The results of this study indicate that an artificial diet of homogenized, de–shelled and gelled brine shrimp nauplii yields similar patterns of gastrovascular transport and stolon growth as a diet of live brine shrimp nauplii. However, the hydractiniids were significantly less likely to ingest the artificial diet blocks than live nauplii. These results bear on the potential use of experimental diets for physiological studies of hydrozoans.
The similar rates of growth and gastrovascular transport indicated no morphogenetic, nor physiological artefacts were associated with consumption of an experimentally generated diet during the first 24 hours. Experimental diets, therefore, may be useful to test the effects of specific compounds on morphogenesis and growth of hydrozoans. It would be possible to generate a dose-response curve for any signal chemical or metabolite that could be included in agar formulation. For instance, it has been suggested that glucose has an important role in colony development (Blackstone, 2003
). Optimizing the protocol for an artificial diet that could systematically vary concentrations of glucose and other metabolites suspected of influencing development could provide direct tests of these hypotheses. Moreover, effects of two compounds could be tested simultaneously by systematically varying their concentration in a factorial combination. Such experiments may provide insight into the physiological regulation of morphological plasticity of hydrozoan colonies.
The analysis to test for the presence of artefacts associated with experimental diets is complicated because it relies on negative evidence (i.e. expectation of similar outcomes in different experimental treatments). However, tests of no effect are not a problem when the power of the experiment to detect the alternative hypothesis is specified a priori. Treatments that reduced rates of gastrovascular transport and growth by 25% in 24 hours were taken to constitute significant experimental artefacts. The power of our experiment to detect significant artefacts associated with the brine shrimp-agar blocks was 0.70 and 0.90 for transport and growth rates, respectively. The power of the experiment dropped to 0.45 and 0.55 for transport and growth rates, respectively, at a resolution of 20% difference in effect between treatments. We inferred that the experimental power was sufficient to reject the null hypothesis. In other words, the experimental treatments did not cause artefacts with respect to growth and vascular transport at a resolution of ≥20% change). Nevertheless, there appears to be a trend towards lower rates of transport in the brine shrimp–agar (BSA) block treatment (), suggesting that we may not be able to detect small differences (<20%), should such differences be biologically meaningful. It is important to point out in this context that future experiments using this protocol to test effects of specific compounds would likely be comparing two treatments using brine shrimp–agar blocks (e.g. a nutrient added to agar blocks in one treatment, but absent in the other). Thus, any (small) effect(s) of the BSA diet would cancel themselves out allowing the effects of the nutrient to be accurately determined.
The low frequency of ingestion of experimental diets in agar cubes is problematic for routine and efficient use in experiments. However, differences in ingestion frequency between different experimental feeding conditions suggest that ingestion frequency could be improved. Our data suggest that dissolved substances, exuded by actively swimming brine shrimp may serve as a cue to polyps for the presence of food. The frequency of ingestion of brine shrimp homogenate in agar cubes was ~60−70% when the polyps were immersed in seawater in which brine shrimp nauplii were recently (within 30 min) cultured, compared to 20% ingestion in unmanipulated seawater. The frequency of ingestion of brine shrimp homogenate agar cubes was similar whether they were served in seawater from brine shrimp culture or in seawater plus a mixture of dNTP (5 μM). Moreover, there was no added effect on ingestion of serving agar cubes impregnated with 5 μM dNTPs in seawater from brine shrimp cultures, suggesting that polyps may cue on the presence of dNTPs released from actively swimming brine shrimp nauplii. Our results are similar to previous work that showed low concentrations of dCDP elicited a feeding response in Podocoryna carnea (D. Lambert, unpublished data).
Our results using P. carnea
differ from those obtained using Hydra littoralis
) and another study using P. carnea
(Doolen et al., 2007
). First, glutathione, which is known to elicit the feeding reflex in Hydra
(up to 5 μM; Lenhoff, 1961
), did not elicit such a response in our trials using P. carnea
using similar concentrations, but Doolen et al. (2007)
observed the feeding response at concentrations exceeding 65 μM. The end of the glutathione response in Hydra
is endogenously controlled rather than oxidation, disappearance or alteration of the glutathione molecule (Lenhoff, 1961
), and evidence suggests a role for arachidonic acid as an endogenous signal (Pierobon et al
., 2003). In P. carnea
, 5 μM dNTPs increased the frequency of ingestion of artificial foods compared to treatments lacking them, but the 60% ingestion frequency was lower than one would expect from an effective elicitor of a feeding reflex. The ingestion response of P. carnea
also was sensitive to whether the seawater in which they were fed contained brine shrimp within the previous 30 minutes. Further experiments systematically comparing seawater ages after brine shrimp removal are required to establish whether the molecules in seawater that elicit feeding reflexes are short-lived, and if other factors (exo- or endogenous) control the feeding reflex of P. carnea
In summary, the use of experimental diets that manipulate the concentrations of one or more molecules show potential as a means to manipulate physiological state and assay the effects on the growth and morphology of hydrozoan colonies. The frequency of ingestion of experimental foods is too low for reliable use in experiments at present, requiring many additional replicates for a sufficient sample size. The technique would benefit from future studies aimed at increasing the frequency of ingestion of experimental foods and streamlining the production of experimental foods. Although experimental foods are ingested too infrequently at present, there are no apparent artefacts associated with such products and rates of growth and vascular transport in experimental colonies are equivalent to that of controls fed an equivalent quantity of live brine shrimp.