Figure 2 . Frequency of push-up behavior under different thermal conditions.

1. A BEHAVIORAL ASSAY OF THERMAL AND DISSOLVED OXYGEN STRESS IN STONEFLY LARVAE (PLECOPTERA: PERLIDAE) Darrell Carson & Scott L. Kight, Dept. of Biology and Molecular Biology, Montclair State University ABSTRACT. We report the results of a preliminary study of environmental stress on stonefly larvae (Acroneuria sp.) collected from a tributary of the Big Flatbrook River in Sussex County, New Jersey. We were primarily interested in whether larval “push-up” behavior, presumably used to increase aeration of gills, could be used as an indicator for multiple sources of stress. Larvae were collected in July 2005 via kick netting and returned to a nearby laboratory at the New Jersey School of Conservation for experimental observations. Specimens were housed and acclimated in two liter aquaria containing heavily aerated stream water and cobble at 22C for 24 h prior to observation. In the first experiment, individuals were placed sequentially in 22C tanks with three different dissolved oxygen conditions (5.7, 5.3, and 4.9 PPM). Stoneflies in the lowest dissolved oxygen condition exhibited more push-ups, on average over a 60 s period, but due to small sample size no significant effect was detected. In the second experiment, dissolved oxygen was maximized with multiple aerators, and individuals were placed sequentially in tanks with three different thermal conditions (22, 25, and 29C). There were significant differences between thermal treatments, and it is important to note that most subjects exhibited no push-ups at all when placed in the warmest thermal conditions for 60 s. It seems likely that these extremely warm conditions caused the animals to enter a state of shock. As these data are preliminary, our conclusions are tentative. However, this behavioral assay appears to be a useful index of oxygen and thermal stress in this indicator species. INTRODUCTION Stonefly larvae (Insecta: Plecoptera) have long been recognized as a useful group of aquatic macroinvertebrates for assessing stream quality. A common measure of stream health is the richness of taxa in three particular groups: the stoneflies, the mayflies (Insecta: Ephemeroptera) and the caddisflies (Insecta: Trichoptera). These organisms are found primarily in cool, oxygen-rich stream riffles and are generally intolerant to pollution, turbidity, organic enrichment, and other forms of environmental impact. This “EPT” metric is in fact an important indicator used in the Environmental Protection Agency’s Rapid Bioassessment Protocols, and hence the stoneflies are of widespread importance in monitoring and policy. Stoneflies are also of economic importance because they can be an important food source for trout. Stonefly larvae respire through external gills located at various positions on the exoskeleton. Oxygen and carbon dioxide are exchanged as rapidly moving stream water flows across the respiratory surface. However, when dissolved oxygen levels are compromised, stonefly larvae exhibit a characteristic compensatory behavioral pattern by moving the thorax and abdomen up and down in what can best be described as resembling “push ups”. This serves, presumably, to increase flow of water over the gill tissue and increases gas exchange. In the present study, we sought to first quantify this behavioral pattern under different dissolved oxygen regimes. Second, we examined whether push up behavior is also associated with other kinds of environmental stress, specifically sub-optimal thermal conditions. METHODS We collected stonefly larvae of the genus Acroneuria (Plecoptera: Perlidae) during the first two weeks of July 2005 in a tributary of the Big Flatbrook River in Stokes State Forest, Sussex County, New Jersey. Genus Acroneuria is characterized by relatively large larvae, and is among a group of plecopteran genera known commonly as “golden stoneflies” for their striking gold and brown mottled coloration. The collection site was located approximately 100 meters south of Lake Oquittunk, and at the time of collection cool (18C) but very low water levels characterized the stream. Specimens were collected by kick netting in riffles and returned to a laboratory at the New Jersey School of Conservation within one hour of collection. Animals were acclimated in heavily aerated 5 L aquaria containing stream cobble from the collection site at 22C for 24 h prior to experimental observations. Figure 1. Frequency of push-up behavior under different dissolved oxygen conditions. One group of larvae was observed under different dissolved oxygen conditions. Specimens were sequentially exposed for 60 s to each of three different conditions: 5.7, 5.3, and 4.9 PPM dissolved oxygen. The order in which animals were treated was randomly assigned: hence any differences between groups would not be attributable to the effects of a particular sequence (i.e. increasingly less 02). During each 60 s focal period, the number of push-ups was recorded. A second independent group of larvae was observed under different thermal conditions. Specimens were sequentially exposed for 60 s to each of three different conditions: 22, 25, and 29C. As before, the order in which animals were treated was randomly assigned: hence any differences between groups would not be attributable to the effects of a particular sequence (i.e. increasing temperature). During each 60 s focal period, the number of push-ups was recorded. RESULTS AND DISCUSSION Stonefly larvae exhibited more push-ups at the lowest levels of dissolved oxygen, but the result was not significant, possibly due to small sample size (Figure 1. N=10, Friedman 2 way AOV F=0.6 P=0.7408). We did detect significant effects in the temperature experiment (Figure 2. N=10, Friedman 2 way AOV F=9.784 P=0.0075), and this effect appears driven by the fact that most subjects exhibited no push-ups at all when placed in the warmest thermal conditions for 60 s. It seems likely that these extremely warm conditions caused the animals to enter a state of shock. As these data are preliminary, and our sample sizes are small, our conclusions are tentative. However, this behavioral assay appears to have the potential as a useful index of oxygen and thermal stress in this indicator species. We intend to investigate push-up behavior on-site in future investigations, to determine if there are differences in the behavior of stonefly larvae in streams that differ in the level of environmental impact. Figure 2. Frequency of push-up behavior under different thermal conditions.