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Syst Parasitol (2010) 76:1–7 Author's personal copy
DOI 10.1007/s11230-010-9229-z
Gut wash, body soak, blender and heat-fixation: approaches
to the effective collection, fixation and preservation
of trematodes of fishes
Thomas H. Cribb • Rodney A. Bray
Received: 6 February 2009 / Accepted: 2 September 2009
Ó Springer Science+Business Media B.V. 2010
Abstract Advice is offered on some effective
methods for collecting and preserving trematodes
from fishes for taxonomy and systematics. Emphasis is
placed on obtaining high-quality specimens that have
reliable data and that are amenable to study by both
morphological and molecular approaches. We empha-
sise the importance of the freshness of the host
specimen, the reliability of its provenance and the
labelling of the specimens. For the collecting itself, we
recommend a ‘gut-wash’ approach for gastro-intesti-
nal species and specific searches for atypical taxa such
as didymozoids, aporocotylids, Saturnius Manter 1969
and transversotrematids. For metacercariae, we rec-
ommend a ‘blender’ approach to release parasites from
host tissues. For fixation, we argue in favour of heat-
killing in fluid at close to boiling temperature. We
recommend against flattening as a routine procedure
for collecting specimens for morphology. Preservation
for morphological study is best in formalin or alcohol,
and alcohol works well for molecular samples. The
importance of reliable labelling and the deposition of
specimens in museums is emphasised.
T. H. Cribb (&)
School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, QLD 4072,
Australia
e-mail: T.Cribb@uq.edu.au
R. A. Bray
Department of Zoology, Natural History Museum,
Cromwell Road, London SW7 5BD, UK
Introduction
There has been no recent estimate of the number of
species of trematodes reported from fishes, but our
own lists contain over 5,000 species and the annual
description of species indicates that there are many
more to be found. For the discovery and description
of such a large fauna to be efficient, a systematic
approach to their collection and preservation is
needed. Following the encouragement of colleagues
who are not expert in trematodes, we present here our
combined experience of effective approaches to the
collection and preservation of trematodes for mor-
phological studies. Key techniques relate to the
treatment of the gut, the collection of trematodes in
other sites, and fixation and preservation methods that
are reliable and repeatable. Although this paper is
directed specifically towards the collection of trem-
atodes of fishes, many aspects should be useful to
workers collecting trematodes from other host
groups. We also direct the attention of workers to
useful contributions in this field (Cooper, 1988;
Gibson, 1984; Pritchard & Kruse, 1982).
Comments and recommendations
Fresh, market or preserved?
Our experience is that fish dissected immediately
after death yield the best specimens. Trematodes,
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even in fish kept on ice, will typically begin to
degenerate significantly (probably both from autoly-
sis and bacterial putrefaction) within 12 h of the
host’s death, sometimes much sooner. Such speci-
mens, even if they are still alive, begin to change
shape and may shed important features, such as
tegumental or oral spines (e.g. Miller & Cribb, 2008).
We have seen many descriptions of samples from
such sources where the condition of the specimens
suggests to us that our science would have been
better-off without the description at all. In addition to
the likely partial degradation of the sample, samples
from fish from markets often have associated uncer-
tainty as to the true provenance of the sample
associated with them. The seller may be poorly
informed or reluctant to identify the true origin of the
fish. We note, for example, that when Yamaguti
(1970) described Opecoelus platycephali Yamaguti,
1970 from the Honolulu Fish Market, the fish was
recorded as ‘‘imported from Australia?’’. We shall
probably never know the origin of this fish. In our
view, science would have been better off without this
description. We might add that recording species
from incompletely identified fishes is equally prob-
lematic. For example, Manter (1963) reported 15
species of trematodes from Fijian fish; for 12 of these,
the host fish was incompletely identified, and in
several cases it was identified only to family. We
consider this to have been a lapse of judgement by
Manter as it is rarely possible to entirely resolve such
issues in later years unless voucher specimens of the
fish have been retained. There is little excuse for such
lapses now, as there are increasing opportunities for
the preservation of host tissue samples for future
molecular analysis where the identity of the host is in
doubt. [While this paper was in review, one referee
disagreed strongly with our view here, taking the
position that any host identification information is
better than none. It was pointed out that fish taxonomy
is always itself evolving; a good example was made of
the cosmopolitan Mugil cephalus, which is now
thought to comprise a species complex (Heras et al.,
2009). We respect these views but stand by our own.
Clearly decisions as to whether it is worth publishing
records from incompletely identified animals should be
made on a case by case basis.] We selected examples
for critique from the work of Yamaguti and Manter
because, overall, their contributions were outstanding
and beyond reproach.
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Syst Parasitol (2010) 76:1–7
Under some circumstances samples are recovered
from preserved fish. Typically the body-cavity has
been opened to allow ingress of either formalin or
alcohol fixative. In our experience, such specimens
are often preserved quite well, but frequently in a
contracted or otherwise distorted shape. Of course,
there are cases where it may never be possible to
examine samples in ideal conditions (perhaps espe-
cially deep-sea fishes), but overall we conclude that
fresh samples give by far the best results.
Gastrointestinal species
We speculate that at least 90% of the trematodes of
fishes live in the gastrointestinal tract. For this reason,
the efficient and effective examination of the gut is the
central activity in the collection of fish trematodes.
We recommend that all aspects of the dissection
should be carried out in a saline solution. We find that
a solution of approximately 0.85% NaCl works well.
This solution works well for actinopterygians, but it
is noteworthy that elasmobranchs may have an
osmolarity much closer to sea-water; however, elas-
mobranchs have relatively few trematodes. For work
with marine fishes we find that an extremely effective
saline solution is created by mixing one part seawater
with three parts freshwater. We use tapwater and
unfiltered seawater with excellent results. Colleagues
have reported to us that carefully prepared solutions
of distilled water and measured amounts of salt and
other reagents have not worked as well!
The entire viscera should be removed from the body-
cavity of the fish. It is worthwhile to try to retrieve the
extreme anterior parts of the pharyngeal region, as some
trematodes, e.g. Derogenes pharyngicola Bray, Cribb &
Barker, 1993, are specific to this site (Bray et al., 1993).
The urinary bladder of teleosts, which may harbour
gorgoderids and opens with the gut at the cloaca in bony
fishes, may be very small and easily left in the body
of the fish. The swim-bladder may also harbour
trematodes.
Once removed, the viscera are best divided into
organs. Separation of organs allows for a cleaner
examination and also for the accurate recording of the
site of infection. Key organs for examination are the
pharynx and stomach, pyloric caeca, intestine, rec-
tum, gall-bladder, urinary bladder and swim bladder.
We find it best to open each of these organs
separately in an appropriately-sized Petri dish and
Syst Parasitol (2010) 76:1–7 Author's personal copy
to examine it progressively under a dissecting
microscope. Worms may be retrieved as they are
observed and placed temporarily (prior to study or
preservation) in a smaller vessel, ideally an excavated
block or small Petri dish, in clean saline solution.
(We advise against storing cucullanid nematodes
and trematodes together, because we have found
these nematodes are capable of punching holes in
flatworms!)
We find that the visual inspection of gastro-
intestinal organs, even under the dissecting micro-
scope and even when the gut appears quite clean, is not
sufficient to find all the trematodes present reliably.
We thus use and strongly recommend a ‘gut-wash’
approach, following initial inspection with the aid of a
dissecting microscope. For this process, we use a wide
mouthed bottle with a tight-fitting lid and a volume of
at least a litre. We place the organ (typically an opened
section of the gut) in the bottle, and fill the bottle about
a third full with saline solution. The lid is put on tightly
and the bottle shaken vigorously for perhaps 20 s. Our
experience is that the shaking never damages trema-
todes, unless perhaps when they are already moribund.
We have found, however, that the process can be
highly destructive for some cestodes, which may
become fragmented or knotted. After shaking, the
bottle is filled with saline. At this stage the tissue
should be recovered from the bottle and re-examined;
it will now be free of ingested matter and mucus and
any parasites that are still attached are likely to be far
more obvious than previously. The gut-wash bottle
should be allowed to settle for at least 1 min. Our
observations show that, depending on the size of the
bottle, 1 min is sufficient to allow all the parasites to
fall to the sediment; periodically, we examine the fluid
that is normally discarded after 1 min of settling and
rarely find any parasites. About three-quarters of the
supernatant should be discarded and then, if suffi-
ciently clean, the sediment can be poured into a Petri
dish for examination. For some fish, the sediment will
need to be washed several times before it is clean
enough to be examined effectively; we find this
especially with herbivores, such as scarids, siganids,
kyphosids and deep-sea macroplanktonivores, such as
alepocephalids. For some fish, where the gut is
especially full of mucus or fine digestate, we do not
bother to inspect prior to the gut-wash. We find that the
‘gut-wash’ is fast and effective and recommend it
especially.
3
An alternative method to dislodge trematodes
attached to the gut is to scrape it, using a blunt edge
such as a scalpel or to run it between the ends of a
pair of forceps. We often find it effective to use this
approach in association with gut-washing.
Many trematode species are specific to the pyloric
caeca. The number of pyloric caeca varies from none
in some fishes, such as labrids, to as few as five or six
in many fishes, and up to perhaps thousands of finely
divided tubules that are interdigitated with the liver in
certain scombrids. In the latter case, it is not
logistically feasible to dissect all the pyloric caeca;
we suggest instead that in such cases they should be
squeezed with forceps or broken up with a blender
(see below) to dislodge the parasites.
A final note on the gut is that in mugilids (mullet),
hemiurids of the genus Saturnius Manter, 1969 may
infect the tissue beneath the lining of the pyloric
stomach (Overstreet, 1977); these must be specifi-
cally sought by peeling off this lining. In addition,
didymozoids may be embedded in the wall of the gut
(see below).
Other sites
Although the gut is by far the most common site for
trematodes in fishes, infections can be found in almost
any site in the body. For many of these all that is
necessary is that the organ be isolated and examined
carefully; this applies to the gills (Syncoeliidae),
gall-bladder (e.g. Fellodistomidae, Lepocreadiidae,
Opisthorchiidae and Zoogonidae), swim-bladder (e.g.
Dicytsarcidae, Isoparorchiidae and Tandanicolidae)
and urinary bladder (Gorgoderidae and Zoogonidae).
Below we comment on three especially difficult taxa
and their sites of infection.
Didymozoids
The Didymozoidae is a major family of trematodes
which have radiated greatly in pelagic fishes, espe-
cially tunas (Scombridae) (Pozdnyakov & Gibson,
2008). A major feature of the family is that they
occur principally in the tissues (mainly the connec-
tive tissues) of their hosts rather than in cavities. They
must therefore be sought specifically by the exami-
nation of the tissues where they reside. Another key
feature of the family is that the adults retain eggs in
the uterus (typically, it is thought, to be dispersed
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only when the host dies), which has the effect of
making most didymozoids bright yellow. Many
didymozoids form pairs and in some cases there is
sexual differentiation into males and females; it is
best to keep such pairs together and separate from
other individuals so that they can be described as a
pair. Many didymozoids are thread-like and may
reach lengths that exceed a metre (e.g. Noble, 1975).
Such species are challenging to work with. They can
only be collected by slow and painstaking dissection
of the surrounding tissues. A special effort should be
made to find the anterior end of the parasite, because
identification is generally impossible without it.
Aporocotylids (syn. sanguinicolids)
Blood flukes live in the circulatory system of a wide
range of fishes. They seem to be most common in the
heart and gills but can also be found in the pectoral
girdle (Montero et al., 2003), the body-cavity (Martin,
1975), the liver (Braicovich et al., 2006) and the kidney
(Bullard et al., 2006); some specialist species probably
occur in other sites. Although we have found a few
individuals of aporocotylids by dislodging them in a
general dissection, in general they must be sought
specifically. Because the collection of aporocotylids is
so challenging, this family is perhaps as poorly known
as any group of trematodes in fishes.
If sanguinicolids are to be looked for, we recom-
mend that the heart should be the first organ dissected,
because coagulation of the blood post mortem makes
recognition of the worms increasingly difficult.
Bullard & Jensen (2008) have described the successful
use of anti-coagulant to reduce this problem.
We have had some success in looking for evidence of
aporocotylids by examining the heart tissue for trapped
eggs as indication of possible infection. Although this
method has not been used extensively, we have found a
number of cases where there are numerous eggs but no
worms to be found; either the infections are long gone or
the worms have eluded us. Something similar can also
apply to didymozoids, where shadows of the former
worms are formed by the eggs.
Transversotrematids
Transversotrematids live beneath the scales of marine
and freshwater fishes of (at least) the Indian and
Pacific Oceans and surrounding land masses. We
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Syst Parasitol (2010) 76:1–7
suspect that they have been frequently missed in
parasitological examinations because they have not
been sought. When we first began to search for them,
we collected them by scraping the surface of the fish
with a scalpel. We have since discovered that this is
unnecessary and, in fact, damages many specimens.
We now find that transversotrematids can be found
easily by simply soaking the body of the fish in
vertebrate saline. Within 30 min most of the trans-
versotrematids will simply have fallen to the bottom
of the container. Although these parasites are in a
partly external site (beneath the free margin of the
scales rather than in a fully internal site), they survive
far better in saline than in seawater or freshwater
(depending on the habitat of the fish).
An advantage of the soaking of a fish body in a
specific search for transversotrematids is that fre-
quently other parasites (perhaps those that have
undergone a post mortem migration) will be found.
Metacercariae in tissue
Most trematode metacercariae in fish occur as
encysted forms in the tissues; large, obvious, unen-
cysted forms, such as those of some clinostomids, are
exceptional. As a result of their size and position in
the host, the recovery of these metacercariae can be
demanding. We use two approaches. The first,
effective on small fish, is direct dissection. Metacer-
cariae can simply be dissected from the tissues using
fine forceps and mounted needles. This is best done
immediately after the fish is killed, while the tissues
are still translucent. For larger fish (e.g. anything
greater than 15 cm long), the volume of tissue makes
this approach increasingly ineffective. For such fish,
we macerate the tissues with an electric blender. We
use either a standard culinary electrical jug blender,
or, even better, a hand-held wand style blender. These
machines allow the fish tissues to be rapidly broken
up to form a slurry with vertebrate saline. In this
process many metacercariae will be released from the
tissues. The slurry of tissue and saline can then
be treated like the ‘gut wash’ described above; often
the largest remaining pieces of tissue should be
removed by a sieve first. We have found that small
metacercariae can be slow to sink and that at least
2 min should be allowed for settling. We find it
useful to blend the head, the fins and their bases, and
the remainder of the fish body separately, as this
Syst Parasitol (2010) 76:1–7 Author's personal copy
gives us some general indication of the site-specific-
ity of the parasites. Blending is certainly a vigorous
method and some metacercariae are destroyed by the
process, but they are often so numerous that this is
not a concern.
Metacercariae localised on the gut wall can often
be found readily by turning the piece of gut-wall over
and scraping it with a heavy scalpel under transmitted
light.
Trematode metacercariae are typically difficult to
identify while still in their cysts, so at least a
proportion of each sample should be excysted. An
advantage of blending in this respect is that often
some of the metacercariae are excysted by the
process. If this does not occur, they may be excysted
mechanically by the use of mounted needles and
forceps or there is an extensive literature on chemical
excystment (e.g. Fried, 1994).
Fixation and preservation
Although there is much to be learned from the study
of live trematodes (especially life-history stages),
ultimately samples must be fixed and preserved if
permanent samples for microscopy and molecular
analysis are to be obtained. There are two issues to be
considered with the preservation of trematodes – how
they are fixed (the process by which the proteins are
coagulated) and in what fluid they are preserved.
There is a long tradition in trematode taxonomy of
fixing specimens with some form of flattening –
typically ‘under a coverglass’ (e.g. Manter, 1934) or
‘under slight pressure’ (e.g. Machida, 2004); this
approach was used extensively by both Manter and
Yamaguti; Yamaguti (1965) described his use of the
‘‘compressarium’’ in some detail. The goal of the use of
such pressure is to control the shape of the specimen at
fixation and render the arrangement of the internal
organs more easily discernible. Although we under-
stand the goal, we do not approve of the outcome. We
think the process, by definition, distorts the specimens
and makes comparison between samples difficult;
slight pressure for one worker is likely to differ from
that of another. In particular, the size and ratios of
suckers can be significantly distorted by flattening and
these are often some of the most useful criteria that we
use in the identification of trematodes. In our view,
‘flattening’ fails the test of reproducibility. We advo-
cate that, if specimens are to be flattened at all, it should
5
be only a proportion of them and that measurements
should be based on unflattened specimens.
We recommend killing and fixing trematodes with
heat as an effective method. We advocate pipetting
worms into vertebrate saline that has been brought to
the boil and then removed from the heat source. We
use 5 or 10 ml Pyrex beakers so that the heating can
be rapid. When this is done, the worms typically
extend and assume a highly uniform and reproducible
shape. Pipetting into hot saline works noticeably
better than pouring hot saline onto the worms in
another container. This is presumably because the
heat dissipates faster in the second approach. Perhaps
the best fixation (and simultaneous preservation) is
achieved by pipetting worms direct into very hot
preservative such as formalin. However, this method
cannot be recommended because of the health risks
associated with the fumes and the loss of opportuni-
ties to use the specimens for both morphological and
molecular studies. In the field, we frequently find
collections of worms in a single fish individual that
may represent more than one species of a given
family but for which the distinctions are not clear in
the living worms. We often find that, after the worms
have been heat-killed, they can be sorted into groups
much more easily than when they were alive.
Trematodes must be preserved after being fixed.
Our standard approach is to preserve some specimens
in 5–10% formalin (the concentration seems to make
little difference) for morphological study and some in
high grade 96% absolute alcohol for molecular
analysis. Importantly, heat-fixation has no apparent
effect on the success with which DNA can be
extracted. A new approach for the field is to heat-
kill the specimens and then preserve them all in 70%
alcohol, so that decisions on the use to be made of the
specimens can be made later; after sorting, specimens
for molecular analysis should be transferred to
absolute alcohol. We find that the quality of slide
specimens produced by alcohol preservation does not
quite match that of formalin preservation, but
certainly the specimens are acceptable and the
flexibility of this method has great advantages.
Berland’s fluid
Berland’s fluid is a solution composed of 1 part
formalin and 19 parts glacial acetic acid (Gibson,
1979) and can be used as a fixing agent when the
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conditions are not conducive to the use of boiling
fluids, such as field collection on research vessels in
moderate to rough conditions. In most cases the
digenean specimens are fixed extended and straight (if
they were alive when fixed). The specimens should be
transferred to a preservation fluid (generally formalin
or alcohol) within a few minutes. Specimens fixed in
this way will not be usable for DNA sequencing.
Also, this method, while adequate for digeneans and
excellent for nematodes, is often less satisfactory for
cestodes where the reaction with calcareous corpuscles
may cause bubbles to form in the tissues and the
microtriches may be stripped off – thus limiting the
use of the material for scanning-electron microscopy.
Labelling
We feel constrained to mention the paramount impor-
tance of careful labelling. We have seen several
instances in the literature of records of parasites that
have clearly arisen from a break-down in the labelling
process and more that we suspect. Cases of clear
mislabelling or label loss were detected and outlined by
Cribb et al. (1999) and Bray & Cribb (2002), but such
clarification after the fact is not always possible. The
label should be as informative as possible placed inside
the container, and a corresponding record (typically
with more information) should be maintained sepa-
rately from the specimen. We find that for internal
labels, pencil writing on ‘goat-skin parchment’ or
waterproof paper works well. We discourage the use of
ink for labelling, even on external labels, as it is almost
inevitable that some fluid will spill on the label at some
stage of the processing of the material, and many inks
run, obscuring or losing the data. Electronically printed
labels should be experimented with before extensive
use; we have seen cases where all the letters have fallen
off the label leaving only an alphabet soup!
Deposition of specimens
It appears to be universally understood that holotypes
and paratypes of new taxa should be deposited in
well-curated collections. Unfortunately, the practice
of lodging voucher specimens of species identified
from novel hosts and localities is not quite as
universal. Although the pressure on museums can
be significant, we encourage our colleagues to lodge
museum voucher specimens of anything that might
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Syst Parasitol (2010) 76:1–7
require future reconsideration, especially representa-
tives from new hosts and new localities.
Acknowledgements The collecting during which the methods
described above were developed was funded mainly by the
Australian Biological Resources Study and the Australian
Research Council. We thank the two anonymous referees for
extensive and thoughtful comments.
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