From conical to spatulate: intra- and interspecific changes in tooth shape in closely related cichlids (Teleostei; Cichlidae: Eretmodini).

The Eretmodini are closely related cichlids endemic to Lake Tanganyika with very divergent oral tooth shapes, ranging from spatulate in Eretmodus to conical in Tanganicodus. To study how closely related cichlids can generate such divergent tooth shapes, we investigated how the enamel organ directs the development of spatulate teeth in Eretmodus cf. cyanostictus (lineage A), both in ontogeny and in adults, and of conical teeth in adult Tanganicodus cf. irsacae, using 3D-reconstructions from serially sectioned tooth germs. The spatulate oral tooth shape that characterizes adult E. cf. cyanostictus (lineage A) is preceded early in ontogeny by a conical tooth shape. We propose two possible hypotheses to account for changes in the folding of the enamel organ (in particular its epithelio-mesenchymal boundary) capable of generating such distinct tooth shapes. Different arguments lead us to favor the hypothesis of an asymmetric growth and differentiation of the enamel organ, such that the tip of a conical tooth corresponds to one "corner" of a spatulate tooth. Applying current molecular models of tooth shape variation, this would imply the existence of asymmetric fields of inhibition. Whether such asymmetric growth reflects the reutilization of a simple mechanism operating in ontogeny has to be clarified.

Fish dentitions as paradigms for odontogenic questions.

Bony fish, and in particular teleosts, represent a morphologically extremely diverse group of vertebrates, well suited to study certain problems in odontogenesis. In this article we address some questions that can benefit much from the use of fish dentitions as paradigms, such as endodermal participation in tooth formation and epithelial primacy in initiation events. Next, we highlight some results recently obtained in our laboratory with respect to two models, the zebrafish (Cyprinidae), and selected species of cichlids (Cichlidae). Finally, we pinpoint some questions that lend themselves admirably to be examined using fish models, such as the factors that control renewed initiation of teeth, and the relationship (or absence thereof) between Hox genes and tooth formation.

Tooth shape differences analyzed by biometric and morphometric approaches: a case study on two morphologically very similar lacustrine cichlid species.

Tooth shape is generally considered to be under strong genetic control, and highly species specific. It is therefore widely used for taxonomic studies. The tooth shape-based morphological classification of the Eretmodini (a tribe of closely related cichlids endemic to Lake Tanganyika), has, however, been shown to be in conflict with recent molecular data, which suggests the occurrence of parallel evolution. A detailed biometric and morphometric analysis of tooth shape has been undertaken for two genetic lineages of the taxon Eretmodus cyanostictus. The use of both landmark-based measurements and elliptic Fourier analysis reveals differences between both lineages and supports the molecular phylogeny. These differences further endorse the interpretation of parallel evolution. In addition, we here present a powerful tool for the analysis of tooth shape.

The accumulation, distribution, and toxicological effects of dietary arsenic exposure in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush).

A 20-day experiment was conducted to compare the accumulation, distribution, and toxicological effects of dietary As, as arsenate, in lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush). Results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed three times per week to lower dietary As doses. In the present study each treatment group was exposed to a combination of one of three doses of As (0, 100, or 1000 microg As/g) and one of two types of diet, no brine shrimp (NS) or with brine shrimp (WS) for a total of eight dosing events. Brine shrimp were added to determine whether their presence enhanced consumption of As-contaminated food. Modified feeding behavior occurred in both fish species fed As contaminated diets, with the exception of the 100 microg As/g NS food. Brine shrimp addition did not affect feed consumption of the As contaminated diets. Significant As accumulation occurred in stomach, pyloric caeca, intestine, liver, kidney, and gallbladder, but not in bile or muscle. As exposure did not have a significant effect on hepatic and renal metallothionein concentrations. Concentrations of lipid peroxides were only significantly elevated in the plasma of LT fed the 1000 microg As/g WS food. Liver somatic indices decreased significantly in both species, whereas hematological parameters were not affected in either species. Histological lesions occurred in gallbladder, liver, kidney, pyloric caeca and intestine from LWF. These lesions were not observed in LT; however, gallbladders were not examined in this species. Weight gain was lower in both species fed As contaminated diets, however, condition factors were not affected.

Altered distribution of lipid-soluble antioxidant vitamins in juvenile sturgeon exposed to waterborne ethynylestradiol.

Studies in mammals have shown that exposure to estrogenic compounds can affect lipid metabolism and plasma concentrations of lipid-soluble vitamins. However, the potential for estrogenic contaminants to induce these effects in fish has not yet been examined. The ability of the estrogen analog ethynylestradiol (EE2) to alter concentrations of the lipid-soluble vitamins A and E in plasma, liver, and kidney was investigated in juvenile lake sturgeon (430 +/- 20 g). The EE2 was delivered to the sturgeon in the tank water at nominal concentrations of 0 (control), 15, 60, or 125 ng EE2/L for a period of 25 d. Concentrations of the egg yolk precursor, vitellogenin, increased dose dependently in plasma. Plasma vitamin E (tocopherol), A1 (retinol), and A2 (dehydroretinol) were elevated by the two highest EE2 treatments compared with the controls. Esterified storage forms of vitamin A were marginally lower in the livers of fish from the highest EE2 dose group, but vitamin E levels in the liver were not significantly affected. Concentrations of vitamin E and vitamin A were significantly depleted in the kidney of fish from the two highest EE2 dose groups. Total lipid content was elevated in the gonad of fish treated with the highest dose of EE2 compared with the controls. Altered lipid and vitamin distribution may be induced by estrogen to facilitate gonadal maturation in sturgeon. Results from these studies indicate that an examination of the implications for vitamin depletion by estrogenic contaminants in juvenile fish is warranted.

Tooth succession in the zebrafish (Danio rerio).

To test whether the formation of replacement teeth in arbitrarily chosen zebrafish follows the same pattern as described for larval and young zebrafish of known age, dentitions of more than 90 animals of different ages and standard lengths were observed under the stereomicroscope. Only the teeth of the ventral tooth row (1V-5V) were considered. Statistical results (G-tests) suggested that tooth replacement does not occur randomly. The most common order of replacement can be represented as the formula 5V-2V-3V-1V-4V and counts for approximately 70% of the observed patterns. Initiation of replacement teeth at positions 4V and 5V is separated by a larger time interval than between any other combination. It is hypothesized that in older juveniles and adults, replacement teeth may be formed during three odontogenic waves.

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush).

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.

A quantitative analysis of pharyngeal tooth shape in the zebrafish (Danio rerio, Teleostei, Cyprinidae).

To test whether successive replacement cycles in the pharyngeal dentition of the zebrafish, a polyphyodont vertebrate model organism, entail overall shape changes in the teeth, a qualitative and quantitative analysis was made of size and shape variables in the five ventral teeth. The following measurements were defined: tooth length, tooth height, neck-crown angle, cusp depth, and crown curvature. Ontogenetic changes in fish, ranging between 6 and 29 mm standard length (SL), were analysed by linear regressions on to SL. The teeth became significantly larger with growth of the fish, through successive replacements and cusp depth also increased over time. Neck--crown angle and crown curvature did not change over time. Position-dependent differences were analysed by Friedman ANOVA and Kendall concordance tests. Measurements differed significantly according to tooth position in the pharyngeal jaws. Tooth 1V was always the smallest, 3V the largest. The neck--crown angle and curvature of the crown increased from 1V to 5V. Cusp depth increased from 1V to 3V, and then decreased again. These results indicate that successive replacement cycles entail a size increase accompanied by shape changes apparently restricted to the crown. These quantitative data lay the basis for further descriptive and experimental studies of tooth shape in this model-species.

A modified mercury saturation assay for measuring metallothionein.

A thorough re-evaluation of assay procedures for a mercury displacement analytical method for metallothionein (MT) described in 1993, was conducted using an analytical standard of rabbit liver MT-II and MT determinations, in liver, kidney, gill and intestine from lake trout (Salvelinus namaycush). These studies indicated that: (1) loss of MT by adherence to polypropylene material occurs with the dilution series; (2) use of a calibration curve using a rabbit liver MT-II analytical standard, is superior to the dilution series for determining unknown MT concentrations; (3) Hg incubation time requirements are less in 20% TCA than in 10% TCA; and (4) employing both 20% TCA and heat at 95 degrees C for 5 min to denature non MT proteins results in the most accurate MT spike recoveries, or recoveries that did not differ significantly from those closest to 100%, in liver, kidney, gill and intestine. Calibration curves with an average r(2) of 0.9998 for 69 trials over different days, while passing through the origin, provided evidence of negligible non-specific binding of mercury. Use of water or saline for preparing tissue homogenates, produced slight, but significant, differences in MT estimates in the intestine and kidney. Comparisons between the original and the modified methods were made on liver, kidney and gill from control, sham-injected and Cd-treated lake trout. Differences in MT estimates obtained between the two methods depended upon the tissue. MT estimates in liver by the modified method were 9, 13, and 10% higher in control, sham-injected and Cd-treated groups, respectively, than those resulting from the original assay. MT estimates in gill by the modified method, were 3.2-fold, 4.0-fold and 69% higher in control, sham-injected, and Cd-treated groups, respectively. Corresponding values for kidney were 37, 75, and 57% higher. Because percent recoveries of MT-II spikes were virtually 100%, in all four tissues, using the modified method, MT estimates obtained using these modifications were more accurate than those obtained using the procedures of the original assay. Application of the modified assay to fish tissues in a Canadian biomonitoring program for metal mining, demonstrated that in 1 week an experienced technologist can complete 90 MT analyses of liver and kidney and 75 analyses of gill and intestine.