our samples of L. patina and published data (Table 4).
The exact number of teeth is not specified in original descriptions of L. patina, and the degree of depth in the grooves between teeth is not shown; therefore, an emended description of L. patina is as follows:
Anterior lip of loricastome either smooth or with 12-17 teeth, which can be disposed at the internal face of the lip; posterior lip always with 9-18 teeth. On both lips, teeth are distributed along the edge. The teeth are delimited by grooves that extend from the edge to the basal area of the lips.
Lagenophrys patina and L. lenticula have been recorded from the continental United States, Canada, Mexico (Puebla), and Uruguay (Clamp, 1990). L. lenticula was also reported from Mexico (Hidalgo state) associated with H. azteca (Aladro-Lubel and Sánchez-Calderón, 2005; Aladro-Lubel et al., 2006). According to proposed biogeographical schemes (Morrone et al., 2002; Morrone, 2005), previous records and the present study place the two species from the Nearctic Region to the Neotropical Region and, for Mexico, in the Mexican Transition Zone at the Transmexican Volcanic Belt Province.
Scanning electron microscopy study of Lagenophrys species is very important for precise description of the species-level characters. Intra- and interspecific variability with respect to characteristics of the lips of the lorica aperture exists in some members of the genus Lagenophrys, and several terms are used to describe any
Figs 5-10. Micrographs of Lagenophrys patina attached to Hyalella azteca. 5-9 - Scanning electron micrographs; 10 - micrograph of empty lorica of L. patina seen by light microscopy. Abbreviations: c-collar, lr- lorica rim, t- teeth; for explanation of other symbols see figures 1-4. Scale bars: 5 - 10 um; 6-
8 - 5 ^m; 9 - 2 ^m; 10 - 13 ^m.
kind ofprotuberance in the lorica aperture lips (rounded tubercles, folds or grooves that extend from the edge to the basal area of the lip, indentations and/or notches). Therefore, we propose that species be categorized into the following general groups: a) species with both lips
smooth, b) species with one of the two lips with some kind of processes, and c) species with both lips carrying some type of processes. The new arrangement proposed here would be useful for all kinds of studies of Lagenophrys species.
Table 3. Morphometric comparison of Lagenophrys lenticula (Kellicott, 1885) specimens from the present study with previously described ones.
Characters Clamp (1991) Present study
Lorica length 44.7-56.6 39.2-бб.б
Lorica width 34.9-47.9 22.4-44.8
Lips of lorica 16.0-18.9 11.2-1б.8
Pseudostalk length 11.1-16.6 8.4-29.б
Pseudostalk width -- 2.8-l.4
Zooid length -- 22.4-51.8
Zooid width -- 19.6-37Ю
Macronucleus length -- 1б.8-30.8
Macronucleus width -- 2.8-5.б
Edge of posterior lip -- Up to eight slight folds irregularly disposed
Host appendages Setae at the joints of the host's sixth and seventh pereiopods Setae of antennae, pereiopods, pleopods, uropods and telson
Morphometric data in pm; the values corresponds to minimum and maximum.
Acknowledgments
We are very grateful to Biól. Armando Zepeda R. (Lab. Microscopía Electrónica, Facultad de Medicina, UNAM), who kindly processed all SEM material and to Biól. Francisco Pasos (Depto. Biología Celular y Tisular, Facultad de Medicina, UNAM) for technical assistance with photographs. We would like to thank Dr. T. Nyborg (Department of Earth and Biological Sciences, Loma Linda University) and Dr. F. Vega (Instituto de Geología, UNAM) for help with English grammar and expression.
References
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Table 4. Morphometric comparison of Lagenophrys patina Stokes, 188l specimens obtained in the present study with previously described ones.
Characters Clamp 1990 Present study
Lorica length 45.6-74.9 42.0-81.4
Lorica width 43.7-77.6 36.4-74.0
Lips of lorica 15.4-23.6 14.0-16.8
Number of teeth on Smooth, occasionally Smooth or with 12 to
anterior lip with several to many 17 teeth across the
shallow evenly entire edge, delimited
spaced indentations by grooves
in edge
Number of teeth on Medial three-fifths of 7-18 teeth distributed
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projections separated
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Macronucleus 9.2-48.0 19.6-36.5
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Macronucleus 2.6-9.7 2.8-11.2
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Host appendages All surfaces of body 12 appendages
Morphometric data in pm; the values corresponds to minimum and maximum.
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Address for correspondence: R. Mayén-Estrada. Ap. Postal 70-374, C.P. 04510, Ciudad Universitaria, México,
D.F. Mexico. E-mail: rme@hp.fciencias.unam.mx
Editorial responsibility: Mark Farmer
