Western North American Naturalist 81(1), © 2021, pp. 54–62 Cold tolerance of mountain stoneflies (Plecoptera: Nemouridae) from the high Rocky Mountains SCOTT HOTALING1,*,†, ALISHA A. SHAH2,†, MICHAEL E. DILLON3, J. JOSEPH GIERSCH4, LUSHA M. TRONSTAD5, DEBRA S. FINN6, H. ARTHUR WOODS2, AND JOANNA L. KELLEY1 1School of Biological Sciences, Washington State University, Pullman, WA 2Division of Biological Sciences, University of Montana, Missoula, MT 3Department of Zoology and Physiology and Program in Ecology, University of Wyoming, Laramie, WY 4Northern Rocky Mountain Science Center, U.S. Geological Survey, West Glacier, MT 5Wyoming Natural Diversity Database, University of Wyoming, Laramie, WY 6Department of Biology, Missouri State University, Springfield, MO ABSTRACT.—How aquatic insects cope with cold temperatures is poorly understood. This is particularly true for high-elevation species, which often experience a seasonal risk of freezing. In the Rocky Mountains, nemourid stoneflies (Plecoptera: Nemouridae) are a major component of mountain stream biodiversity and are typically found in streams fed by glaciers and snowfields, which are rapidly receding due to climate change. Predicting the effects of climate change on mountain stoneflies is difficult because their thermal physiology is largely unknown. We investigated cold tolerance of several alpine stoneflies (Lednia tumana, Lednia tetonica, and Zapada spp.) from the Rocky Mountains, USA. We mea- sured the supercooling point (SCP) and tolerance to ice enclosure of late-instar nymphs collected from a range of thermal regimes. SCPs varied among species and populations, with the lowest SCP measured for nymphs from an alpine pond, which was much more likely to freeze solid in winter than flowing streams. We also show that L. tumana cannot survive being enclosed in ice, even for short periods of time (<3 h) at relatively mild temperatures (−0.5 °C). Our results indi- cate that high-elevation stoneflies at greater risk of freezing may have correspondingly lower SCPs, and despite their common association with glacial meltwater, these stoneflies appear to be living near their lower thermal limits. RESUMEN.—No se comprende bien sobre cómo los insectos acuáticos pueden sobrellevar las temperaturas muy bajas. Esto es particularmente cierto para especies que habitan en elevaciones muy altas y que con frecuencia experi- mentan un riesgo de congelamiento estacional. En las montañas rocosas, las moscas de la roca (Plecóptera: Nemouri- dae), son un componente importante de la biodiversidad de los arroyos de montaña y se encuentran típicamente en arroyos alimentados por glaciares y campos de nieve que, debido al cambio climático, están retrocediendo rápidamente. El predecir los efectos del cambio climático en esta especie de plecópteros es difícil puesto que su fisiología térmica es en gran parte desconocida. Hemos investigado la tolerancia al frío de varias moscas de roca alpinas (Lednia tumana, Lednia tetonica, and Zapada spp.) de las montañas rocosas de los Estados Unidos. Hemos medido un punto de sobreen- friamiento y la tolerancia al hielo en confinamiento de ninfas en estadio tardío colectadas de un rango de regímenes térmicos. Los puntos de sobreenfrimiento variaron entre las especies y las poblaciones, las más bajas en los puntos de sobreenfrimiento fueron las ninfas de un estanque alpino, que era más susceptible a congelarse en el invierno que los arroyos de agua corriente. También encontramos que L. tumana no puede sobrevivir estando encerrada en hielo, aún en pequeños periodos de tiempo (<3 h) en temperaturas relativamente moderadas (−0.5 °C). Nuestros resultados indican que las moscas de roca en elevaciones prominentes con mayor riesgo de congelamiento pueden tener correspondiente- mente un bajo punto de sobreenfrimiento, y a pesar de su asociación común con el agua de deshielo glacial, estas moscas parecen habitar cerca de sus límites bajos térmicos. Freshwater habitats in cold regions typically or more thermally stable microclimates or by experience winter ice cover and freezing tem- modifying local conditions (e.g., through case- peratures, which can be injurious or lethal to making; Danks 1971). Others withstand cold aquatic insects (Danks 2008). Resident species conditions in situ through supercooling (i.e., may avoid extreme cold by moving to warmer maintaining internal water in liquid form below *Corresponding author: [email protected] †Contributed equally SH orcid.org/0000-0002-5965-0986 AAS orcid.org/0000-0002-8454-7905 54 HOTALING ET AL. ♦ COLD TOLERANCE OF MOUNTAIN STONEFLIES 55 its freezing point) or freeze tolerance (Zachari- nymphs; Danks 2008) and for species living assen 1985, Danks 2008, Sinclair et al. 2015). at high latitudes and elevations, where taxa Organisms living in wet environments are likely are most likely to experience freezing. Aquatic to encounter external ice when their body tem- insects generally employ an array of physio- peratures are already at or near freezing. Thus, logical and biochemical strategies to mitigate inoculative freezing, in which contact with cold stress (reviewed by Lencioni et al. 2004). external ice induces the formation of internal These include production of cryoprotectants ice, is likely the main driver of organismal freez- to lower the temperature of internal ice forma- ing in these habitats (Frisbie and Lee 1997). tion (Duman 2015); cryoprotective dehydra- At high elevations, aquatic habitats are typ- tion, in which body water is lost to lower the ically distributed across a mosaic of streams amount available for freezing (e.g., Gehrken and ponds that are fed by an array of hydro- and Sømme 1987); and resistance to anoxic logical sources (e.g., glaciers, perennial snow- conditions induced by ice enclosure (e.g., fields, subterranean ice; Hotaling et al. 2017, Brittain and Nagell 1981). Eggs of the stonefly Brighenti et al. 2021). These habitats remain Arcynopteryx compacta (Perlodidae) from the snow covered for much of the year (Lencioni mountains of southern Norway avoid freez- 2004). Mountain streams do not freeze solid ing by supercooling to −31 °C, a trait medi- because of their steep gradients and year- ated by the loss of approximately two-thirds of round flow, with minimum temperatures often the eggs’ water content (Gehrken and Sømme close to 0 °C in the main channel (Giersch et 1987, Gehrken 1989). Nymphs of another high- al. 2017, Shah et al. 2017, Tronstad et al. latitude stonefly, Nemoura arctica (Nemour - 2020). Resident taxa are therefore predicted to idae), survive freezing to well below 0 °C by experience minimum temperatures of ~0 °C. preventing intracellular ice formation through This prediction rests on the assumption that the production of a xylomannan-based glyco - overwintering nymphs remain in the unfrozen lipid. This glycolipid inhibits inoculative freez- primary stream channel throughout the winter ing through the inactivation of ice nucleating season. However, because of the inherent varia- agents (Walters et al. 2009, 2011). However, it tion in aquatic habitats, nymphs inhabiting is unlikely that glycolipid production repre- different regions of a stream (e.g., littoral zone, sents a singular “magic bullet” that confers bed sediments) may experience subfreezing freeze tolerance in stoneflies, or aquatic insects temperatures, even when the rest of the stream broadly, given the diversity of known freeze- remains unfrozen (Lencioni 2004). Alpine protecting molecules (Toxopeus and Sinclair ponds, in contrast, have little or no flow and 2018). Like N. arctica, high-alpine nemourids are often shallow, making them susceptible to in North America appear to overwinter as freezing solid (Wissinger et al. 2016). As melt- early-instar nymphs (Supplementary Material 1) induced flows are reduced in the future under and may be exposed to a similar risk of freez- climate change (Huss and Hock 2018), head- ing. How they cope with such risks, however, water streams will become shallower with less has not been investigated. flow velocity, raising the risk of freezing in all In this study, we investigated cold toler- seasons for resident organisms. ance across populations of late-instar nymphs Stoneflies (order Plecoptera) inhabit every from at least 3 species—Lednia tumana (Ricker continent except Antarctica and, when pres - 1952), Lednia tetonica Baumann & Call 2010, ent, often represent a substantial portion of and Zapada spp.; Nemouridae—in the north- aquatic biodiversity (DeWalt et al. 2015). After ern Rocky Mountains, USA. For all popula- hatching, stoneflies follow a 2-stage life cycle, tions, we measured the dry supercooling point with development through multiple instars as (SCP), which is the temperature at which an aquatic larval nymphs followed by emergence organism releases latent heat indicative of ice as winged terrestrial adults. Whether develop- formation (Sinclair et al. 2015) in the absence ment occurs in a single year or spans multiple of ice. Enclosure in ice may also be a major seasons, eggs and larvae of high-elevation stone - risk for mountain stoneflies, especially in flies are exposed to near freezing temperatures. slower-flowing shallow streams. Ice enclosure Cold tolerance of stoneflies and of aquatic can cause mortality through inoculative freez- insects in general is not well known, particu- ing, hypoxia, mechanical damage, or a combi- larly for nonadult life stages (i.e., eggs or nation of these factors (Conradi-Larsen and 56 WESTERN NORTH AMERICAN NATURALIST (2021), VOL.