T. H. Cribb (&)
School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, QLD 4072, Comments and recommendations
Australia
e-mail: T.Cribb@uq.edu.au Fresh, market or preserved?
R. A. Bray
Department of Zoology, Natural History Museum, Our experience is that fish dissected immediately
Cromwell Road, London SW7 5BD, UK after death yield the best specimens. Trematodes,
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even in fish kept on ice, will typically begin to Under some circumstances samples are recovered
degenerate significantly (probably both from autoly- from preserved fish. Typically the body-cavity has
sis and bacterial putrefaction) within 12 h of the been opened to allow ingress of either formalin or
host’s death, sometimes much sooner. Such speci- alcohol fixative. In our experience, such specimens
mens, even if they are still alive, begin to change are often preserved quite well, but frequently in a
shape and may shed important features, such as contracted or otherwise distorted shape. Of course,
tegumental or oral spines (e.g. Miller & Cribb, 2008). there are cases where it may never be possible to
We have seen many descriptions of samples from examine samples in ideal conditions (perhaps espe-
such sources where the condition of the specimens cially deep-sea fishes), but overall we conclude that
suggests to us that our science would have been fresh samples give by far the best results.
better-off without the description at all. In addition to
the likely partial degradation of the sample, samples Gastrointestinal species
from fish from markets often have associated uncer-
tainty as to the true provenance of the sample We speculate that at least 90% of the trematodes of
associated with them. The seller may be poorly fishes live in the gastrointestinal tract. For this reason,
informed or reluctant to identify the true origin of the the efficient and effective examination of the gut is the
fish. We note, for example, that when Yamaguti central activity in the collection of fish trematodes.
(1970) described Opecoelus platycephali Yamaguti, We recommend that all aspects of the dissection
1970 from the Honolulu Fish Market, the fish was should be carried out in a saline solution. We find that
recorded as ‘‘imported from Australia?’’. We shall a solution of approximately 0.85% NaCl works well.
probably never know the origin of this fish. In our This solution works well for actinopterygians, but it
view, science would have been better off without this is noteworthy that elasmobranchs may have an
description. We might add that recording species osmolarity much closer to sea-water; however, elas-
from incompletely identified fishes is equally prob- mobranchs have relatively few trematodes. For work
lematic. For example, Manter (1963) reported 15 with marine fishes we find that an extremely effective
species of trematodes from Fijian fish; for 12 of these, saline solution is created by mixing one part seawater
the host fish was incompletely identified, and in with three parts freshwater. We use tapwater and
several cases it was identified only to family. We unfiltered seawater with excellent results. Colleagues
consider this to have been a lapse of judgement by have reported to us that carefully prepared solutions
Manter as it is rarely possible to entirely resolve such of distilled water and measured amounts of salt and
issues in later years unless voucher specimens of the other reagents have not worked as well!
fish have been retained. There is little excuse for such The entire viscera should be removed from the body-
lapses now, as there are increasing opportunities for cavity of the fish. It is worthwhile to try to retrieve the
the preservation of host tissue samples for future extreme anterior parts of the pharyngeal region, as some
molecular analysis where the identity of the host is in trematodes, e.g. Derogenes pharyngicola Bray, Cribb &
doubt. [While this paper was in review, one referee Barker, 1993, are specific to this site (Bray et al., 1993).
disagreed strongly with our view here, taking the The urinary bladder of teleosts, which may harbour
position that any host identification information is gorgoderids and opens with the gut at the cloaca in bony
better than none. It was pointed out that fish taxonomy fishes, may be very small and easily left in the body
is always itself evolving; a good example was made of of the fish. The swim-bladder may also harbour
the cosmopolitan Mugil cephalus, which is now trematodes.
thought to comprise a species complex (Heras et al., Once removed, the viscera are best divided into
2009). We respect these views but stand by our own. organs. Separation of organs allows for a cleaner
Clearly decisions as to whether it is worth publishing examination and also for the accurate recording of the
records from incompletely identified animals should be site of infection. Key organs for examination are the
made on a case by case basis.] We selected examples pharynx and stomach, pyloric caeca, intestine, rec-
for critique from the work of Yamaguti and Manter tum, gall-bladder, urinary bladder and swim bladder.
because, overall, their contributions were outstanding We find it best to open each of these organs
and beyond reproach. separately in an appropriately-sized Petri dish and
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only when the host dies), which has the effect of suspect that they have been frequently missed in
making most didymozoids bright yellow. Many parasitological examinations because they have not
didymozoids form pairs and in some cases there is been sought. When we first began to search for them,
sexual differentiation into males and females; it is we collected them by scraping the surface of the fish
best to keep such pairs together and separate from with a scalpel. We have since discovered that this is
other individuals so that they can be described as a unnecessary and, in fact, damages many specimens.
pair. Many didymozoids are thread-like and may We now find that transversotrematids can be found
reach lengths that exceed a metre (e.g. Noble, 1975). easily by simply soaking the body of the fish in
Such species are challenging to work with. They can vertebrate saline. Within 30 min most of the trans-
only be collected by slow and painstaking dissection versotrematids will simply have fallen to the bottom
of the surrounding tissues. A special effort should be of the container. Although these parasites are in a
made to find the anterior end of the parasite, because partly external site (beneath the free margin of the
identification is generally impossible without it. scales rather than in a fully internal site), they survive
far better in saline than in seawater or freshwater
Aporocotylids (syn. sanguinicolids) (depending on the habitat of the fish).
An advantage of the soaking of a fish body in a
Blood flukes live in the circulatory system of a wide specific search for transversotrematids is that fre-
range of fishes. They seem to be most common in the quently other parasites (perhaps those that have
heart and gills but can also be found in the pectoral undergone a post mortem migration) will be found.
girdle (Montero et al., 2003), the body-cavity (Martin,
1975), the liver (Braicovich et al., 2006) and the kidney Metacercariae in tissue
(Bullard et al., 2006); some specialist species probably
occur in other sites. Although we have found a few Most trematode metacercariae in fish occur as
individuals of aporocotylids by dislodging them in a encysted forms in the tissues; large, obvious, unen-
general dissection, in general they must be sought cysted forms, such as those of some clinostomids, are
specifically. Because the collection of aporocotylids is exceptional. As a result of their size and position in
so challenging, this family is perhaps as poorly known the host, the recovery of these metacercariae can be
as any group of trematodes in fishes. demanding. We use two approaches. The first,
If sanguinicolids are to be looked for, we recom- effective on small fish, is direct dissection. Metacer-
mend that the heart should be the first organ dissected, cariae can simply be dissected from the tissues using
because coagulation of the blood post mortem makes fine forceps and mounted needles. This is best done
recognition of the worms increasingly difficult. immediately after the fish is killed, while the tissues
Bullard & Jensen (2008) have described the successful are still translucent. For larger fish (e.g. anything
use of anti-coagulant to reduce this problem. greater than 15 cm long), the volume of tissue makes
We have had some success in looking for evidence of this approach increasingly ineffective. For such fish,
aporocotylids by examining the heart tissue for trapped we macerate the tissues with an electric blender. We
eggs as indication of possible infection. Although this use either a standard culinary electrical jug blender,
method has not been used extensively, we have found a or, even better, a hand-held wand style blender. These
number of cases where there are numerous eggs but no machines allow the fish tissues to be rapidly broken
worms to be found; either the infections are long gone or up to form a slurry with vertebrate saline. In this
the worms have eluded us. Something similar can also process many metacercariae will be released from the
apply to didymozoids, where shadows of the former tissues. The slurry of tissue and saline can then
worms are formed by the eggs. be treated like the ‘gut wash’ described above; often
the largest remaining pieces of tissue should be
Transversotrematids removed by a sieve first. We have found that small
metacercariae can be slow to sink and that at least
Transversotrematids live beneath the scales of marine 2 min should be allowed for settling. We find it
and freshwater fishes of (at least) the Indian and useful to blend the head, the fins and their bases, and
Pacific Oceans and surrounding land masses. We the remainder of the fish body separately, as this
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gives us some general indication of the site-specific- be only a proportion of them and that measurements
ity of the parasites. Blending is certainly a vigorous should be based on unflattened specimens.
method and some metacercariae are destroyed by the We recommend killing and fixing trematodes with
process, but they are often so numerous that this is heat as an effective method. We advocate pipetting
not a concern. worms into vertebrate saline that has been brought to
Metacercariae localised on the gut wall can often the boil and then removed from the heat source. We
be found readily by turning the piece of gut-wall over use 5 or 10 ml Pyrex beakers so that the heating can
and scraping it with a heavy scalpel under transmitted be rapid. When this is done, the worms typically
light. extend and assume a highly uniform and reproducible
Trematode metacercariae are typically difficult to shape. Pipetting into hot saline works noticeably
identify while still in their cysts, so at least a better than pouring hot saline onto the worms in
proportion of each sample should be excysted. An another container. This is presumably because the
advantage of blending in this respect is that often heat dissipates faster in the second approach. Perhaps
some of the metacercariae are excysted by the the best fixation (and simultaneous preservation) is
process. If this does not occur, they may be excysted achieved by pipetting worms direct into very hot
mechanically by the use of mounted needles and preservative such as formalin. However, this method
forceps or there is an extensive literature on chemical cannot be recommended because of the health risks
excystment (e.g. Fried, 1994). associated with the fumes and the loss of opportuni-
ties to use the specimens for both morphological and
Fixation and preservation molecular studies. In the field, we frequently find
collections of worms in a single fish individual that
Although there is much to be learned from the study may represent more than one species of a given
of live trematodes (especially life-history stages), family but for which the distinctions are not clear in
ultimately samples must be fixed and preserved if the living worms. We often find that, after the worms
permanent samples for microscopy and molecular have been heat-killed, they can be sorted into groups
analysis are to be obtained. There are two issues to be much more easily than when they were alive.
considered with the preservation of trematodes – how Trematodes must be preserved after being fixed.
they are fixed (the process by which the proteins are Our standard approach is to preserve some specimens
coagulated) and in what fluid they are preserved. in 5–10% formalin (the concentration seems to make
There is a long tradition in trematode taxonomy of little difference) for morphological study and some in
fixing specimens with some form of flattening – high grade 96% absolute alcohol for molecular
typically ‘under a coverglass’ (e.g. Manter, 1934) or analysis. Importantly, heat-fixation has no apparent
‘under slight pressure’ (e.g. Machida, 2004); this effect on the success with which DNA can be
approach was used extensively by both Manter and extracted. A new approach for the field is to heat-
Yamaguti; Yamaguti (1965) described his use of the kill the specimens and then preserve them all in 70%
‘‘compressarium’’ in some detail. The goal of the use of alcohol, so that decisions on the use to be made of the
such pressure is to control the shape of the specimen at specimens can be made later; after sorting, specimens
fixation and render the arrangement of the internal for molecular analysis should be transferred to
organs more easily discernible. Although we under- absolute alcohol. We find that the quality of slide
stand the goal, we do not approve of the outcome. We specimens produced by alcohol preservation does not
think the process, by definition, distorts the specimens quite match that of formalin preservation, but
and makes comparison between samples difficult; certainly the specimens are acceptable and the
slight pressure for one worker is likely to differ from flexibility of this method has great advantages.
that of another. In particular, the size and ratios of
suckers can be significantly distorted by flattening and Berland’s fluid
these are often some of the most useful criteria that we
use in the identification of trematodes. In our view, Berland’s fluid is a solution composed of 1 part
‘flattening’ fails the test of reproducibility. We advo- formalin and 19 parts glacial acetic acid (Gibson,
cate that, if specimens are to be flattened at all, it should 1979) and can be used as a fixing agent when the
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conditions are not conducive to the use of boiling require future reconsideration, especially representa-
fluids, such as field collection on research vessels in tives from new hosts and new localities.
moderate to rough conditions. In most cases the
digenean specimens are fixed extended and straight (if Acknowledgements The collecting during which the methods
described above were developed was funded mainly by the
they were alive when fixed). The specimens should be
Australian Biological Resources Study and the Australian
transferred to a preservation fluid (generally formalin Research Council. We thank the two anonymous referees for
or alcohol) within a few minutes. Specimens fixed in extensive and thoughtful comments.
this way will not be usable for DNA sequencing.
Also, this method, while adequate for digeneans and
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