Lagenophrys lenticula and L. patina (Peritricha), epibionts of ...Lagenophrys lenticula and L....
Transcript of Lagenophrys lenticula and L. patina (Peritricha), epibionts of ...Lagenophrys lenticula and L....
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© 2007 by Russia, Protistology
Protistology 4 (4), 339�345 (2006/7) ProtistologyProtistologyProtistologyProtistologyProtistology
Lagenophrys lenticula and L. patina (Peritricha),epibionts of Hyalella azteca (Amphipoda). A studyusing scanning electron microscopy to reveal detailsof the lorica aperture
R. Mayun�Estrada and M. A. Aladro�Lubel
Laboratorio de Protozoolog|a, Depto. Biolog|a Comparada, Fac. Ciencias,
Universidad Nacional Autsnoma de Muxico
Summary
The peritrich ciliates Lagenophrys lenticula and L. patina are epibionts of the amphipodHyalella azteca, from Pwtzcuaro Lake, Michoacwn, Mexico. The two species werestudied with scanning electron microscopy, enabling fine characterization of the lipsof the lorica aperture and complete description of each species. Prevalence anddistribution of each lagenophryid species on the basibiont body and theirbiogeographical record are updated.
Key words: lorica aperture, lagenophryids, ciliated epibionts, amphipods
Introduction
Members of the genus Lagenophrys are ciliatedprotozoa adapted to living as epibionts on crustaceans.Species of Lagenophrys spend most of their life cycleattached to their host's exoskeleton by means of theventral surface of their distinctive lorica or, in somecases, by means of a pseudostalk formed from anarrowed region of the lorica. The free�swimming phaseof the life cycle of Lagenophrys is the migratory larva,or telotroch, that is characteristic of all sessilineperitrichs. It is ephemeral, having the function ofseeking out and settling on a new host once it is released.
Extra telotrochs are produced when the host undergoesecdysis, as part of the process of abandoning the loricaeon the host's old exoskeleton. Some characters thatdefine species of Lagenophrys are the structure of thelorica and the lips of the lorica aperture.
To date, 62 species of Lagenophrys have beendescribed (Clamp and Kane, 2003), of which 26 usemarine and freshwater amphipods as substrates. Only10 species of this genus have been studied with scanningor transmission electron microscopy (Couch, 1973;Felgenhauer, 1979, 1982; Schadel, 1983, 1985, 1986;Walker et al., 1986; Clamp, 1988; Claps and Sampsns,1994; Veltkamp et al., 1994; Roberts and Chubb, 1998;
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· R. Mayun�Estrada and M. A. Aladro�Lubel340
Mayun�Estrada and Aladro�Lubel, 2000). Of thesestudies, 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 amphi�pods. Only optical microscopy has been used to describethe morphology of L. lenticula (Felgenhauer, 1979).The structure of the lips of the lorica aperture is a keydiagnostic feature used to identify species of Lageno�phrys. The purpose of this paper is to add newinformation that will improve diagnostic features ofthese species with emphasis on the details of the lips asrevealed by scanning electron microscopy. In addition,morphometric data, biogeographical record anddistribution on the host's body are provided.
Material and Methods
The crustacean amphipod Hyalella azteca wascollected from Pwtzcuaro Lake, Michoacwn, Mexicowith the aid of a 5 mm�mesh net over a five monthperiod through the years 2000�2002 (January andNovember, 2000; February and June, 2001, andFebruary 2002). In order to observe living ciliatesattached to the exoskeleton, amphipods were mainta�ined in laboratory aquaria at room temperature, usingunfiltered water from the lake with submerged plantsadded. To record distribution on the host's body,preferential habitat, and prevalence, each amphipodwas 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) andtelson (T). Each part was fixed with 5% formaldehydeand stained or impregnated with Harris hematoxylinand protargol in order to reveal the cytologicalcharacters of epibionts. For scanning electron micro�scopy, material was fixed in 1% glutaraldehyde,transferred to 2.5% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.2), critical point dried, andcoated with carbon and gold. Morphometric data of
lagenophryid peritrich cilates were recorded, and themean, maximum, minimum and standard deviationcalculated.
Results
MORPHOLOGICAL ASPECTS
Morphometric data of both species of Lagenophrysare shown in Table 1. Details and disposition of bothlips bordering the lorica aperture in L. lenticula areshown in figures 1�4. The lips were observed only in theclosed position. Viewed laterally, the posterior lipshowed only fine projections (Fig. 2), but seen fromabove (Fig. 3), projections were irregularly disposed andup to eight in number, with a folded appearance. Thesefolds 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. Theanterior lip carried from 12 to 17 indentations (Fig. 7),but specimens with few (Fig. 6) or no teeth (Figs. 5 and10) were also observed. Teeth were observed across theentire edge. The posterior lip always had 7 to 18 teeth(Figs 5�10) distributed along its edge. In somespecimens, these teeth were different in size, but inothers, all were of the same size. Teeth of both lips wereseparated from one another by grooves or folds thatpenetrated the lips to different depths. In the case ofthe posterior lip, this can be seen easily with opticalmicroscopy when the loricastome is open (Fig. 10).Observation of teeth on the anterior lip is difficult withoptical microscopy, either because of the shallow depthof the grooves that delimits each one or because the teethare disposed on the inner face of the loricastome.
DISTRIBUTION AND PREVALENCE
Altogether, 584 individuals of Hyalella azteca werecollected, of which 67.6% had Lagenophrys patina and10.9% 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
length Lorica width Lips of
lorica Pseudostalk
length Pseudostalk
width Zooid length
Zooid width
Macronucleus length
Macronucleus 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 µm.
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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 appendagesof H. azteca but with differences in prevalence. Ciliateswere recorded on the coxal plates with the highestfrequency (70%) and on the head with the leastfrequency (0.6%). L. lenticula showed the greatestprevalence on setae of pereiopods (63%) and was foundattached to only five appendages (antennae, pereiopods,pleopods, uropods and telson).
Discussion
According to Clamp and Kane (2003), speciesincluded in the genus Lagenophrys have a lorica aperturebounded and closable by two opposing lips, which areformed from folds of lorica material. Edges of one orboth 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 bothlorica aperture lips are several characters confirmingthat the specimens we collected should be placed withinthe genus Lagenophrys and identified as L. lenticula andL. patina.
We confirmed, as Clamp (1991) stated in theemended description of L. lenticula, that the loricaaperture lips are moderately arched and smooth,without indentations. However, we also observed severalfolds in the edge of posterior lip, not easily distingui�shable under optical microscopy. With respect tomorphometric data, we found that some features differfrom previously reported data (Table 3). According toClamp (1991), L. lenticula attaches only to setae at thejoints of the sixth and seventh pereiopods. Additionally,we found that L. lenticula also attaches to setae of fouradditional regions of the body (Tables 2 and 3). Thisdata allows for an emended description as follows:
Figs 1�4. Micrographs of Lagenophrys lenticula attached to amphipod Hyalella azteca. 1�3 � Scanningelectron micrographs; 4 � live individual. Abbreviations: al� anterior lip, l� lorica, lal� lorica aperturelips, pl� posterior lip, ps� pseudostalk, s� setae of basibiont. Scale bars: 1� 10 µm; 2, 3 � 5 µm; 4 � 13µm.
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· R. Mayun�Estrada and M. A. Aladro�Lubel342
anterior lip smooth; posterior lip with slight folds on itsedge. The epibiont attaches to setae of several appendages.
For L. patina, Clamp (1990) reported morpholo�gical variability of the lips. Clamp (1990) observed thatthe edge of anterior lip could be both smooth and withmany shallow evenly spaced indentations, and that onlythe medial three�fifths in the edge of posterior lip hadseveral large blunt tooth�like projections, separated byshallow notches, which were absent in some individuals.In describing L. patina (misidentified as L. labiata),Felgenhauer (1979) documented that the borders ofboth lips were crenated and irregular. We assume thatthese observations correspond to the teeth. We confirmvariation of the two aperture lips of L. patina,exemplified in this case among individuals that wereattached to hosts from a population of H. azteca inMichoacwn, Mexico. On both lips we observed thepresence of variable number of teeth. Lips of L. patinavary as follows: the anterior lip was either smooth orwith indentations (number of teeth ranged from 12 to17), and each one was delimited by grooves on the innerface of the loricastome. The posterior lip showed 9�18teeth, whose size increased from the lateral edge to themedial edge. We also found some morphometricdifference between our samples of L. patina andpublished data (Table 4).
The exact number of teeth is not specified inoriginal descriptions of L. patina, and the degree ofdepth in the grooves between teeth is not shown;therefore, an emended description of L. patina is asfollows:
Anterior lip of loricastome either smooth or with12�17 teeth, which can be disposed at the internal faceof the lip; posterior lip always with 9�18 teeth. On bothlips, teeth are distributed along the edge. The teeth aredelimited by grooves that extend from the edge to thebasal area of the lips.
Lagenophrys patina and L. lenticula have beenrecorded 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 Swnchez�Caldersn, 2005; Aladro�Lubel et al., 2006). Accordingto proposed biogeographical schemes (Morrone et al.,2002; Morrone, 2005), previous records and the presentstudy place the two species from the Nearctic Regionto the Neotropical Region and, for Mexico, in theMexican Transition Zone at the Transmexican VolcanicBelt Province.
Scanning electron microscopy study of Lageno�phrys species is very important for precise descriptionof the species�level characters. Intra� and interspecificvariability with respect to characteristics of the lips ofthe lorica aperture exists in some members of the genusLagenophrys, and several terms are used to describe any
Ta
ble
2.
Occ
urr
en
ce a
nd
dis
trib
utio
n o
f sp
ec
ies
of L
ag
en
op
hry
s in
the
am
ph
ipo
d H
. a
zte
ca
fro
m P
atzc
uar
o L
ake
, Mic
ho
acan
, M
exi
co
.
Dat
e N
N
H
Sp
eci
es
H
A
Mp
G
C
P
e P
n
Un
P
P
l U
T
Jan
200
0 69
44
47
L.
lent
icul
a L.
pat
ina
0 0 0
2 (
2.8
) 0
0
0
0
0
46
(66.
6)
0 7
(10
.0)
0
3 (
4.3
) 0
7
(10.
0)
44
(63.
0)
7 (1
0.0
) 0
3
(4
.3)
11 (
16.0
) 1
(1
.4)
8 (
11.6
) 0
No
v 2
000
42
0 11
L. le
ntic
ula
L. p
atin
a 0 0
0 1
(2.
3)
0
0
0
0
0 1
0 (2
4.0)
0
1 (
2.3
) 0
1
(2
.3)
0
2 (
4.8
) 0
4
(9
.5)
0
1 (
2.3
) 0
2 (
4.8
) 0 0
Feb
200
1 1
64
13
85
L. le
ntic
ula
L. p
atin
a 0 0
0 0 0
0
0
0
0
76
(46.
0)
0 8
(4.
8)
0
22
(13.
0)
0
16
(10.
0)
13
(8
.0)
15
(9
.0)
0
0
1 (
0.6
) 3
(1
.8)
0 0 Ju
n 2
001
134
0
122
L.
lent
icul
a L.
pat
ina
0 0 0
12
(9
.0)
0
0
0
1 (0
.7)
0 9
2 (6
9.0)
0
28 (
21.0
) 0
5
1 (3
8.0
) 0
6
2 (4
6.0
) 0
4
7 (3
5.0
) 0
0
0
10
(7
.8)
0 6
(4
.4)
Feb
200
2 1
75
7 1
30
L. le
ntic
ula
L. p
atin
a 0
1 (
0.6
) 2
(1.
1)
7 (
4.0
) 0
5
(2.8
) 0
10
(5.
8)
0 122
(70.
0)
0 20
(11
.0)
0
27
(15.
0)
0
32
(18.
0)
6 (
3.4
) 3
7 (2
1.0
) 1
(0
.6)
2 (
1.1
) 0
9 (
5.1
) 0
3 (
1.8
)
Ja
n=
Jan
ua
ry, N
ov=
Nov
em
ber
, Fe
b=
Feb
ruar
y, J
un=
Ju
ne, N
= n
umb
er
tota
l of h
ost
s in
the
sam
ple
, NH
= n
umb
er o
f ho
sts
with
lag
enop
hry
ids.
The
dat
a co
rres
pon
ds
resp
ect
ivel
y to
num
be
r an
d
fre
que
ncy
% (
in p
are
nth
esis
) of
hos
ts th
at h
arb
ore
d e
pib
ion
ts. H
= h
ead
, A=
ant
enn
ae,
Mp
= m
outh
pa
rts,
G=
gna
thop
od
s, C
= c
oxae
, Pe=
pe
reio
nite
s, P
n=
ple
oni
tes,
Un
= u
roni
tes,
P=
per
eio
pod
s,
Pl=
ple
opo
ds,
U=
uro
pod
s, T
= t
els
on.
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· 343ProtistologyProtistologyProtistologyProtistologyProtistology
kind of protuberance in the lorica aperture lips (roundedtubercles, folds or grooves that extend from the edge tothe basal area of the lip, indentations and/or notches).Therefore, we propose that species be categorized intothe following general groups: a) species with both lips
smooth, b) species with one of the two lips with somekind of processes, and c) species with both lips carryingsome type of processes. The new arrangement proposedhere would be useful for all kinds of studies of Lage�nophrys species.
Figs 5�10. Micrographs of Lagenophrys patina attached to Hyalella azteca. 5�9 � Scanning electronmicrographs; 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 µm; 6�8 � 5 µm; 9 � 2 µm; 10 � 13 µm.
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· R. Mayun�Estrada and M. A. Aladro�Lubel344
ACKNOWLEDGMENTS
We are very grateful to Bisl. Armando Zepeda R.(Lab. Microscop|a Electrsnica, Facultad de Medicina,UNAM), who kindly processed all SEM material andto Bisl. Francisco Pasos (Depto. Biolog|a Celular yTisular, Facultad de Medicina, UNAM) for technicalassistance with photographs. We would like to thank Dr.T. Nyborg (Department of Earth and BiologicalSciences, Loma Linda University) and Dr. F. Vega(Instituto de Geolog|a, UNAM) for help with Englishgrammar and expression.
References
Aladro�Lubel M.A. and Swnchez�Caldersn G.2005. Ciliados epibiontes de la vegetacisn sumergida y
de los invertebrados de la Laguna de Tecocomulco. In:La Laguna de Tecocomulco. Geo�Ecolog|a de unDesastre. (Eds. Huizar A.R., Jimunez F. E. and JuwrezC.). Publ. Esp. 3. I. Geolog|a, UNAM. pp. 169�178.
Aladro�Lubel M. A., Mayun�Estrada R. andReyes�Santos M. 2006. Registro actualizado de ciliados(Agosto, 2004). Listados faunisticos de Muxico. XI.Inst. Biol. UNAM.
Clamp J.C. 1988. A new species of Lagenophrys(Ciliophora: Peritrichia: Lagenophryidae) ectocom�mensal on the wood�boring isopod Limnoria (Flabel�lifera: Limnoridae). Trans. Am. Micros. Soc. 107, 12�16.
Clamp J.C. 1990. Redescription of three speciesof Lagenophrys (Ciliophora: Peritricha: Lagenophry�idae) and a new North American species of Lageno�phrys from hypogean amphipods. Trans. Am. Microsc.Soc. 109, 1�31.
Clamp J.C. 1991. Revision of the family Lageno�phryidae Bztschli, 1889 and description of the familyUsconophryidae n. fam. (Ciliophora, Peritricha). J.Protozool. 38, 4, 355�377.
Clamp J.C. and Kane J.R. 2003. Redescription offour species of lagenophryid peritrichs (Ciliophora)from Australia and New Guinea, with descriptions oftwo new species. Rec. Aust. Mus. 55, 153�168.
Claps M.C. and Sampons M.R. 1994. First recordof Lagenophrys discoidea Kellicott (Ciliophora,Peritrichia, Lagenophryidae), ectocommensal ofArgentinian ostracods. Iheringia, Ser. Zool. PortoAlegre. 76, 167�170.
Couch J.A. 1973. Ultrastructural and protargolstudies of Lagenophrys callinectes (Ciliophora: Peritri�chida). J. Protozool. 20, 638�647.
Felgenhauer B.E. 1979. A note on the scanningelectron microscopy and hosts of the widespreadperitrich ciliate Lagenophrys labiata. Trans. Amer.Micros. Soc. 98, 4, 591�595.
Felgenhauer B.E. 1982. A new species of Lageno�phrys (Ciliophora: Peritrichida) from the freshwatershrimp Palaemonetes kadiakensis. Trans. Am. Microsc.Soc. 101, 2, 142�150.
Mayun�Estrada R. and Aladro�Lubel M.A. 2000.First record of Lagenophrys dennisi (Ciliophora:Peritrichia) on the exoskeleton of crayfish Cambarelluspatzcuarensis. J. Eukaryot. Microbiol. 47, 57�61.
Morrone J.J. 2005. Biogeographic areas andtransition zones of Latin America and the Caribbeanislands based on pangeobiographic and cladisticanalyses of the entomophauna. Ann. Rev. Entomol. 51,467�494.
Morrone J.J., Espinosa D. and Llorente J. 2002.Mexican biogeographic provinces: preliminary scheme,general characterizations and synonymies. Acta Zool.Mex. (n. s.). 85, 83�108.
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�66.6 Lorica width 34.9�47.9 22.4�44.8 Lips of lorica 16.0�18.9 11.2�16.8 Pseudostalk length 11.1�16.6 8.4�29.6 Pseudostalk width �� 2.8�7.4 Zooid length �� 22.4�51.8 Zooid width �� 19.6�37.0 Macronucleus length �� 16.8�30.8 Macronucleus width �� 2.8�5.6 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 µm; the values corresponds to minimum and maximum.
Table 4. Morphometric comparison of Lagenophrys patina Stokes, 1887 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 anterior lip
Smooth, occasionally with several to many
shallow evenly spaced indentations
in edge
Smooth or with 12 to 17 teeth across the
entire edge, delimited by grooves
Number of teeth on posterior lip
Medial three�fifths of edge with several
large, blunt tooth like projections separated
by shallow notches
7�18 teeth distributed along its edge,
delimited by grooves
Zooid length �� 33.6�66.6 Zooid width �� 28.0�52.2 Macronucleus length
9.2�48.0 19.6�36.5
Macronucleus width
2.6�9.7 2.8�11.2
Host appendages All surfaces of body 12 appendages Morphometric data in µm; the values corresponds to minimum and maximum.
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Address for correspondence: R. Mayun�Estrada. Ap. Postal 70�374, C.P. 04510, Ciudad Universitaria, Muxico,D.F. Mexico. E�mail: [email protected]
Editorial responsibility: Mark Farmer
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Scha del H. 1985. Epizoische Einzeller aufFlohkrebsen. 2. Besiedler der Gammaridenbeine.Mikrokosmos. 74, 269�273.
Schadel H. 1986. Sesshapte Wimpertiere aufWasserasseln. Mikrokosmos. 75, 293�301.
Veltkamp C.J., Chubb J.C., Birch S.P. and EatonJ.W. 1994. A simple freeze dehydration method forstudying epiphytic and epizoic communities using thescanning electron microscope. Hydrobiologia. 288, 33�38.
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