- Thiamine (vitamin B₁) is required by all living organisms for carbohydrate metabolism
and synthesis of amino acids. Thiamine deficiency is responsible for several related
classes of early life stage mortality disorders in salmonines, including Thiamine
Deficiency Complex (TDC) in the Laurentian Great Lakes, Cayuga Syndrome in the
Finger Lakes, and the M74 in the Baltic Sea. TDC is caused not by lack of sufficient
intake of thiamine but rather by a diet rich in prey that contain thiaminase, a
thiamine-degrading enzyme. TDC caused by ingestion of thiaminase-containing
prey occurs in these wild fish populations and has also been observed in mink,
foxes, seals, alligators, chickens, and ruminants.
TDC is one of several impediments to rehabilitation of Lake Trout (Salvelinus
namaycush), the native apex predator in the Great Lakes. The proximate cause of
TDC is known to be the ingestion of prey fish containing high levels of thiaminase,
specifically Alewife (Alosa pseudoharengus) and Rainbow Smelt (Osmerus mordax);
however, the ultimate source of thiaminase in thiaminase-containing fishes remains
unknown. Evaluating potential sources of thiaminase in fishes is essential for
understanding thiaminase trophodynamics and developing management strategies
for mitigating TDC and facilitating rehabilitation of Lake Trout populations.
Thiaminase in prey fishes is hypothesized to originate from two possible sources:
either thiaminase-containing fishes acquire thiaminase from their diet (dietary
acquisition hypothesis) or thiaminase-containing fishes make the thiaminase enzyme
themselves (de novo synthesis hypothesis); these two hypotheses are not mutually exclusive. Chapter 1 describes the causes of TDC and why characterization of the
sources of thiaminase in aquatic food webs is necessary.
The three primary data chapters presented herein describe whole food web
investigations that seek to determine the source of thiaminase in Great Lakes fishes.
In Chapter 2, zooplankton were evaluated as a potential source of dietarily acquired
thiaminase activity by comparing the thiaminase activity in bulk zooplankton to the
zooplankton community composition. Three types of multivariate analysis revealed
one candidate source of thiaminase activity, Ploesoma, an omnivorous loricate
rotifer. Despite the apparent correlation between Ploesoma biomass and thiaminase
activity in bulk zooplankton, Ploesoma spp. constituted sufficiently low biomass that
its thiaminase activity would have to be extraordinarily high (i.e., at unprecedented
levels) to constitute the major source of thiaminase in Great Lakes food webs.
Furthermore, Ploesoma was never observed in the diet of thiaminase-containing fish.
Therefore Ploesoma is an unlikely source of thiaminase. No other component of the
zooplankton community was consistently related to thiaminase activity.
In Chapter 3, the thiaminase activity in fishes was assessed in relation to species,
season, depth, and location of capture, and the dietary acquisition hypothesis was
assessed directly using stomach content and fatty acid signature analysis.
Thiaminase activity was higher in spring than in summer and fall. Round Goby
(Neogobius melanostomus) thiaminase activity was higher than previously reported,
and Slimy Sculpin (Cottus cognatus) thiaminase activity was highly variable,
sometimes exceeding that of Rainbow Smelt. The dietary acquisition hypothesis
was evaluated by comparing the thiaminase activity in fish viscera to both the diets
and fatty acid signatures of fishes. No compelling evidence that thiaminase activity
of fish viscera was consistently the result of consumption of any specific prey taxa
was found using three types of multivariate analyses. Indicator Species Analysis
suggested Bythotrephes as a potential candidate source of thiaminase, but
Bythotrephes was not consumed by some fishes with high thiaminase activity. If the
source of thiaminase activity is dietary, the source may be Bythotrephes for some
fishes, but those that did not consume Bythotrephes would need to obtain thiaminase
from a different source. Fatty acid analysis suggested a moderate tendency for
higher thiaminase activity in pelagically-feeding rather than benthically-feeding fishes, but several pelagically feeding fishes contained undetectable thiaminase
activity and some benthically feeding fishes contained thiaminase activity
comparable to or greater than pelagically feeding fishes. Together, this evidence
indicated that no specific prey item was consistently related to thiaminase activity.
In Chapter 4, the de novo hypothesis was assessed directly using two approaches.
The first approach compared the biochemical characteristics of thiaminases in
Alewife, Carp (Cyprinus carpio), quagga mussels (Dreissena rostriformis bugensis),
and a bacterium (Paenibacillus thiaminolyticus) that has been isolated from the
intestines of Alewife and is known to produce thiaminase. Thiaminases in these four
organisms vary in their mass, migration characteristics, tolerance to denaturation,
and isoelectric points, suggesting that the source of thiaminase differs in these four
taxa. The second approach identified candidate thiaminase genes in fishes using
existing information from small peptide fragments from two fish thiaminases from
partially purified from Carp and Red Cornetfish (Fistularia petimba). Candidate
genes in Carp, Zebrafish (Danio rerio), and Alewife that were homologous to the
known peptide fragments were identified, synthesized, and overexpressed. The
Zebrafish candidate gene produced an active thiaminase enzyme. This finding
confirms de novo synthesis and represents the first report of a thiaminase-encoding
protein in any multicellular organism. The candidate gene identified for Alewife is
predicted to produce a protein product with biochemical properties that match those
The dietary acquisition hypothesis was not well-supported, and findings from
Chapters 2 and 3 did not converge as would be expected if the source of thiaminase
was dietarily acquired. The potential for de novo synthesis by fishes was confirmed
experimentally, which represents the first report of a gene for a thiaminase enzyme
from a multicellular animal. Future research should focus on confirming that de novo
production accounts for the thiaminase activity in fishes and understanding the
physiological factors that lead to increased thiaminase production. This work is
relevant to fishery managers in affected ecosystems (Great Lake, Baltic Sea, Finger
Lakes, NY) and to biochemists and nutritionist interested in thiamine metabolism.