-
© 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;
-
· 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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341ProtistologyProtistologyProtistologyProtistologyProtistology
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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·
345ProtistologyProtistologyProtistologyProtistologyProtistology
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
Roberts G.N. and Chubb J.C. 1998. The distributionand location
of the symbiont Lagenophrys aselli on thefreshwater isopod Asellus
aquaticus. Freshwater Biol.40, 671�677.
Scha del H. 1983. Three new peritricha onGammaridae. Arch.
Protistenk. 127, 115�126.
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.
Walker M.H., Roberts E.M. and Usher M.L. 1986.The fine structure
of the trophont and stages in telotrochformation in
Circolagenophrys ampulla (Ciliophora,Peritrichida). J. Protozool.
33, 2, 246�255.
