Micromastigotes scottae sp. nov. (Parabasalida: Spirotrichonymphina): a new twist on the hypermastigont condition

2008·4.2 MB·English

by  Stephen L Cameron

MICROMASTIGOTESSCOTTAES?. NOV. (PARABASALIDA:
SPIROTRICHONYMPHINA): A NEW TWIST ON
THE HYPERMASTIGONT CONDITION.
STEPHEN L. CAMERON, CORALYN D. TURNER AND PETER J. O^DONOGHUE
Cameron, S.L., Turner, C.D. & O'Donoghue, P.J. 2008 04 30: Micromastigotes scottae
sp. nov. (Parabasalida: Spirotrichonymphina): A New Twist on the Hypermastigida.
Memoirs of the Queensland Museum 52(2): 127-138. Brisbane. ISSN 0079-8835.
This study redescribes the genus Micromastigotes, Hollande and Carruette-Valentin,
1971, a parabasalid flagellate symbiotic in termites, on the basis of light and electron
microscopy and erects a new species, M. scottae. The genus Micromastigotes is characterised
by possessing flagella bands which spiral around the anterior portion of the cell, the location
of the nucleus and Golgi bodies at the base of the anterior flagellated area, and an axostyle
which runs the length of the cell. Indi\ iduai flagella are derived from the central axis of the
ceil exiting perpendicularly, each is offset from the preceding flagellum by 12 — 18® thus
forming a spiral band. Ultrastructurally, the flagella bases form a structure resembling a spiral
staircase. Peltoaxostylar and prcaxostylar fibres arise from the anterior most kinetosomes and
the striated lamina forms a weakly undulating sheet directed posteriorly from each flagellum.
Dense lamina and parabasal fibres arc absent. Micromastigotes was originally classified
as part of the Spirotrichonymphina and there are some similarities to other genera in this
group, but Micromastigotes lacks a flagellar gutter, a U-shaped band at the base of the flagella
composed of the striated and dense lamina, which is diagnostic of the spirotrichonymphines.
The spiralisation pattern in Miewmastigotes is not consistent with previous schemes for
the development of a polymastigont condition in spirotrichonymphines suggesting that
Micromastigotes may represent an independent derivation of a polymastigont condition
from a trichomonad-like ancestor.n Parabasalia, Spuvtrichonymphina, Micromastigotes,
Privileged-Flagella Hypothesis, Schedorhinoiermes.
Stephen L Cameron, Australian National Insect Collection <& CSIRO Entomology’, PO Box
1700 Canberra. ACT, 2601 (email: [email protected]): Coralyn D. Turner &
Peter J. O’Donoghue. Department of Microbiology’and Parasitology, The University of
Queensland, Brisbane, QLD, 4072; 26 November 2007.
The flagellate phylum Parabasalida includes polyphyletic collection of groups which have
the most diverse ranges of cell structures of any independently adopted a multiflagellated or poly-
protist group. Most species occur as anaerobic mastigont condition (Brugerolle & Patterson,
symbionts in a range of hosts including 2001). Even though molecular phylogenies
mammals, reptiles and birds but the greatest of the parabasalids consistently recover the
diversity of species occurs within tennites and polymastigont Trichonymphidae as the earliest
wood-eating cockroaches (Yamin, 1979). Much diverging branch of the parabasalid tree, this result
of this diversification has been associated with is almost certainly artefactual and it should not
increases in cell size and in the complexity of the be interpreted that the ancestral parabasalid was
flagellar structures and their associated fibrous polymastigont (Hampl et al., 2004).
support organelles. The simplest parabasalids,
Recent molecular phylogenies of the parabas¬
the Trichomonadida have 3 anterior flagella and
alids (Gerbod et al., 2001, 2002; Hampl et al.,
a recurrent flagellum, and this arrangement, 3
2004; Keeling, 2002; Okhuma et al., 2000,2005)
+ R, forms the basis of the ‘privileged flagella
have suggested that there have been up to five
hypothesis'which considers that the more complex
acquisitions of the polymastigont condition,
arrangements are all derived stales arising by
addition and elaboration of one or more of the six if Calonymphidae is polyphyletic (Gerbod
‘privileged’ elements (Brugerolle, 1991). The el al., 2002) (Fig. I). Four groups have been
more complex parabasalids usually have vastly traditionally united as the Order Hypermastigia:
greater numbers of flagella and have been the Trichonymphidae, Eucomonymphidae
collectively classified as the Hypermastigida and Staurojoeninidae (collectively the Sub¬
although this taxon is almost certainly a order Trichonymphina); the Suborder
128 MEMOIRS OF THE QUEENSLAND MUSEUM
Monocercomonadidae (Free Living)
Trichomonadinae
Trichomonadid
Monocercomonas (Symbiotic)
Dientamoeba + Histomonas + Tritrichomonas
Calonympha + Snyderella
Metadevescovina + Joenina
Cristamonadida
Devescovina + Metadevescovina
Coronympha + Metacoronympha
Foaina
Spirotrichonymphidae Spirotrichonymphida
Hypotrichomonas + Trichomitus Trichomonadida
<s> Eucomonymphidae + Hoplonymphidae
Trichonymphida
<i> Trichonymphidae + Staurojoeninidae
FIG. 1. Phylogeny of the Parabasalia and acquisitions of a multimastigont condition, redrawn after Gerbod et
al., (2002) & Ohkuma et al. (2005). I, Calonymphid type: replication of a karyomastigont; 2, Lophomonad
t^e: replication of flagellum 1 into an anterior flagella plate; 3, Spirotrichonymphid type: spiral flagella
bands, flagellar gutter surrounding the kinetosome base; 4, Eucomonymphid type: slightly spiralised
flagella bands, each kinetosome base connected to a striated, sinusoidal root; 5, Trichonymphid type:
meridional flagella bands, derived from a complex anterior rostrum.
Spirolrichonymphina (Spirotrichonymphidae) parallel evolution (Dolan et al., 2000a, 2000b;
and the Suborder Lophomonadina. The fiftli group, Dolan & Kirby, 2002). The lophomonads also
the Calonymphidae, has long been classified within retain the privileged flagella and the polymastigont
the Trichomonadida but has recently been grouped condition is achieved by vast replication of flag¬
with the devescovinids and lophomonads as the ellum 1 into an anterior flagella plate (Brugerolle
Order Cristamonadida on the basis of molecular & Patterson, 2001; Brugerolle & Bordereau,
phylogenies and reassessment of morphological 2003). More complex arrangements which are
structures (Brugerolle & Patterson, 2001). Each less readily interpreted in terms of modification
of these five acquisitions of the polymastigont of the privileged flagella occur in the other three
condition has been accompanied by a distinctive groups. Eucomonymphids are characterised by
subcellular architecture of tubules and fibres to their flagella being grouped into slightly spiral¬
support the profusion of locomotory flagella. The ised, longitudinal rows and each flagellum
simplest system is seen in the calonymphids where possesses a striated, sinusoidal microtubular
polymastigery is achieved by simple multiplication root which was lenned the parabasal filament by
of the privileged karyomastigont, the privileged Hollande & Carruette-Valentin (1971) although
flagella plus associated nucleus (Dolan et ah, its relationship to the parabasal filament
2000a, 2000b). The simplicity of this structure of trichomonads is unclear (cf. Brugerolle,
and its’ clear relation to flagella structure in the 1999, 2000; Cameron & O’Donoghuc, 2003).
monomastigont devescovinids is what has lead to Trichonymphids have a very complex
the grouping of calonymphids initially within anterior rostrum from which meridional lines
Trichomonadida (Brugerolle & Lee, 2000) of flagella arise, the rostrum itself is a very
and more recently within Cristamonadida complex structure with multiple interacting
(Brugerolle & Patterson, 2001). Even if the layers of fibrils (Hollande & Carruette-
calonymphids are polyphyletic as suggested Valentin, 1971). Spirotrichonymphina, as the
by Gerbod et al. (2002), the two calonymphid name suggests, have spiral rows of flagella
groups have both achieved the polymastigont radiating from the anterior end, each row has
condition in the same way, by replication of the as its base a structure termed the ‘gutter of the
karyomastigont, and constitute an example of flagella band’ by Brugerolle (2001) composed
MICROMASTIGOTES SCOTTAE SR NOV. 129
FIG. 2. Morphology of Micwmasligotes scottae sp, nov. A, Line diagram. B, Scanning electron micrograph.
C, Light micrograph of protargol stained specimen. Scale bars = 10 pm. Ax, axostyle; FB, flagella band;
GB: Golgi body; N, nucleus.
of outer dense lamina and an inner striated pers observ.). Micromastigotes apparently has
lamina. The flagellar gutter thus has the fonn of a the simplest morphology of all; the flagella are
U-shaped ribbon spiraling around the cell close apparently derived directly from a central core,
to the base of the kinetosomes. This arrangement axial within the cell and in the description by
occurs in the widespread and speciose genera of Hollande & Carruette-Valentin (1971), lacking
spirolrichonymphines, Spirotrichonynipha and both striated roots and the striated lamina. The
Holomasfigotoides (Brugerolle, 2001; Lingle possibility that Micromastigotes represents the
& Salisbury, 1995) but variations occur in the simplest spirotrichonymphine led us to undertake
simpler genera Microjoenia, SpiwmcbonymphcUa a complete redescription of the genus and a
and Micromastigotes Hollande 8l Carruette- detailed study of its flagella structures following
Valentin (Brugerolle, 2001; Hollande & Carruette- the discovery of a new species of this genus
Valentin, 1971). In Microjoenia, the flagellar in the northern Australian pest termite species
gutter is poorly formed, the dense and striated Schedorhinotermes intermedins.
lamina being frequently separated and not
forming a continuous ribbon connecting adjacent MATERIALS AND METHODS
kinetosomes (Brugerolle, 2001). The flagella of
Spirothchonymphella are derived from a central Thirteen colonics of Schedorhinotermes
core or columella, each flagellum is connected intermedins were collected from southeastern
to the columnella by a pair of striated roots, Queensland (QLD) Australia, from Joyner (3
internal to the striated roots is a striated lamina colonies), Deception Bay (I), Samsonvale
which spirals downward to follow the path (3), Samford (4), Ferny Hills (I) and Moggill
of the flagella band (Brugerolle, 2001; SLC Creek (1). Colonies were collected from under
130 MEMOIRS OF THE QUEENSLAND MUSEUM
FIG. 3. Ultrastmcture of Micwmastigotes scottae sp. nov. A, Longitudinal section, whole cell; B-D, Serial
longitudinal sections through a single cell from axial plane (6) to peripheral plane (8); E-F, Anterior
mastigont system. Scale bars: 5: 2 pm; 6-8: 500 nm; 9: 1 pm; 10: 500 nm. Ax: Axostyle; B: Bacteria; FB:
Flagella bands; GB: Golgi body; Pr: Preaxoslyle; Pt: Peltoaxostyle; SL: Striated Lamina.
fallen timber, within dead fallen branches ethanol which were used to identify the termite
and from within tubular galleries within species collected. Nest material was collected
the bark of living trees. Individual termites along with termites and each colony was provided
representing the worker, major soldier and with tissue paper soaked in water as a moisture
minor soldier castes were collected from each and food source to maintain the colony in the
laboratory. Workers were examined shortly
colony. Voucher collections were made of each
after collection by dissecting the hindgut
colony by preserving five of each caste in 70%
MICROMASTIGOTES SCOTTAE SR NOV. 131
into a small drop of invertebrate saline (0.6% solutions (5%, 10%, 15%, 20%, 25%, 30%, 40%,
NaCI). Some workers were removed directly 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%)
from the nest and examined, these are referred for 10 min each. Cells were gradually infiltrated
to as ‘dirty’ specimens. ‘Cleaned’ specimens with Epon resin (25%, 50%, 75% Epon in 100%
were generated by isolating individual workers acetone for 1 hour each, 100% Epon overnight)
from nest material and then rearing them on and embedded in fresh 100% Epon, pelleted
water-soaked tissue paper for several days to by gentle centrifugation and cured for 1 day at
purge them of dirt and coarse wood fibres in 60"C. Semi-thin survey sections were cut with
their guts. glass knives, stained with 1% loluidine blue and
used to orientate sections. Ultra-thin sections (70
Light microscopic observations were per¬
nm and 90 nm) were cut with diamond knives,
formed on Giemsa-stained and protargol-
mounted on formvar-coated copper slot grids,
impregnated specimens. Giemsa staining was
stained with 5% uranyl acetate in 50% methanol
performed on partially air-dried smears fixed
for 2 min., washed in distilled water for 30 sec.
with methanol. Slides were examined without
and dried. The sections were then counter stained
coverslips by bright-field microscopy under
with Reynold’s lead solution (2% lead citrate)
immersion oil. Specimens from both ‘cleaned’
for 1 min., washed in distilled water for 30 sec.
and ‘dirty’ termites were prepared for light
and dried prior to examination. Sections were
microscopy to determine if cleaning caused
examined in a JEOL 1010 transmission electron
artefactual changes to the flagellates. Protargol
impregnation was perfonned according to the microscope.
method of Foissner (1991) on specimens fixed
RESULTS
with Schaudin’s fluid. Cells were drawn using a
camera lucida, and measured in each dimension
The soldiers and workers from 5 of the 13
using a calibrated eye-piece micrometer.
colonies (38%) were found to harbour a small
Measurements are presented as a range of values
hypermastigid flagellate belonging to the genus
followed by the average in parentheses.
Micromastigotes in addition to other parabasalid
Specimens for electron microscopy were coll¬ flagellate species. This Micromastigotes species
ected exclusively from ‘cleaned’ termites dissected appeared to be novel and its morphology and
into Locke’s fluid (composition in mM: 136 NaCl, ultrastructure are described below.
5.6 KCl, 1.2 MgCI2, 2.2 CaC12, 1.2 NaH2P04,
14.3 NaHC03 and 10 dextrose, final pH 7.3- Micromastigotes scottae sp. nov.
7.4) and fixed in a vast excess, typically at least
10 volumes, of 3% glutaraldehyde in 0.066M TYPE HOST. Schedorhinotermes intermedins (Isoptera:
Rhinotermitidae)
cacodylate buffer (pH 7.2) for 30 min. Cells
for scanning electron microscopy were washed HABITAT. Termite hindgut
in O.IM cacodylate buffer for 1 hour, post-
TYPE LOCALITY, by Moggill Creek, Brisbane, QLD
fi.xed with 1% osmium tetroxide in 1.5% pota¬
{2T3TS \52°5TE)
ssium ferricyanide for 1 hour, washed three times
in distilled water and stored in 70% ethanol. OTHER LOCALITIES. Joyner, Pine Rivers, QLD
Specimens were dehydrated in a graded series of (27®17'S 152°56'E); Ferny Hills, Pine Rivers, QLD
ethanol solutions (80%, 90%, 100%, 100?/o) for (27°23'S I52°56'E)
10 min each. Samples were critical point dried
TYPE MATERIAL. Holotype deposited with the
in C02 between 8nm polycarbonate filters in a Queensland Museum (Brisbane, Australia), accession
Millipore Swinnex filter holder. Dried samples number: G463727.
were mounted on stubs with double sided tape,
DESCRIPTION. (Fig. 2A-C). Body elongate;
sputter coated with platinum and examined in
rounded anterior rostrum grades into an ovoid
a JOEL 6400 scanning electron microscope.
For transmission electron microscopy, fixed mid-body, tapers posteriorly into a long tail;
samples from several termites were pooled and 16-40 (28) pm long by 6.4-13 (9) pm wide;
washed three times in Soerenson’s phosphate shape index (length to width ratio) 2.3-4.4 (3.1).
buffer (pH 6.8) for 30 min each. Cells were Anterior rostrum 1.6-6.4 (4) pm long, bears 4
post-fixed in 4% osmium tetroxide for 1 hour flagella bands which spiral clockwise around
and washed three times in distilled water (10 the rostrum, approximately 1.5 gyres per band;
min., 10 min. and overnight). Specimens were flagella 8-16 (9.8) pm long, not adherent to the
then dehydrated in a graded series of acetone cell, confined to the rostrum giving the cell an
132 MEMOIRS OF THE QUEENSLAND MUSEUM
anterior tuft-like appearance. Single nucleus, 3D). The axostyle proper runs posteriorly from
spherical 1.6-3.2 (2.1) pm diameter, consistently the base of the nucleus and is composed of a
located centrally in the cell at the level of base rolled sheet of microtubules surrounding a low-
of the rostrum, 1.6-6.4 (4.2) pm from anterior density cytoplasm (Fig. 2A). It extends almost to
of cell. Two parabasal bodies, ovoid to spherical the posterior end of the cell but does not project
flank the nucleus, level with base of the rostrum. beyond the cell (Fig. 2A). There are 2 Golgi
Axostyle extends from nucleus to posterior end bodies which flank the nucleus, posterior to the
of cell, slightly curved along its length, non¬ bottom-most flagella; the parabasal fibre which
projecting. connects the Golgi bodies to the kinctosomes is
absent (Fig. 2A). Electron-dense bacteria appear
DIFFERENTIAL DIAGNOSIS. Whilst the
to be scattered randomly within the cell, several
original description of M grassei Hollande are located near the flagellar bands whereas
& Carruelte-Valentin, 1971 is quite lacking in
others occur throughout the body (Figs 3C, F).
detail as to what are the diagnostic features of
Food-vacuoles are mostly present within the
the species, the accompanying photos clearly
posterior ‘tail-like’ portion of the cell and do not
show that it is a squat species almost oval in
appear to contain whole wood fibres (Fig. 2A).
outline with a short, narrow posterior tail. In
contrast M. scottae is elongate, the cell being REVISED GENERIC DIAGNOSIS. Micro-
approximately 3 times as long as it is wide, mastigotes Hollande & Camiette-Valentin, 1971.
and has a posterior tail almost as wide as the Polymastigont flagellates with flagella arranged
anterior portion of the cell. Additionally, in A/. into bands which spiral around an anterior rostrum
grassei the flagellar bands extend posterior to in a clockwise direction. Flagella bands arc derived
the nucleus, are connected to parabasal bodies from the centre of the cell and radiate like a spiral
and individual flagella tire adherent in the proximal stair-case. A flagellar gutter connecting individual
portion. None of these features occur in M. scottae. flagella in each band is absent. Axostyle present,
derived from the striated lamina which extends
ETYMOLOGY. M scottae is named in honour of a
from the posterior of each kinetosome. Nucleus
friend whom I (SLC) failed, Kirsten Scott.
and Golgi bodies located immediately posterior to
Ultrastmeture. (Figs 3A-F). Individual flagella the flagella bands.
are derived from a central core which runs
axially within the cell, each flagellum is angled DIFFERENTIAL GENERIC DIAGNOSIS.
approximately 12-18° relative to the previous It is difficult at this time to definitely assign
flagellum in the band (Figs 3B, E, F), one full Microniastigotes to any parabasalid family and
rotation around the cell thus consists of about we propose to leave it as a Spirotrichonymphina
20-30 flagella with increasing numbers of ifisertae sedis pending a better understanding
flagella in the posteriormost bands (Fig. 3F), of relationships amongst these groups, Micro-
each band wraps around the cell about one and mastigotes is most readily confused with
a halftimes and there are 4 independent bands. members of the Spirolrichonymphinae Grassi,
Each flagellum extends out perpendicularly 1917; namely Spirotrichonympha, Microjoenia
or nearly so from the central core, thus more and Spirotrichonymphe/Ia. Microniastigotes is
posteriorly located flagella are surrounded by more readily distinguished from the first two genera
cytoplasm than anterior ones. Each kinetosome by the absence of the flagellar gutter and the
does not appear to be anchored to any particular origin of the flagella in the central axis of the
structure, each is close to a centre tubular structure cell. Microniastigotes is distinguished from
but not connected to it nor intimately associated Spirotrichonympliella by the restriction of the
with the striated lamina. Peltoaxostylar and flagella bands to the anterior portion of the cell; in
preaxoslylar fibres arise from the anterior most Spirotrichonymphella the bands cover the whole
kinetosoines and are directed towards the anterior cell.
membrane of the cell (Fig. 3E). Tlie peltoaxostyle
is external to the preaxostyle and is much longer. DISCUSSION
The dense lamina is absent. A striated lamina is
derived from the bottom edge of the kinctosomes
TAXONOMIC STATUS
and extends posteriorly, approximately paralleling
the course of the flagellar band towards the nucleus The original description of Microniastigotes by
(Figs 3D, E). The striated lamina surrounds the Hollande & Carruette-Valentin (1971) provided
nucleus and gives rise to the axostyle (Fig. a basic description of the species and of what set
MICROMASTIGOTES SCOTTAE SR NOV. 133
this genus apart Irom other parabasalid flagellates. M. scottae; the flagellar bands of M. grassei are
There was no line diagram of the whole cell but accompanied by parabasal bodies whereas there
the series of photographs of silver-stained cells is no connection between the parabasal bodies
and several transmission electron micrographs and flagellar bands of M scottae; there is no
serve to illustrate the most significant features of striated lamina recorded in M grassei but it is
the genus, i.e. flagella arising from an anterior prominent in M scottae and finally the basal
rostrum and their derivation from a central bodies of each flagellar band are connected by
spiral within the core of the cell. The features of electron dense material in M. grassei whereas
Micwmostigotes grassei included: this doesn't appear to be the case in M scottae.
Of these differences the first two, distribution
• spiral flagellar bands arising on an anterior
of the flagellar bands and adherant flagella are
rostrum and which do not forni an anterior
known to vary within parabasalid genera (Lingle
column or converge in the posterior of the
& Salisbury, 1995; Radek, 1997) and so are not
cell;
sufficient to justify erection of a new genus for
• large dictyosomes which are widely M scottae. The published electron micrographs
separated; of M. grassei (Hollande & Carruette-Valentin,
1971 henceforth H & C-V, 1971) make it difficult
• individual flagella are proximally adherant;
to interpret the fonn of the parabasal bodies. The
• nucleus is apical within the cell; longitudinal tEM section of M grassei (Fig. 51, H
& C-V, 1971) depicts only the anterior of the cell
• axostyle resembling a compact rod and and does not extend to the level of the parabasal
projects posteriorly; bodies which flank the nucleus in M. scottae.
The written description of M grassei, however,
• anteriormosl region has a clear cytoplasm,
refers to dictyosomes, the form of which is con¬
without inclusions, fibrils or tubules;
sistent with the bodies found in M scottae.
• flagellar bands, under the cap are enclosed Additionally, in M. grassei there is a long cistern
by morphoplasm and are interconnected of the endoplasmic reticulum which follows
with each other in 2 independent groups. On the flagellar band (Fig. 52a, H & C-V, 1971).
its origin each band has the same structure This feature may have been misinterpreted by
as the those in Spirotrichonympha. An Hollande & Carruette-Valentin (1971) as it
ergoplasmic cistern (reticulum?) runs along occupies the same position as the striated lamina
the flagellar bands and it is between them, in M scottae and the published micrographs
where the dictyosomes are in place. include ribbon like structures labeled in one
figure parabasal lamina (lames parabasales in the
• basal bodies have their cavity filled with
original French) which is simply another term
glycogen and are connected by electron
for the striated lamina (Fig. 51, H & C-V, 1971)
dense material
but labelled endoplasmic reticulum in another
• the anterior basal bodies give rise to a (Fig. 52a H & C-V, 1971). The electron dense
preaxostyle that induces the microtubules material which connects the basal bodies of
of the pelta and the axostyle A'/, grassei doesn’t seem to be present in M.
scottae but the density of this material seems to
• axostyle fibrilles enclose a cytoplasm that
vary considerably between the three published
contains glycogen
micrographs of M. grassei so it is hard to deter¬
• dictyosomes are piled up in a dozen very mine the significance of this feature.
long cisterns. These contain an electron
The key similarity between M. grassei and
dense mass. Frequently there are multiple
M. scottae is the overall structure of the kinety
successive sacks, which contain a dense
system - flagella bands which spiral from a
material at the same level, 6-8 saccules
central core which is oriented axially within the
are present and are associated with each
cell. Furthermore, the absence of any similar
other by a dense substance.
arrangement in other hypermastigids makes
There are, however, differences between M. us confident that these two species belong to the
grassei and M. scottae. The flagellar bands of M same genus. Most of the characters which we have
grassei extend posterior to the nucleus whereas found to vary between these two species are either
in M scottae they are anterior to it; the flagella known to vary between species in other parabasalid
are adherent in M. grassei but non-adherent in genera (i.e. flagellar band length, adherence of
134 MEMOIRS OF THE QUEENSLAND MUSEUM
flagella) or are ditTicuIl to interpret on the basis of flagellar band (Hollandc & Carructte-Valentin,
published electron micrographs alone (parabasal 1971) whereas in each of the Spirotrichonympha
body shape and distribution, striated lamina and species examined the parabasal bodies arc discrete
electron dense connections between the basal ovoid structures composed of a stack of multiple
bodies). Additionally, none of these features are cistemae, and several separate parabasal bodies are
consistently used for generic level discrimination distributed down the length of the flagellar band.
within parabasalid flagellates whereas the arrang¬ Spirotrichonympha is, by parabasalid standards,
ement of the flagella structures has proven to be a large genus with 27 recognised species
very valuable and consistent (e.g. Brugerolle, 2001; (Radek, 1997; Yamin, 1979) in two subgenera,
Brugerolle & Lee, 2000). Whilst it is probable so there is clearly scope for variation within
that a thorough reinvestigalion of A/, grassei may this genus. Furthermore, Spirotrichonympha
clarity many of these issues, at present we prefer has recently been shown to be polyphylclic with
to adopt a conservative taxonomic approach, respect to the genus Holomastigotes (Ohkuma
assigning M. scottae to Micromastigotes rather et al, 2005). Detailed ultrastructural studies are
than erect yet another monotypic genus whose lacking for the species for which molecular data is
relationships to other parabasalid flagellates is available and vice versa, precluding resolution of
poorly understood. this matter at the present time, but it seems at least
plausible that Spirotrichonympha must consist of
A complicating factor is Spirothchonympha
at least 2 genera with divergent morphologies -
minor, Radek, 1997. In his revision of the
one represented by S. mirabilis, S. grandis and S.
ultrastructure of the Spirotrichonymphidae,
elongata which possess the features defined by
Brugerolle (2001) suggested that S. minor may be
Brugerolle (2001) and the other represented by S.
a member of Micromastigotes in which case the
minor and defined by the diflerent combination
characteristics of this species must also be taken
of features outlined in Radek (1997). Additional
into account in revision of the latter genus. The
studies will help to clarify their relationships,
description of S. minor by Radek (1997) does
but none of the four Spirotrichonympha species
not include clear evidence for the presence of a
which have been examined ullrastruclurally are
flagellar gutter which may give the impression
sufllciently similar to Miewmastigotes to warrant
of similarity to M. grassei, particularly if one
their inclusion in this genus or the inclusion of
accepts that the striated lamina is absent in A/.
M. scottae in Spirotrichonympha.
grassei. We have demonstrated the presence of
a striated lamina in A/, scottae in the present BIOGEOGRAPHY AND
study and it is possible that it is also present in
HOST ASSOCIATIONS
M. grassei but has been misinterpreted as being
part of the endoplasmic reticulum. Furthermore, The description of a second species of Micro¬
S. minor lacks the arrangement of the llagellar mastigotes raises an interesting issue about
bands which we have found to be diagnostic of the geographic distribution of this genus.
Micromastigotes. With the exception of lacking The type species, A/, grassei, was collected
a flagellar gutter the structure of the flagellar from Postelectrotermes praecox on the island
bands in S. minor is much more similar to that of Madeira, off the Atlantic coast of Africa.
of other Spirotrichonympha spp. in that the Micromastigotes scottae was found at almost the
band is composed of peripheral basal bodies opposite end of the globe, in Schedorhinotermes
which are interlinked by electron dense material intermedins in Queensland, Australia. This seem¬
(Brugerolle, 2001; Radek, 1997). Other shared ingly widely disparate distribution is easier
ultraslructural features include peripheral para¬ to understand when the distribution of the
basal bodies (5. mirabilis, S. grandis, S. elongata), respective hosts is taken into account. While
adherent flagella (5. grandis), and flagella bands both host species have comparatively small
which extend to the posterior of the cell {S. distributions in North Africa and adjacent islands
mirabilis, S. grandis, S. elongata). Of these and northeastern Australia respectively, the
features, peripheral, ovoid parabasal bodies distri¬ genus Postelectrotermes is widely distributed,
buted along the length the flagellar bands are its 13 species spread over Africa, the Middle
particularly important. In A/, grassei, the parabasal East and India, whereas Schedorhinotermes
bodies associated with the flagellar bands are broadly overlaps this distribution, its 36 species
described (if one does not accept our contention stretching from Melanesia, to Australia, through
that this structure is probably the striated lamina) the Indonesian archipelago, Asia and into north
as a single, narrow cistemae which parallels the Africa (Constantino, R. website: http://www.unb.
MICROMASTIGOTES SCOTTAE SR NOV. 135
br/ib/zoo/docenle/constant/catal/catnew.html; permanence of the axostyle in cytokinesis; and
Myles, T. website: hllp;//www.utoronto.ca/forest/ possible mode of nutrition. Unfortunately, most
termite/termite.hlm). Thus, there is considerable of these characteristics appear to be of little
scope for local mixing between one or several value in imposing a familial classification upon
species from each genus possibly resulting in host the spirotrichonymphines. The rostral column,
switching. This, combined with the fact that North as seen in light microscopy, is an artefact of the
African and Asian tenniles have been the least cell shape and thus is plastic even at the species
investigated for their flagellate faunas (Yamin, level. Tlie columella, a defined fibrillar structure
1979), makes it probable that there are undescribed is not always responsible for the appearance
species of Micmmasiigotes occurring through this of a rostral column under light microscopy.
interv'cning area. Flagellar bands are a feature of all genera, but if
restricted to denote those species which possess
Even more interesting than the geographic
a llagellar gutter, this does split Micromastigotes
distribution Micromastigotes is the broad taxon¬ and Spirotrichonymphella and some from the
omic range of the hosts w'hich it infects;
remaining genera. This feature is, however, variable
Postelectrotermes is a kalotermitid whereas within Spirotrichonympha\ flagellar gutters occur
Schedorhiuotermes is a rhinotermitid. These two in three species examined by Brugerolle (2001),
families of tennites have radically different life¬
S. mirabilis^ S. grandis and S. elongata but are
styles. Kalotermitids construct colonies within
absent in S. minor (Radek, 1997). Tlie numbers of
sound dry wood (including living trees) whereas flagellar bands are knowTi to be variable within the
rhinotermitids are subterranean, the main colony genera Spiwtrichonympha and Holomastigotoides
being constructed in fallen timber and foraging (Radek, 1997), and it is thus unlikely to be valuable
trails radiating out under the ground and up above the generic level. As emphasised above, the
trees within tubular galleries made of cemented difl'erenlialion ofthe parabasal (= striated) lamina is
soil (Watson & Gay, 1991). These differences in a useful character in understanding the difterences
host life-style have been frequently mirrored in between simpler genera such as Micromastigotes
differences in the taxa of llagellates which occur and the more complex ones. The position of the
within them e.g. devescovinids. calonymphids nucleus is of limited variability within the group
and oxymonads are confined to kalotermids, and and is generally located at the base of the llagellar
eucomonymphids and holomastigotoidids to bands or at the point where the cell expands in
rhinotermitids (Brugerolle & Lee, 2000; Dolan diameter. In both cases it is close to the flagellar
et al., 2000a, 2000b; Yamin, 1979). Groups bases so the fibrous support structures can reach
which have wider distributions across multiple it. The presence of the axostyle throughout cell
termite families include the trichonymphids and division has been emphasised as a significant
spirotrichonymphines (Yamin, 1979) suggesting character by Cleveland et al. (1934). It is,
that Micromastigotes may be related to one of however, difficult to apply generally as complete
these. cell cycles are known for only a handful of genera
and we currently have no idea how variable this
SYSTEMATIC PLACEMENT character is within genera. The only characters
OF MICROMASTIGOTES which show consistent and interprelable variation
across the spirotrichonymphines are those related
Hollande & Carruette-Valentin (1971) noted
to the mastigont system and its support fibres.
the possible relationship of Micromastigotes to
other spiral-form hypermastigids and proposed Flagella structure in the spirotrichonymphids
that they formed a natural group without was reviewed by Brugerolle (2001) who homo-
assigning the new genus to an existing or novel iogised the anterior end ofthe spirotrichonymphid
family but rather placing it as a genus insertac flagella band with the privileged flagella. Each
sedis within the suborder Spirotrichonymphina. band has a complete set of privileged flagella al
They did, however, propose several characters its anterior end and the majority of the band is
upon which the spirotrichonymphines could be composed of the linearly replicated flagellum 3.
split into families (and presumably could be used In addition, each genus within the family could
to place Micromastigotes) including: presence be diagnosed on the basis of modifications of
or absence of a rostral column and llagellar the flagellar gutter - the complex of striated and
bands; numbers of llagellar bands; whose dense lamella which fonned a U-shaped band at
portion is not differentiated out of the parabasal the base ofthe kinetosomes. Brugerolle (2001),
lamellae; position of the nucleus; contingent however, noted that Spirotrichonymphella lacked
136 MEMOIRS OF THE QUEENSLAND MUSEUM
FIG. 4. Schematic diagram of flagellar band structure in the Spirotrichonymphina; each represents a single
band for simplicity, all extant species have multiple bands arranged with radial symmetry. A, Spiralised
linear band of nearly parallel basal bodies as found in Spimtnchonvmpha, Microjoenia, Holomastigotoides.
B, Spiral stair case, basal bodies each rotated several degrees relative to the preceding body as found in
Micwmasiigotes.
a flagellar gutter, the kinetosomes were connected suggests that the privileged flagella are oriented
to a central core-like structure, the columella, by perpendicularly to the cell membrane, the mutli-
striated roots and the striated lamella formed a flagellate condition is achieved by serial replication
weak band at the base of the kinetosome. This of flagellum 3 and spiralisation is then achieved
is a broadly similar arrangement to that seen in by torsion of the band (kinetosomes plus flagellar
Micromasfigotes, however the striated roots are gutter) around the cell, individual flagella are
absent. In both cases, the kinetosomes radiate from roughly parallel to each other at their bases (Fig.
a central core, the kinetosome bases are buried deep 4A). Spiralisation in Micromastigotes has clearly
in the cytoplasm, the dense lamina is absent and the not been achieved in this fashion. Flagella are
striated lamina forms a sheet rather than a ciirv^ed oriented perpendicularly to the axis of the cell
half of the U-shaped flagellar gutter. Conversely, and spiralisation is achieved by rotation of kine¬
SpirotrichonympheUa lacks an axostyle, flagellar tosomes around this central axis, by about 12-
bands extend the entire length of the cell and the 18° per flagellum in A/, scottae (Fig. 4B).
basal bodies are not anchored directly together in The distinction here is not simply related to size.
the axial centre of the cell but rather arc anchored Whilst most spirotrichonymphids are quite large
to the columella by striated roots. This suggests species for which peripheral flagellar bands are the
that Micromastigotes should probably be class¬ only functional option (flagella radiating from a
ified within the spirotrichonymphines and that central core may embed too much of the flagellum
the similarities to SpirotrichonympheUa are shaft in cytoplasm for them to beat eftectively)
probably not indicative of a close relationship the peripheral location of the flagellar bands
between the two genera. occurs in even the smallest spirotrichonymphids.
Aherojoenia antemdepressa is only two thirds the
Brugerolle's (2001) homologisation of the size of Micromastigotes scottae and yet displays
anterior ends of the flagella bands of spiro¬ the classical spirotrichonymphid arrangement
trichonymphines with the privileged flagella of of peripheral flagella bands bounded inter¬
trichomonads semu lato provides ftirther evidence nally by a flagellar gutter (Brugerolle, 2001).
of the distinction between Micromastigotes and the The evolutionar)' relationships of the spiro¬
other Spirotrichonymphidae. The arrangement of trichonymphid genera are currently unknown, so
the flagella bands in most spirotrichonymphines it is impossible to discern whether Microjoenia