Research Papers
Institute for Creation Research
February 2, 2005


ABSTRACT

Microscopic heterophyid trematode parasitic worms of the Genus
Ascocotyle infect certain amnicolid and hydrobiid snails and certain
cyprinodont and poeciliid estuarine fishes as first and second
intermediate hosts. Adult trematode worms are found to mature in the
intestines of particular definitive hosts, most often piscivorous
birds, but also certain mammals. A survey of these parasites,
harvested from fish hearts and gills collected in Mississippi, Texas
and California, shows that they are obligated to complex life cycles
requiring at least three disparate and different hosts to achieve
fecundity. Methods of infection, host infection site and host
specificity are often unique to each different species of these
parasites. Additionally, Ascocotyle worms demonstrate highly
specialized structures such as HCl resistant cysts, HCl sensitive
penetration glands and sensory organs which may serve to guide them
to the specific infection site. These heteroecious life cycles and
specialized structures are shown to be too complex to have developed
by chance, therefore, evolutionary mechanisms appear insufficient to
explain them. A creationist design argument for the presence of such
parasites is promulgated.

INTRODUCTION

Arthur Looss [1] erected the genus Ascocotyle with A. coleostomum
(Looss, 1896) as the genotype for the Ascocotyle complex. Excluding
synonyms, or identical organisms with differing names, there are at
present over 30 named species comprising the Ascocotyle complex as
defined per Travassos [2], Stunkard and Uzmann [3], Burton [4],
Sogandares-Bernal and Bridgman [5], and Sogandares-Bernal and
Lumsden [6]. The adult worms (Fig #1) are non-pathogenic, intestinal
parasites of piscivorous, or fish-eating, birds and mammals [7].

It is in the intestines of these definitive hosts that the parasite
matures and produces eggs which are passed with feces into the marsh
or estuary [8]. Because these organisms are hermaphroditic, or
self-fertilizing, one parasite can populate a marsh with eggs [9].

Each fertilized egg has been shown to be able to produce a redia
capable of growing 6000 swimming larvae over a one year period [10].

The first intermediate hosts are amnicolid and hydrobiid snails,
which take up the eggs while feeding over the bottom surface of the
estuary [11]. Infection within the particular snail type
specifically depends upon the particular species of Ascocotyle
present, and cross infection has been shown not to occur in many
experimental trials [12, 13, 14, 15]. The eggs develop into a redia
or brood stage (Fig #2), and often travel from the the snail
digestive gland to the gonads or hepatopancreas where further
development ensues. The redia grows in length and bears up to 50
cercariae in various stages of development. When mature, these
cercariae (Fig #3, Fig #4) leave the redia through a birth pore,
mostly during daylight hours (but often at dawn), and swim out of
the snail towards illuminated areas of the water [16, 17]. The
second intermediate hosts are centrarchiid, cyprinodontid, mugilid,
and poeciliid fishes, and, in at least one instance, anuran tadpoles
[18]. Cercariae swim near these fish and are taken up in the
respiratory current. Some Ascocotyle will, at this point, attach to
and penetrate the gill filaments of the fish, dropping their tails
before or soon after penetration [19, 20, 21]. Some species will
immediately encyst in the gill, by the production of a multi-layered
hyaline, collagen-like cyst [22, 23], while others will search for
and enter the efferent blood vessels supplying blood to the gills
[24, 25]. The metacercariae will then travel within the blood
system, either against or with blood flow, to the heart, liver,
brain and other organs, where cysts will be produced and further
development is arrested [ibid]. Other species of cercariae do not
attach to the fish gills, but rather, are swallowed by the fish.
They produce cysts in the stomach and intestine or penetrate the
intestine and encyst in the liver and mesentary [26, 27].

As mentioned previously, host infection and even organ infection
site is Ascocotyle species characteristic [28, 29, 30, 31]. It is at
this point in the life cycle, that predation upon the infected fish
by herons, egrets, or raccoons and other mammals, must occur in
order that the definitive host can become infected and the worm can
sexually mature. Metacercarial cysts, which are HCl resistant,
successfully pass the definitive host stomach and dissolve only
within the intestine, freeing the worm to fertilize and eject eggs.
These life cycles are typically completed in brackish water
estuarine marsh habitats, but some second intermediate hosts have
been shown to swim out to sea [32]. In this paper, specific, complex
life cycles and morphological structures of Ascocotyle leighi, A.
pachycystis and A. diminuta, collected from fish hearts in
Mississippi, Texas and California are evaluated in the light of the
creation/evolution paradigms.

MATERIALS AND METHODS Sheepshead minnow, (Cyprinodon variegatus),
Sailfin Molly, (Poecilia latipinna), Mosquitofish (Gambusia affinis)
(Fig #5) and Killifish (Fundulus parvipinnis) (Fig #6) specimens
were collected by seine, dip net and trap method from Pine Island,
Mississippi, South Padre Island, Texas, and Newport Beach and Point
Mugu, California, and of 60 fish collected, only 7 were uninfected.
The hearts, gills and livers of these fish were harvested and
examined under a dissecting microscope for the presence of
metacercarial cysts, indicating a possible Ascocotyle infection.

Some of the hearts and gills were fixed in glutaraldehyde, embedded
in plastics and thin-sectioned [33]. Some cysts were removed from
the heart and gill tissue for mechanical and enzymatic excystment
and further study under scanning electron microscopy [34], (Fig #7).
Worms which were enzymatically excysted only exited their cysts
under conditions of a 7.4 PH adjusted mixture of saline, RPMI media
and trypsin in an incubator at 370C for 3-5 hours. Using fine needle
forceps, other cysts were forcibly popped open releasing the live
metacercaria for observation under high magnification light
microscopy. Whole mounts were made of some of these excysted worms
(Fig #8). 2-5 micron thick sections of fish hearts and gills were
cut with diamond and glass knives, stained with Methylene Blue and
Azure II and coverslipped (Fig #9, Fig #10, Fig #11).

Trematodes for SEM study were processed through osmium,
thiocarbohydrazide and a graded series of alcohols to absolute
alcohol. Dehydration by the critical point drying method was
attempted resulting in the loss of many specimens. Air dried worms
were transferred by hand under the dissecting microscope to SEM
stubs and were sputter coated for 4 minutes at 30mA deposition.
Stubs were then transferred to a Jeol JSM 35 Scanning Electron
Microscope and were observed and photographed at 100-2000X
magnification at 15 and 25 KV.

DISCUSSION

These parasites are an evolutionary enigma for several reasons and
their presence raises more questions than are answered. The
evolutionary paradigm for r-strategists (or organisms which are
small, fast-growing and which have short, highly-populated
generation times), calls for them to employ a rapidly developing,
independent life cycle which allows them to exploit their
environment quickly, achieve sudden fecundity and bear the most
possible offspring with a minimum of exposure to survival hazards
and expenditure of energy. In the light of that definition,
Ascocotyle breaks all the rules. For one, Ascocotyle manifests a
life cycle which may take up to a year or much more to complete, if
the second intermediate host (bearing many such parasites) can
escape predation or death. As mentioned previously, many examples
exist of infected first and second intermediate hosts being kept
alive in laboratory aquaria for long periods of time with no
ill-effect, showing that the parasite is capable of surviving (or
enduring) a lengthy hiatus before it can pass on its genes. This
type of life cycle is uncommon for many microscopic organisms which
typically bear offspring quickly, and do not expend energy on many
intermediate stages. This may run counter to some thinking that
evolution selects those r-strategists which develop into fitter
populations faster than others in order to exploit the available
environmental resources [35]. Conversely, some authors feel that
evolutionary selection may run along a continuum from r-strategists
to K-strategists (slow-growing organisms with long generation times
and few offspring), where, "In the ecological void the optimal
adaptive strategy channels all possible resources into survival and
production of a few offspring of extremely high competitive
ability." [36] Clearly ascocotylids are not k-strategists in the
sense that many cercariae can be produced from one snail bearing a
redia. By placing most of those cercariae into one or a few hosts
which may or may not become predated upon, Ascocotyle certainly
seems to minimize its chances for success.

Secondly, Ascocotyle is anything but independent, being strictly an
obligate parasite. In the world of parasites, this feature is not
unique as many completely depend upon other organisms to survive
(many organisms would vanish tomorrow if the host population upon
which they live died out).

The enigma for a 'survival of the fittest' interpretation of life
cycles is that Ascocotyle, like most trematodes is obligated to
three hosts, and therefore its chances of reaching sexual maturity
are several times smaller than other, more independent organisms.
Again, this does not seem to square with the typical r-strategy
scenario within which ascocotylids should operate.

This parasite requires a snail as the first intermediate host, but
not just any snail will do. Some 160 types of snails inhabit these
estuaries [37], yet the rediae do not develop in most of them. What
is it about the "right" snail which causes the egg to develop into a
redia, and what happens to eggs which are ingested by snails within
which no development takes place? It would seem best to develop an
evolutionary plan which would allow many types of intermediate hosts
to serve as appropriate vehicles for development.

It also appears, based on the life histories referenced herein that
the redia only develop in a certain part of the snail [38]. What
mechanism guides the redia to the hepatopancreas or for that matter
the gonads for completion of that stage?

These parasites require a 2nd intermediate fish host and not just
any fish. As mentioned previously, laboratory studies have shown
that Ascocotyle is very selective about the fish hosts which it
infects.

What is not clear is what mechanism guides these cercariae to the
proper fish for encystment. Do redial and cercarial spines and
sensory papillae around the oral sucker and along the ventral
tegument (Fig #12) play a role in host/organ detection and
emplacement? When one would expect sibling species to be most alike,
why do some sibling species (like A. leighi and A. pachycystis) only
infect certain, but not the same fish in the same locality?

In addition, ascocotylids are not only host specific but they are
organ specific within the 2nd intermediate host. Some metacercariae
will travel with blood flow within the 2nd intermediate host and
always end up in the same organ or site, others swim against the
efferent flow and encyst within the heart. What mechanism or sensory
apparatus indicates to the parasite which organ they are in or how
to get there? Since they are generally non pathogenic and since a
heavy parasite burden (even to the heart) has a minimal impact upon
the 2nd intermediate host [39] one wonders if these parasites may
confer some advantage to the host.

Finally, they require a 3rd host, which really begs the evolutionary
question. Concern over this is expressed by some authors, in an
attempt to supply an evolutionary explanation [40, 41]. Here the
definitive host must digest the fish, while the cyst must pass this
process unscathed. If the goal is to quickly survive and reproduce,
why tie survival to the (potentially lethal) digestive process of a
mammal or bird? It would seem to be "safer" to infect, say the shell
of a shellfish which may be discarded by a raccoon or a bird after
the meal is complete.

A. sexidigita and A. mcintoshi both appear to go the digestive route
[42, 43], not once, but twice; first within the fish and then the
bird. What allows this cercaria to resist digestion within the fish
stomach if it does not encyst within a protective capsule before
burrowing through the intestinal submucosa to the visceral organs?
Cysts ingested by the definitive host also do not dissolve in HCl
which is found normally within the host stomach, but only break down
in a 7.4 pH adjusted solution of saline and media (nutrient broth)
with trypsin (a digestive enzyme) at a temperature of 370C. These
are very close to the conditions found within the definitive host
intestine. Biochemical and ultrastructural cyst studies have shown
that there is a small collagen content to the cyst wall, but of more
value is the lipid-protein complex, which would definitely assist
the passage of the metacercariae through the digestive tract. [44,
45, 46, 47, 48, 49, 50]. A. mcintoshi cercarial penetration glands,
which assist the cercaria to enter an encystment site, do evert for
penetration in weak solutions of HCl, as shown by Leigh [51] but
otherwise do not. This indicates a mechanism which is sensitive to
gastric juices which would be encountered when swallowed by the
fish.

The question which must be asked at this point, is: "What if these
relationships, behaviors and specialized structures were designed? "
If they were designed, what would constitute a design feature or
structure for Ascocotyle or any other parasite, and could we
recognize it if we saw it? Michael Behe, in his seminal book, [52],
goes to great lengths to show this by illustrating the bewildering
complexity of the bacterial flagella, the chemical-electrical basis
for vision, and the cascade system of blood clotting. Using the
phrase "irreducibly complex", he deftly shows that these complex
systems are comprised of components, or sub-parts, any of which, if
not present, would prevent the entire system from working, making it
worthless.

He also shows the utter foolishness of expecting that a
gradualistic, Darwinian mechanism could have produced such elegant
systems, by chance, using the trial and error method, "The impotence
of Darwinian theory in accounting for the molecular basis of life is
evident not only from the analysis in this book, but also from the
complete absence in the professional scientific literature of any
detailed models by which complex biochemical systems could have been
produced...the scientific community is paralyzed. No one at Harvard
University, no one at the National Institutes of Health, no member
of the National Academy of Sciences, no Nobel prize winner - no one
at all can give a detailed account of how the cilium, or vision, or
blood clotting, or any complex biochemical process might have
developed in a Darwinian fashion."

That Michael Behe can recognize intelligent design in bacteria,
blood and vision is evident, "There is an elephant in the roomful of
scientists who are trying to explain the development of life. The
elephant is labeled 'intelligent design.' To a person who does not
feel obligated to restrict his search to unintelligent causes, the
straightforward conclusion is that many biochemical systems were
designed. They were designed not by the laws of nature, not by
chance and necessity; rather they were planned." (Emphasis in the
original). If Behe can see intelligent, planned design in a
bacterial flagella, then clearly he would see intelligent, planned
design in the HCl sensitive cercarial penetration glands and the HCl
resistant, yet trypsin and pH sensitive metacercarial cyst of
Ascocotyle which requires it to be HCl resistant at one point in its
fantastic voyage, and yet precisely trypsin and pH sensitive at
another. These and other features found within the members of the
Ascocotyle complex can be no less objects of intelligent, planned
design than Behe's bacterial flagella.

Finally, the "limitations" of hermaphrodism as understood by an
evolutionary system which would seek to amplify genetic mixing to
every extent possible does not seem to fit the ascocotylids. Every
member of this diverse group is a hermaphrodite, yet many
significant differences between species exist. Often these
difficulties are so dramatic that many new Genus and subgenus levels
have been established and discarded [53, 54, 55].

CONCLUSION

Complex obligate life cycles, as shown within the Ascocotyle complex
require that all of the special structures and features be in place
or the system will fail and the organism will not live to bear
offspring. Eggs must be ingested by a snail which will not digest
them, but rather, which will provide sanctuary for the cercaria to
develop in close proximity to the next host in the life cycle. The
second intermediate host must as well, readily accept the infection
and also be able to support a large parasite burden with no ill
effects. The cercaria must be able to penetrate that host tissue,
with a minimum of tissue response, and find the appropriate organ
for encystment. Some cercariae must have the ability to resist
gastric digestion on their way to encystment within the intestinal
lining. The metacercaria must produce an HCl resistant, yet trypsin
sensitive cyst. Finally, the hermaphroditic adult must be able to
survive in the definitive host and produce eggs which will fall
exactly where the first host may ingest them. Chance alone cannot
account for this system or the structures it displays. An argument
from intelligent design, however, might be made on the basis of the
"irreducible complexity" of the structures and features found in
this group.

ACKNOWLEDGEMENTS

I thank the reviewers for their suggestions, the late Dr. Richard D.
Lumsden for his guidance and friendship, Dr. Les Eddington of Azusa
Pacific University for free use of the EM suite, Mr. Ronnie Palmer
of the Gulf Coast Research Lab for A. pachycystis and A. leighi
specimens, Mr. Brandt Darby for help with collection of specimens in
California, Mr. Tom Keeney, Environmentalist, Point Mugu Naval Air
Weapons Station and finally, my family, especially Patti, my wife
for putting up with an 'in-home' laboratory.

FIGURES:

Figure 1. Ascocotyle mcintoshi, after Lumsden, 1963. Scale bar = 10
microns
Figure 2. Typical redia, after Stein, 1968. Scale bar = 10 microns
Figures 3, 4. Typical cercaria, after Stein, 1968. Scale bar = 10
microns
Figure 5. Gambusia affinis, with penny for reference.
Figure 6. Fundulus parvipinnis, with penny for reference.
Figure 7. A. diminuta, SEM micrographs.
Figure 8. A. angrense, whole mount, brightfield.
Figure 9. A. leighi, encysted in fish heart, thin section.
Figure 10. A. pachycystis, encysted in fish heart, thin section.
Figure 11. A. diminuta, encysted in fish gill, thin section.
Figure 12. A. pachycystis, showing tegumental papillae.

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