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C. CARAMELO, E. MARTÍNEZ-ANSEMIL
1
Turk J Zool2012; 36(1): 1-14© TÜBİTAKdoi:10.3906/zoo-1002-25
Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
Carlos CARAMELO, Enrique MARTÍNEZ-ANSEMIL*Departamento de
Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Universidade da
Coruña, 15071 A Coruña - SPAIN
Received: 09.02.2010
Abstract: Over the past several years, the authors of this work
have investigated external structures of microdrile oligochaetes
using scanning electron microscopy (SEM). Both published and
still-unpublished data have revealed new structures of interest,
especially those associated with mating, ciliate sense receptors,
chaetae, and dorsal pores. Mating systems can be classifi ed in 3
categories: grasping, coupling, and embracing. Ciliate sense
receptors can be classifi ed as poorly defi ned (unelevated),
sensory buds, or papillae, and are provided with blunt cilia and/or
sharp cilia. Th ey are present along the whole body (including
clitellum, budding, and regeneration zones), scattered on the
prostomium, peristomium, and pygidium, arranged in transversal rows
and scattered in chaetal segments. Somatic chaetae show great
variability when observed by SEM. Hair chaetae are at least
potentially provided with denticulations. Dorsal pores were
observed in aquatic microdriles. SEM proves to be an invaluable
tool to discover important structures associated with functional
anatomy in microdriles.
Key words: Functional anatomy, microdrile oligochaetes,
morphology, SEM, ciliate sense receptors, mating structures,
somatic chaetae, dorsal pores
Research Article
* E-mail: [email protected]
IntroductionBefore the year 2000, the observation by
scanning
electron microscopy (SEM) of external structures in microdriles
was uncommon. Only a few studies have been published, most of them
referring to observations on ciliate sense receptors (Chapman,
1979; Farnesi et al., 1982a, 1982b; Smith, 1983; Römbke and
Schmidt, 1999) and somatic chaetae (Harman and McMahan, 1975;
Milbrink, 1983; Smith, 1985; Chapman and Brinkhurst, 1986; Grimm,
1986, 1987, 1988; Finogenova and Poddubnaja, 1990; Römbke and
Schmidt, 1990; Ohtaka, 1995, Bouché et al., 1999).
A descriptive study dealing with the external structures
involved in attachment and sperm transfer in some freshwater
microdriles using SEM was published by Cuadrado and
Martínez-Ansemil (2001). More recently, Yáñez et al. (2006) and
Caramelo and Martínez-Ansemil (2010) contributed to the knowledge
of ciliate sense receptors in microdriles, providing a signifi cant
amount of new data as well as analyses of the typology and patterns
of distribution in the main taxonomic groups.
Th e aim of the present work was to provide an overview of the
functional anatomy of microdrile oligochaetes as revealed by SEM.
New data are
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Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
2
provided mainly on systems related with mating, somatic chaetae,
and dorsal pores.
Materials and methodsTh e biological material used in the
present study
was examined previously in the studies by Cuadrado and
Martínez-Ansemil (2001), Yáñez et al. (2006), and Caramelo and
Martínez-Ansemil (2010). Most of the samples were collected from
habitats in Galicia (NW Iberian Peninsula); detailed information
about each species discussed in this paper is distributed among the
aforementioned articles and the current one (i.e. when dealing with
individuals for which new data are provided). All the fi gures in
this paper are original.
Material studiedTh e material studied comprises a total of
36
species belonging to the microdrile families Naididae (9
Naidinae, 2 Pristininae, 5 Rhyacodrilinae, 1 Phallodrilinae, and 9
Tubifi cinae), Phreodrilidae (1), Parvidrilidae (1), Lumbriculidae
(4), and Enchytraeidae (4), as well as Eiseniella tetraedra
(Savigny, 1826)—a lumbricid very common in freshwaters.
Material providing new dataMost specimens were collected in the
2 following
streams of A Coruña (Galicia): Porto do Cabo stream at Moeche
and Sar stream at Codesido (Rois). In the list below we will refer
to them, respectively, as Porto do Cabo and Sar.
Naidinae: Nais alpina, 2 specimens: Porto do Cabo, 07/02/07.
Nais elinguis, 3 specimens: Sar, 10/09/03. Ophidonais serpentina, 3
specimens: Sar, 15/05/08. Stylaria lacustris, 3 specimens: Sar,
02/03/02. Vejdovskyella comata, 3 specimens: Sar, 02/03/02.
Pristininae: Pristina aequiseta, 6 specimens: Porto do Cabo,
07/02/07. Pristina longiseta, 6 specimens: Sar, 12/11/08.
Rhyacodrilinae: Bothrioneurum vejdovskyanum, 2 specimens: Sar,
06/09/07. Branchyura sowerbyi, 3 specimens: Cazalegas reservoir,
Alberche river, Spain, leg. N. Prat. Peristodrilus montanus, 3
specimens: Porto do Cabo, 07/02/07. Protuberodrilus tourenqui, 1
specimen: Porto do Cabo, 07/02/07. Rhyacodrilus
falciformis, 2 specimens: Porto do Cabo, 02/04/83. Tubifi cinae:
Krenedrilus realis, 1 specimen: Arbón reservoir, Navia river,
Asturias, Spain, leg. M. Real, 07/08/88. Limnodrilus udekemianus, 2
specimens: Porto do Cabo, (1 specimen), 07/02/07; Sar, (1
specimen), 06/09/07. Potamothrix bavaricus, 2 specimens: Ebrón
stream, Valencia, Spain, leg. S. Pérez, 12/05/96. Potamothrix
heuscheri, 1 specimen: Ebrón stream, Valencia, Spain, leg. S.
Pérez, 08/02/96. Psammoryctides barbatus, 2 specimens: Ebrón
stream, Teruel, Spain, leg. S. Pérez, 16/11/95. Spirosperma
velutinus, 3 specimens: Porto do Cabo, 07/02/07. Phreodrilidae:
Insulodrilus sp., 3 specimens: Knockmoyle, Ireland, leg. Wisdom,
11/08. Parvidrilidae: Parvidrilus spelaeus, 1 specimen: Pajsarjeva
cave, Vhrnika, Slovenia, leg. B. Sambugar and F. Gasparo, 26/05/97.
Lumbriculidae: Lumbriculus variegatus, 5 specimens: Sar, 06/09/07.
Stylodrilus heringianus, 4 specimens: Sar, 17/06/08. Enchytraeidae:
Enchytraeus albidus, 2 specimens: culture original from Bull
Island, Dublin, Ireland, leg. R. Schmelz and R. Collado, 06/95.
Fridericia monochaeta, 2 specimens: A Zapateira, A Coruña, Spain,
leg. R. Schmelz, 27/01/09. Lumbricillus sp., 2 specimens: culture
original from littoral of North Sea, Tjärnö, Sweden, leg. R.
Schmelz and R. Collado, 09/94. Lumbricidae: Eiseniella tetraedra, 2
specimens: Porto do Cabo, (1 specimen), 07/02/07; Sar, (1
specimen), 17/07/08.
MethodsSpecimens collected in the fi eld were brought
alive to the laboratory and identifi ed using a light microscope
equipped with diff erential interference contrast (mounted in a
drop of water). Individuals were anesthetized with 7.5% magnesium
chloride added progressively to water or with ethanol and fi xed
with 2% glutaraldehyde and 1% paraformaldehyde in 0.1M cacodylate
buff er for 2 h. Aft er fi xation, specimens were washed 3 times in
0.1M cacodylate buff er for 10 min, postfi xed with 1% osmium
tetroxide for 2 h in the same buff er, and dehydrated through
increasing acetone series. All specimens were mounted on stubs, and
sputter-coated with gold. Observations were carried out using a
Jeol JSM-6400 SEM at the SAI (Universidade da Coruña).
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C. CARAMELO, E. MARTÍNEZ-ANSEMIL
3
Results and discussionSystems related with mating (Figures 1 and
2)In observations conducted using SEM, Cuadrado
and Martínez-Ansemil (2001) described some structures and
mechanisms used by naidids for attachment and sperm transfer. Some
of these systems appear to be similar to those already known in
earthworms (see Jamieson, 2006) whereas others represent anatomical
novelties that could be found exclusively in microdriles. All
mating systems revealed by Cuadrado and Martínez-Ansemil (2001)
imply the apposition of male and spermathecal pores. Aft er
observing by SEM several other species belonging to the families
Enchytraeidae, Lumbriculidae, and Phreodrilidae, and aft er
comparing these observations with previous results, we now propose
that the way to achieve the apposition of genital pores and to
maintain the attachment of partners during the sperm transfer in
microdriles can be included in 1 of the following 3 categories:
Grasping. Th e most elaborate system described by Cuadrado and
Martínez-Ansemil (2001) belongs to this category. It is the system
utilized by the rhyacodriline Peristodrilus montanus (Figure 1A-C),
in which the combined action of a complex system of muscles allow
the penial chaetae (placed between male pores) to enter the lateral
sides of a singular ‘anchorage bridge’ located between the
spermathecal pores. Th e apposition of the male and the
spermathecal pores, and the fi rm holding of the 2 partners, would
easily allow sperm transfer to occur. A dense ciliated area on the
prominent outer part of the tegument surrounding the penial chaetal
bundles may facilitate the fi nding of the correct anchorage
place.
According to Cuadrado and Martínez-Ansemil (2001), similar
systems could be present in at least 4 of the 5 species of the
tubifi cine genus Krenedrilus Dumnicka, and in the phallodriline
Bathydrilus rohdei (Jamieson, 1977). In Krenedrilus the bundles of
penial chaetae are arranged with the tips close together and
directed towards each other, and a small epidermal papilla is
present in the mid-ventral line of the spermathecal segment. B.
rohdei is provided with an ‘X-shaped mid-ventral slit’ that,
according to Erséus (1981), corresponds to the location of the tips
of the penial chaetae of the mate. Many aquatic
naidids have similar penial chaetae that—based on form, location
and orientation—might also have an anchorage function. Although
they could anchor directly on the body wall, new anchorage
structures can be expected in these species when more extensive
observations through SEM are performed.
Coupling. Many microdriles are provided with complementary
mating structures that fi t each other and are used both for
attachment and sperm transfer. Th is seems to be the case for
Rhyacodrilus falciformis, whose falciform penial chaetae fi t into
the lateral oblong spermathecal pores. Similarly, in
Protuberodrilus tourenqui, the male porophore brings the 2 male
pores (close to the tip) to fi t with the 2 spermathecal pores of
the partner, which are placed near the median ventral line of the
body in a depression fl anked by prominent chaetophores (Cuadrado
and Martínez-Ansemil, 2001). Th e prominent pendant penes of
Stylodrilus heringianus, oriented towards the rear part of the
worm, could also help in holding partners when they penetrate
deeply into the large spermathecal pores (Figure 1E).
Erséus (1979) and Erséus and Baker (1982) interpreted the giant
penial chaetae of Adelodrilus Cook and Inanidrilus Erséus as
structures involved in sperm transfer. Cuadrado and
Martínez-Ansemil (2001) consider their probable participation also
in the holding of the partners. When protracting penes are long and
surrounded by cuticular sheaths (e.g., Limnodrilus Claparède,
Aktedrilus Knöllner), their intrusion far into the spermathecal
ducts might help, not only in the sperm transfer, but also in the
attachment of the mates (Cuadrado and Martínez-Ansemil, 2001).
Whether or not attachment is the primary function, anatomically all
these structures of the male genitalia somehow seem to play a role
in the holding of the partners, especially when they are strongly
curved and the spermathecal pores occupy a lateral or a dorsal
position (e.g., Aktedrilus). However, besides their eventual
participation in sperm transfer, another possible role could be
associated with sperm competition (i.e. the emptying of full
spermathecae from a previous mating—prior to sperm transfer by the
new mate), a phenomenon already known in other taxonomical groups
(e.g., Cordero et al., 2004; Velando et al., 2008).
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Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
4
Some of the coupling systems also seem to be aided by gland
secretions, such as in P. tourenqui (see Cuadrado and
Martínez-Ansemil, 2001).
Embracing. Th e embrace seems to be a common procedure to
achieve and maintain the apposition of male and spermathecal pores
during the sperm transfer in many of the microdriles, especially
those that are devoid of grasping penial chaetae or elaborated
coupling structures. Embrace allows the alignment of male and
spermathecal pores even if the latter have a lateral position and
it can be performed by at least 2 diff erent mechanisms. Th e fi
rst mechanism involves a great fl exibility of the body wall and a
complex muscular system at the genital region, that permit the
protrusion and retraction of diff erent areas to ensure the
apposition of the pores during sperm transfer, as has been
described by Cuadrado and Martínez-Ansemil (2001) for some tubifi
cines. Similarly, it has now been clearly shown for the enchytraeid
Enchytraeus albidus (Figure 2A, B) and presumed for Insulodrilus
sp. (Figure 2C, D).
Th e second mechanism, described by Schmelz (2003) for
enchytraeids in the genus Fridericia Michaelsen, consists of the
eversion of the bursae, embracing the partner as 2 narrow claspers,
thus allowing the contact of male and lateral spermathecal pores
during mating. According to Schmelz (2003), most of the glandular
secretions of the male copulatory organs are probably adhesive
slimes. Figure 2E-G illustrates this embrace system.
According to Cuadrado and Martínez-Ansemil (2001), the typical
gutter-shaped spermathecal chaetae (Figure 1D), present in many
aquatic microdriles, would not participate directly in the
attachment and sperm transfer, but would act as piercing chaetae
involved in some mechanical or chemical stimulation during sperm
transfer. If some mechanical stimulation occurs, the secretions of
the large glands associated with these genital chaetae would serve
to fi rmly attach the partners while they protract and retract.
Until biochemical and physiological studies are performed, the
exact
A
100 m
sp
20 m
C
10 m
D
100 m
B
pc pc
mp
100
m
E
p
sp sp
ab
Figure 1. Mating structures. (A-C) Peristodrilus montanus: A,
anchorage bridge; B, male pores and penial chaetae; C, detail of
the penial chaetae and the ciliature. (D) Protuberodrilus
tourenqui: genital chaeta. (E) Stylodrilus heringianus: general
view of the genital region. ab, anchorage bridge; mp, male pore; p,
penis; pc, penial chaetae; sp, spermathecal pore.
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C. CARAMELO, E. MARTÍNEZ-ANSEMIL
5
role of these structures remains unknown. Th us, Koene et al.
(2005) proposed that the chaetal glands in earthworms may produce
an allohormone that manipulates the reproductive physiology of the
mating partner.
Ciliate sense receptors (Figure 3)A summary of the typology and
distribution
of the external ciliated sense receptors according to Yáñez et
al. (2006) and Caramelo and Martínez-Ansemil (2010) is provided
below. Th e only novelties
E
co
bs
gb gb
vd
mp
ag
10 m
100 m
mp
fp
A
2 m
F
10 m
D
20 m
C
mp
100 m mb G
B
Figure 2. Mating structures. (A, B) Enchytraeus albidus: A,
general view of the genital region; B, detail of a male pore. (C,
D) Insulodrilus sp.: C, general view of the genital region; D,
detail of a male pore. (E, F) Fridericia monochaeta: E, general
view of genital region; F, spermathecal pore and intersegmental
furrow. (G) Fridericia: schematic drawing of the male copulatory
organs (modifi ed from Schmelz, 2003). ag, area glareosa; bs,
bursal slit; co, copulatory organs; fp, female pore; gb; glandular
body; mb, medial side of bursa; mp, male pore; vd, vas
deferens.
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Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
6
are some micrographs helping in the interpretation of the diff
erent categories and patterns of distribution proposed by the
aforementioned authors.
TypologyTh e observations by SEM allow identifi cation of
diff erent types of receptors according to the shape of the
cilia and to the shape of the receptors.
Types according to the shape of the ciliaReceptors of blunt
cilia, generally multi-ciliated
and shorter than 6 μm long (Figure 3A-C);Receptors of sharp
cilia, with 1-5 cilia more than 6
μm long (mostly 8-14 μm long) (Figure 3E);Composed receptors,
with a variable number of
blunt and sharp cilia together (Figure 3D, F).Both blunt and
sharp cilia are present in all
microdriles studied, with the only exception in the amphibiotic
family Enchytraeidae. Out of the 8 species studied in this family
(Römbke and Schmidt, 1999; Caramelo and Martínez-Ansemil, 2010),
only Cognettia sphagnetorum seems to have sharp cilia in addition
to the characteristic receptors of very short blunt cilia of this
family.
Types according to the shape of the receptorPoorly defi ned
(unelevated) (Figure 3A, E, F, I, J)Sensory buds (epithelial bumps)
(Figure 3B, C, G,
H) Papillae (Figure 3D)Th e most common type of sense receptor
in
microdriles is the poorly defi ned one. Sensory buds are common
among the Enchytraeidae. Ciliate papillae appear to be restricted
to the chaetal segments of a few species of Naididae. Th e
characteristic multi-ciliated receptors of blunt cilia of the
enchytraeids deserve special mention (Figure 3C). Papillae are
always composed receptors, with the sharp cilia occupying the inner
part. Many sensory buds of Eiseniella tetraedra, the most common
lumbricid found in freshwaters, are also composed receptors, but
here the sharp cilia are located at the periphery of the organ
(Figure 3F).
Patterns of distributionExternal ciliate sense receptors are
scattered on
the prostomium (including proboscis when present), on the
peristomium and pygidium, and arranged in
a transversal chaetal row and scattered or organized in other
transversal rows in each chaetal segment (Figure 3G-I). Ciliate
sense receptors persist in the clitellar region at maturity and are
present in budding and regeneration zones (Figure 3J-M). Th e
density of ciliate sense receptors is greater at the anterior
region (especially on the prostomium and peristomium), less in the
subsequent chaetal segments, and increases again in the posterior
segments.
Except in the case of the composed receptors, the aquatic
families and subfamilies of microdriles show the same types of
ciliated sense receptors and with a similar distribution along the
body. However, a longer size in blunt and sharp cilia and a smaller
number of cilia in the multi-ciliated receptors of blunt cilia
could be related to a more epibenthic way of life (see Caramelo and
Martínez-Ansemil, 2010, Table 1). Th e number of cilia per
receptor, at least in the anterior body region, is generally
greater in the amphibiotic family Enchytraeidae and especially in
the lumbricid E. tetraedra. Higher numbers have been observed in
terrestrial megadriles (see Aros et al., 1978; Moment and Johnson,
1979).
Although diff erent kinds of cilia and ciliate sense receptors
are presumed to play diff erent functions, additional
ultrastructural and physiological data are needed to accurately
assign a role to a particular kind of sense receptor. Th e limited
information available to date generally suggests that the
penetrative uniciliate sensory cells act as mechanoreceptors and
the multiciliate sensory cells with emergent cilia act as
chemoreceptors (see Welsch et al., 1984). Nonetheless, penetrative
multiciliate sense receptors can be produced by uniciliated and/or
multiciliated sensory cells. According to Yáñez et al. (2006), the
distribution in rows of most of the multiciliated receptors of
blunt cilia at the chaetal segments could be related to a tactile
function in relation to locomotion, whereas the presence of
receptors with long cilia in freshwater families of microdriles
together with their absence in earthworms could indicate that they
act as rheoreceptors. Th e recent observation of long sharp cilia
in aquatic or semiaquatic worms of terrestrial origin (E. tetraedra
and probably also C. sphagnetorum) is interpreted by Caramelo and
Martínez-Ansemil (2010) as the expression of an ancient
character.
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C. CARAMELO, E. MARTÍNEZ-ANSEMIL
7
C 2 m
E
I
20 m
dp
2 m
A
20 m
G
2 m
D 2 m
F 20 m
H
2 m
B
50 m
L
2 m
J 5 m
ec M K
20 m
2 µm
Figure 3. Ciliate sense receptors. (A) Scarcely defi ned sense
receptor of blunt cilia: Lumbriculus variegatus; (B, C) Sensory
buds: B, Protuberodrilus tourenqui; C, Lumbricillus sp.; (D)
Papilla: Ophidonais serpentina; (E) Sense receptor of sharp cilia:
Insulodrilus sp.; (F) Composed receptor: Eiseniella tetraedra; (G).
Sense receptors on prostomium and peristomium: Pristina longiseta;
(H) Sense receptors on pygidium: Enchytraeus albidus; (I)
Transversal chaetal row: Peristodrilus montanus; (J) Cilia at the
clitellum: Stylodrilus heringianus; (K) Sense receptors at a
budding zone: P. longiseta; (L, M) Sense receptors on a
regeneration zone: Lumbriculus variegatus, L, general view; M,
detail of sense receptors. dp, dorsal pore; ec, emerging
chaeta.
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Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
8
Somatic chaetae (Figure 4)Somatic chaetae are generally used to
identify
microdriles at the family level, and sometimes also at the
subfamily, generic, or specifi c levels. Th us, the type of the
chaetae, their particular shape, and number in dorsal and ventral
bundles are always included in the description of oligochaete taxa.
Most of the information about the somatic chaetae in microdriles
has been provided by optical microscopy. Our observations by SEM,
combined with the available literature, reveal particular traits
that we briefl y summarize and discuss in the following 3 sections
devoted to hair chaetae, crotchets, and needles. Th e presence or
absence of denticulations in hair chaetae and the presence or
absence of intermediate teeth in those chaetae currently considered
to be bifi d crotchets are the most relevant questions.
Hair chaetae (Figure 4A-H)Hair chaetae are present in many
aquatic
oligochaetes, commonly in dorsal bundles and, rarely, in ventral
ones (Capilloventridae, Parvidrilidae). Traditionally, the hair
chaetae of microdriles were reported to be smooth; thus, with only
a few exceptions, the mentions of denticulations in hair chaetae
were limited to the Naidinae (Ripistes Dujardin, Stylaria Lamarck,
and Vejdovskyella Michaelsen), and Pristininae (Pristina
Ehrenberg), but even in these groups (the formerly family Naididae)
smooth hair chaetae were considered the general rule (Sperber,
1948).
A limited number of studies have dealt with the observation of
hair chaetae by means of SEM, but, interestingly, they generally
report the observation of denticulations in hair chaetae of taxa
that were formerly considered devoid (e.g., Smith, 1985; Chapman
and Brinkhurst, 1986; Grimm, 1987; Ohtaka, 1995). We have also
found some kind of denticulations (Figure 4A-H) in at least some
chaetae in most of the genera studied (Nais Müller, Stylaria,
Vejdovskyella, Pristina, Peristodrilus Baker and Brinkhurst,
Krenedrilus, Potamothrix Vejdovský and Mrázek, Psammoryctides
Hrabĕ, Spirosperma Randolph, and Parvidrilus Erséus). Although the
treatment of the samples might sometimes infl uence the appearance
of denticulations, it seems now that probably all hair chaetae have
at least the potential to develop denticulations according to a
more or less
defi ned pattern (Figure 4A-H). Th us, the view of the hair
chaetae as generally smooth structures must be challenged by that
of hair chaetae as structures at least potentially provided with
some kind of denticulations.
Crotchets (Figure 4K-O)A number of more recent references have
reported
the presence of intermediate teeth in chaetae that historically
were considered bifi d (mostly ventral crotchets) (e.g.,
Brinkhurst, 1963; Brinkhurst and Jamieson 1971; Smith, 1985;
Martínez-Ansemil and Giani, 1986; Chapman and Brinkhurst, 1987;
Grimm, 1987). Similarly, our observations (Figure 4K-O) confi rm
that pectination is more widespread than had been previously
thought. Currently, pectination in bifi d chaetae has already been
documented in at least one species in each of the following genera:
Nais, Dero Oken, Branchiura Beddard, Peristodrilus, Rhyacodrilus
Bretscher, Potamothrix, Psammoryctides, Tubifex Lamarck, and
Varichaetadrilus Brinkhurst and Kathman.
In most cases, the presence of intermediate teeth in typical
bifi d chaetae is not characteristic of a particular species but
rather appears to occur under certain environmental conditions (see
Chapman and Brinkhurst, 1987). In our opinion, the development of
intermediate teeth in typical bifi d crotchets reveals a certain
degree of plasticity in the phenotypic expression of the
chaetoblasts, particularly in the transition between typical bifi d
crotchets and typical pectinate chaetae when following the
evolutionary models shown in Brinkhurst (1984).
Needles (Figure 4H, I)Needles are acicular dorsal chaetae whose
bifi d
or simple-pointed condition is sometimes diffi cult to
ascertain. Yet, when observed using SEM, minute teeth can show
remarkable diff erences (Figure 4H, I).
Some variability in the shape of the chaetae was generally
accepted in the literature, but aft er the insightful study by
Loden and Harman (1980) “Ecophenotypic variation in setae of
Naididae (Oligochaeta)”, the shape of the chaetae as a valid
criterion to distinguish species from one another was seriously
questioned. Furthermore, the environment infl uences not only the
shape but also the presence and number of a particular kind of
chaetae as has
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C. CARAMELO, E. MARTÍNEZ-ANSEMIL
9
been demonstrated by Chapman and Brinkhurst (1987) in their work
eloquently entitled “Hair today, gone tomorrow: induced chaetal
variation in tubifi cid oligochaetes”. Th e SEM could help to
reveal
some morphological diff erences between taxa, as could be the
case of diff erent types of denticulations in hair chaetae.
However, due to the high plasticity and homoplasy concerning the
expression and the
1 m
A D
2 m1 m
B C
5 m 1 m
E
5 m
F2 m
J
2 m
K1 m
M
N
1 m
O 2 m2 m
L
5 m
H
2 m
I
2 m
G
Figure 4. Somatic chaetae. (A-G) Denticulations on hair chaetae:
A-B, Pristina aequiseta; C-D, Pristina longiseta; E, Nais elinguis;
F, Vejdovskyella comata; G, Peristodrilus montanus. (H, I). Bifi d
needle chaetae: H, V. comata; I, P. longiseta. (J) Bifi d hair
chaeta: Stylaria lacustris. (K-O) Pectination on crotchets: K,
P.montanus, ventral crotchet; L, Branchiura sowerbyi, dorsal
crotchet; M-N, Rhyacodrilus falciformis ventral crotchets; O, Nais
alpina ventral crotchets.
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Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
10
evolution of the chaetae among microdriles, SEM would probably
be a more useful tool to detect abnormalities produced by
environmental stresses as shown by Milbrink (1983) than to reveal
phylogenetic relationships.
Dorsal pores (Figures 3I and 5)In many oligochaetes, the
coelomic cavity
communicates with the exterior through small pores located in
the mid-dorsal line of the body. Th ese unpaired structures are
named dorsal or coelomic pores. Until now, dorsal pores were
generally considered present only in earthworms (megadriles) and
the enchytraeids of the genus Fridericia (Stephenson, 1930;
Brinkhurst and Jamieson, 1971; Bouché, 1972; Schmelz, 2003).
Nevertheless, the presence of dorsal pores had already been
reported in 2 aquatic species of microdriles: the rhyacodrilines
Monopylephorus auklandicus (Benham, 1909) and Bothrioneurum
vejdovskyanum (see Chekanovskaya, 1962; Timm, 1979).
Th e observation of species belonging to the families
Enchytraeidae, Naididae, Lumbriculidae, Lumbricidae, Phreodrilidae,
and Parvidrilidae revealed the presence of dorsal pores—not only in
taxa where they should be expected (the lumbricid Eiseniella
tetraedra and the enchytraeid Fridericia monochaeta) (Figure 5A-D)
but also in some species of typical freshwater microdriles.
Although not all the material was observed in dorsal view, it must
be pointed out that the only freshwater microdriles where we have
found dorsal pores are the rhyacodrilines B. vejdovskyanum,
Peristodrilus montanus, and Protuberodrilus tourenqui. Th e study
of a poorly preserved specimen of Parvidrilus spelaeus
(Parvidrilidae) also revealed the presence of pores in the
mid-dorsal line of the body, but these are probably not truly
dorsal (coelomic) ones but the pores of the mid-dorsal glandular
pouches characteristic of the family (see Martínez-Ansemil et al.,
2002). Th e distribution and diameter of the dorsal pores that we
observed in the 3 species of rhyacodrilines are as follows:
Bothrioneurum vejdovskyanum. (Figure 5G-I). Dorsal pores present
from 3/4 to the posterior end. Diameter, 8-13 μm.
Peristodrilus montanus. (Figure 3I, Figure 5J, K). Dorsal pores
observed only at 3/4 and 4/5. Diameter, 13-20 μm.
Protuberodrilus tourenqui. (Figure 5E, F). Dorsal pores present
from 5/6 to at least 7/8. Diameter, 8-12 μm.
SEM proves to be a useful tool for the observation of dorsal
pores in microdriles. Interestingly, all 4 aquatic species of
microdriles showing dorsal pores belong to the Rhyacodrilinae, an
ancient subfamily of naidids. Historically, it has been accepted
that the functions of dorsal pores were connected with their
presence in species from terrestrial habitats and their absence in
aquatic species (i.e. protection against desiccation, keeping the
surface wall moist for respiratory exchanges, protection against
bacteria and parasites) (see Stephenson, 1930). Before introducing
new biological hypotheses related to the presence of dorsal pores
in aquatic microdriles, we think it appropriate to investigate
their presence in other microdriles, especially other rhyacodriline
genera and their closest relatives. It is worth highlighting the
diff erent number and distribution of dorsal pores in the 4 species
of rhyacodrilines, and that the fi rst dorsal pore is located in a
more anterior segment than had been reported previously in
megadriles and Fridericia.
Other structures (Figure 6)In addition to the structures
described above,
we have occasionally observed some structures that could also
provide additional information from a taxonomical or a biological
perspective if they were systematically observed. Th us, it is
noteworthy that the accurate position and diameter of the
nephridial pores is easily observed by SEM and could be informative
(Figures 6A, B). Furthermore, it is interesting to note the
porosity observed at the body wall (e.g., Lumbriculus variegatus:
Figures 6C, D). Finally, we note the observation of female marks
(depressions in the body wall) with diff erent shapes in mature
(well developed male pores and clitellum) enchytraeids (Fridericia
monochaeta) (Figures 6E, F) and naidids (Limnodrilus udekemianus)
(Figure 6G); these depressions correspond with the position of the
female pores. Perhaps the shape of female marks is highly variable
and consequently not very useful as a taxonomical character (e.g.,
comparison of the
-
C. CARAMELO, E. MARTÍNEZ-ANSEMIL
11
diff erent shape of the 2 marks of the same individual in L.
udekemianus: Figure 6H, I). Regardless, these female marks—devoid
of female pores when male
pores and clitellum are well developed—provide us with insight
into how protandric worms likely avoid self-fertilization.
H
5 m
E6/7
7/8
50 m
A 500 m
B
20 m
C
30 m
D
5 m
G
100 m J
200 m
K
10 m
I
10 m
6/7
F
10 m
Figure 5. Dorsal pores. (A, B) Eiseniella tetraedra: A, dorsal
pores at anterior body region; B, detail of a pore. (C, D)
Fridericia monochaeta: C, pore at 6/7; D, detail. (E, F)
Protuberodrilus tourenqui: E, pores at 6/7 and 7/8; F, detail.
(G-I) Bothrioneurum vejdovskyanum: G, dorsal pores at anterior body
region; H, detail of the pore 37/38; I, coelomocytes fl owing
through a pore. (J, K) Peristodrilus montanus: J, pores at 3/4 and
4/5; K, detail of the pore 4/5.
-
Morphological investigations of microdrile oligochaetes
(Annelida, Clitellata) using scanning electron microscopy
12
ConclusionSEM is an invaluable tool that has increased the
knowledge of the functional anatomy on microdriles,
and has resulted in the broader understanding and discovery of
several important structures. However, additional studies and
observations using SEM are still
F 5 m
G
mpfm
50 m H 10 m I 10 m
E
co
fm
50 m
B 5 m
C
50 m
D
2 m
A
50 m
Figure 6. Nephridial pores. (A, B) Lumbricillus sp.: A,
nephridial pores at anterior body region; B, detail of the
nephridial pore of VII. Epidermal pores. (C, D) Lumbriculus
variegatus in regeneration: C, general view; D, detail. Female
marks. (E, F) Fridericia monochaeta: E, general view of the genital
region; F, detail of a female mark. (G-I) Limnodrilus udekemianus:
G, general view of the genital region; H-I, detail of the 2 female
marks from the same individual. co, copulatory organ; fm, female
mark; mp, male pore.
-
C. CARAMELO, E. MARTÍNEZ-ANSEMIL
13
needed, especially with regard to mating structures.
Furthermore, the results obtained during this study encourage us to
pursue new investigations using other techniques (e.g., CLSM, TEM,
microscopic sections) in order to broaden our knowledge on the
muscular complex and glands involved in the diff erent mating
systems, the relationships between the ciliate sense receptors and
the nervous system, and the muscular and nervous systems that
control the opening of the dorsal pores.
AcknowledgementsTh is work was supported by the Spanish
Ministry
of Education and Science and FEDER (Project No. CGL.2006-13417).
Th e authors wish to thank R. Schmelz for providing enchytraeids
and phreodrilids, S. Cuadrado for his scientifi c collaboration, M.
Quintela for proofreading, and Mark J. Wetzel for valuable
scientifi c comments and revising the text. Two anonymous reviewers
improved the quality of the paper.
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