Contributions to Zoology, 70 (2) (2001)Henry C. Merchant; Ritindra N. Khan; Robert E. Knowlton: The effect of macrophytic cover on survival of Palaemonetes pugio and P. vulgaris (grass shrimp) in the presence of predatory Fundulus heteroclitus (killifish)
Materials and methods

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Laboratory methods

In the laboratory, macrophytes and substrates were placed in separate 20-l plastic buckets containing aerated seawater. Shrimp were sorted by species and placed into separate 350-l flow-through aquaria equipped with undergravel biological filters. Aeration was provided by air stones that were connected via plastic tubing to a constant air supply. In these stock holding tanks, salinity (35 ppt), temperature (22°C), pH (8.2), dissolved oxygen (18mg/l), and photoperiod (12L:12D) were held constant. Photoperiod was maintained via automatic timers. In an attempt to minimize the influence of possible conditioning during holding, both species of shrimp were held without any cover or vertical structure (including macrophytes) over a level layer of colorless, commercially obtained aquarium gravel composed of quartz fragments. The gravel fragments were not homogeneous in size. The largest particles were 0.95 cm in diameter and were retained on a ♯5 USA Standard Testing Sieve; the smallest were retained on a ♯21 sieve. One percent (by weight) of the gravel was retained on the ♯5 sieve; 72% on a ♯10 sieve; 18% on a ♯12 sieve; 9% on the ♯21 sieve. While awaiting use in experiments, shrimp were fed daily surfeit amounts of commercially obtained fish food (Tetra-Min®, manufactured by TetraWerke, Melle, Germany). Food was not offered during the 12-hour period immediately preceding selection of shrimp for use in the experiments.

In a separate holding aquarium, Fundulus heteroclitus (80 ± 5 mm in length) were maintained over bare gravel in the same physico-chemical conditions as the grass shrimp. Killifish were fed a diet of Palaemonetes daily except that no prey were provided for 48 hours prior to their use as predators in experiments.

All experiments took place in four separate aquaria arranged linearly on a bench within the same room and at the same temperature and photoperiod as the holding tanks. Preliminary experiments indicated that there were no position effects associated with the location of the tanks with respect to each other. The experimental aquaria were each 60 × 30 × 35 cm (approx. 63-l) and were half filled with artificial sea water (35 ppt; pH 8.2) prepared by dissolving Instant Ocean® sea salts in deionized water. There was no flow-through system in the experimental aquaria. Air stones connected to the same air supply as the holding tanks provided constant aeration except that in an attempt to remove a possibly confounding source of procedural variation, the stones were removed 12 hours prior to and during the experiments. Earlier experiments (Khan et al., 1995) established species differences in the number of grass shrimp resting on the commonly occurring natural substrates (wood, sand, shell, and mud). Since the use of macrophytic cover in response to the presence of the predator might be influenced by an association with a particular type of natural substrate, the same artificial (and therefore more likely to be neutral) substrate used in the holding tanks (colorless quartz aquarium gravel) was used in the present experiments. Four plastic trays (each 30 × 15 cm) were tightly fitted into the bottom of each aquarium; into each tray a level (2-cm thick) layer of aquarium gravel was placed. In one of the aquaria, all four trays were left without macrophytic cover. In the other three aquaria, individuals of one type of macrophytic cover were randomly selected from the stock tanks and added to all four trays. One of the three aquaria received Codium; another, Ulva; the third, a synthetic ”plant” made of a plastic material (polypropylene) superficially resembling Ambulia. Within each aquarium enriched with macrophytes, approximately 90% of the area was covered by individuals of the appropriate macrophyte. Ten gram lead weights held the macrophytes in position. Highly branched Codium offered cover that was a more-or-less erect loose tangle of semi-rigid thalli at various levels above the substrate. The individuals of Codium were densely covered with periphyton. The broad, flat thalli of Ulva “flopped” over, providing a broad sheet of cover appressed at the unanchored edge against the gravel substrate. Periphyton on Ulva, if present, was not noticeable. The synthetic “Ambulia” provided structure characterized by stiff upright “stems” with regular broader “leaves” at regular vertical intervals. No periphyton was present on the “Ambulia.”

In all of the experiments reported here, counts of shrimp were made during daylight hours under ambient room lighting. Shrimp tended to be sedentary for sufficiently long times to allow careful counting. When macrophytic covers were provided, their nature and density did not interfere with accurate counting of shrimp. Codium was openly branched and the loose tangle of thalli permitted seeing into the cover. Erect portions of Ulva provided passages between the thalli that allowed the observer to readily see into the cover, and although Ulva tended to flop over on itself, it did so such that shrimp could congregate within the spaces between the bends and folds. Shrimp tended not to push themselves between the appressed edge of the Ulva thalli and the substrate, and therefore remained visible to the experimenter. “Ambulia” was upright and rigid with many spaces between “stems” and “leaves” that permitted seeing into the cover.

After the experimental aquaria were established, 20 individual Palaemonetes pugio (30 ± 5 mm in length) and two Fundulus heteroclitus (80 ± 5 mm in length) were selected at random from the stock aquaria and added to each of the experimental aquaria. Numbers of prey and predator individuals used per replicate experiment were based upon pilot studies conducted over bare gravel (as used in the present study) which indicated that one killifish of this size could consume five Palaemonetes of either species in 24 hours. If this exploitation rate prevailed in the present study, a starting population of 20 prey individuals would leave 10 live individuals after 24 hours exposure to the predator. The aquaria were left undisturbed for 24 hours after which the Fundulus were removed from the aquaria, and the number of surviving prey was counted and recorded. Counts are known to be accurate because at the time of counting, all shrimp were removed, recounted, and discarded. Killifish, live and plastic plants, and gravel also were removed from the aquaria and discarded. This entire experimental procedure was repeated once using different individuals of the same prey species, different individuals of the predator (Fundulus), different individuals of live and plastic macrophytic cover, and different gravel.These procedures were repeated twice more using the second prey species, Palaemonetes vulgaris (30 ± 5 mm in length), instead of P. pugio.

The experimental protocol was repeated two more times, but in each of these replicates, 15 individuals of each prey species were substituted for the 20 individuals of a single prey species. In these “mixed-species” experiments, distinguishing the two species was made easier by prior staining of one of the species with Alcian blue. It has been established that this procedure affects neither the viability nor the behavior of stained individuals (Coen et al., 1981). In the first of these mixed species replicates, P. vulgaris was stained; in the second, P. pugio. In the present studies, the similarity in the number of each species consumed by the predator when the prey was stained vs. unstained suggests that vulnerability to predation was also not affected by staining.