0-{ 0 H Freeze Tolerance ~~~~~~~~~~~~~~~~~~~~~~~4-1 0- in the GoldenrodGall Fly Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/29/339930/4451922.pdf by guest on 28 September 2021 4. LUKEH. SANDRO RICHARDE. LEE,JR. BJirds migrate. Bears hibernate. Turtles and One aspect of goldenrod gallmakers that has frogs retreatto the bottom of lakes. Most animals must received little attention in the science education litera- avoid harsh winter conditions; few can survive freezing. ture is the winter biology of these unusual insects. In Larvae of the goldenrod gall fly (Eurosta solidaginis), autumn, the overwintering larva enters a state of dor- can survive freezing to -40?C or below. The study of mancy, called diapause, and gradually acquires the survival at low temperatureis called cryobiology. This capacity to survive freezing to temperatures of -40?C article provides an introduction to the winter biology of and below (Baust & Lee, 1981). In contrast, a beetle this widely distributed and unusual species, and sug- larva and two parasitic wasps that also overwinter in gests classroom activities that illuminate principles of goldenrod galls are intolerant of freezing and must cryobiology through insect overwintering. avoid internal ice formation. A variety of opportunities for educational activities are found in the complex, yet easy-to-manipulate, Life Cycle trophic relationships between goldenrod plants, insects that induce gall formation, and the natural ene- Only a single generation of the goldenrod gall fly mies of these gallmakers.Gall collection, measurement, occurs each year, with more than 11 months of the and observation (exit holes, larval response, tempera- insect's life spent inside the gall. Adults emerge in late ture, etc.) can help students develop scientific process spring. Mating occurs on the apex of the plant where skills including observation, classification, measure- the male waits to attractfemales soon after they emerge ment, inference, prediction, control of experimental from the gall (Abrahamson& Weis, 1997). After exten- variables, and material manipulation (Peard, 1994). sive inspection of the goldenrod plant for a suitable site, Galls can also be studied to learn about insect oviposit- the female deposits her eggs (usually singly) into the ing behavior and plant responses to three types of leaves surrounding a bud. Each female deposits about gallmakers-each with its own distinct gall type 20-25 eggs in her life (at least under laboratory condi- (Newell, 1994). Likewise, classroom activities can focus tions). on the collection and study of galls to discover princi- Eggs hatch within 5-8 days, and the larvae imme- ples of ecology and insect life cycles (Kahn, 1997). diately tunnel inward to the meristematic bud tissue where a chamber is created as the larvae feed on the plant matter found there. The presence of the larvae LUKE H. SANDRO is a biology teacherat SpringboroHigh School induce the plant to form a spherical stem gall, approxi- in Springboro, OH; e-mail: 1sandro@springboro. mately 15-30 mm in diameter. The precise mechanism K12.oh.us.RICHARD E. LEE,JR. is a P-ofessortin the Department of induction remains a mystery, but it clearly involves of Zoology, Miami University, Oxford, OH; e-mail: leere@ complex control of the developmental processes with- muohio.edu. in the plant (Abrahamson & Weis, 1997). GOLDENRODGALL FLY 29 Gall tissue is the only food for the growing larvae,and An interesting aspect of supercooling is that as the the adults are nonfeeding. Within the gall, the larvae volume of liquid decreases, its capacity to supercool undergo two molts. The first (from first to second instar) increases-this is one reason that insects, being essential- usually occurs in mid-July,and the second (from second to ly small bags of fluid, often supercool extensively. In third and final instar) in mid-August. In September, the addition, during the autumn, freeze-avoiding insects third instar larva excavates an exit tunnel that extends up often prepare for winter by decreasing their Tc even fur- to the epidermis of the gall. The following spring, the adult ther through the production of cryoprotectants, such as fly will use this tunnel to emerge from the gall. glycerol, sorbitol, and trehalose-which act as "antifreeze" - and through the elimination of potential ice nucleating Insect Overwintering: Two agents, such as grains of soil, certain crystals, and even bacteria that inhibit supercooling (Lee & Costanzo, Strategies for Survival 1998). In temperate climes, both active and dormant insects are sometimes exposed to subzero temperatures,increasing Freeze-TolerantInsects the chance that their body fluids could freeze. In response Insects that survive freezing must overcome two major to this threat, insects have evolved two primary strategies: problems. First, ice may cause mechanical damage due to freeze-avoidanceand freeze-tolerance(Lee, 1989). intracellularfreezing or due to the formation of ice crystals Downloaded from http://online.ucpress.edu/abt/article-pdf/68/1/29/339930/4451922.pdf by guest on 28 September 2021 between layers of tissues/organs that forces the layers Freeze-AvoidingInsects apart. The second is cellular dehydration.When ice forms Most insects cannot survivefreezing of body fluids;con- extracellularly,water molecules are gradually taken out of sequently, they must avoid severe cold by moving to warm solution to join the growing ice lattice; this process is microhabitatsand/or physiologicallyenhancing their capaci- termed freeze concentration. Solute concentration is ty to avoid freezing.The freezingpoint may be defined as the increased in the remaining extracellularwater, thus creat- temperaturebelow which existing ice crystalswill grow and ing an osmotic gradient which causes increasing amounts equals the melting point. For an insect, the temperatureof of water to leave the cell. If excessive, this dehydration crystallization(Ta) is the temperatureat which spontaneous causes lethal injury. ice nucleationoccurs in body water and the ice latticebegins Comparatively few insects survive the freezing of to grow. Since many insects have an extensive capacity to their body water. These species avoid freezing injury, in supercool(i.e., remainliquid at temperaturesbelow the freez- part, by increasing their Tc through the synthesis of ice- ing point of their body fluids), the Tcmay be many degrees nucleating agents that function to inhibit supercooling. below the freezingpoint. In the laboratory,an insect's T, is Since freezing occurs more gradually at higher tempera- measuredby placing a thermocoupleon the body to detect tures, the insect gains more time to physiologically adjust the exothermicheat of crystallizationthat is releasedas body to ice formation. Freeze-tolerant insects also synthesize water freezes (visible as the "spike"in Figure 1). high levels of the cryoprotectants glycerol, sorbitol, and trehalose. These compounds act in a number of ways. As mentioned, they function as "antifreeze"by colligative- \4 - BodyTemperature ly depressing the freezing point and 0 MeltingPoint of BodyFluids decreasing the total amount of ice formed. I1vX Temperatureof Crystallization(TJ For example, one mole of cryoprotectants decreases the freezing point of a solution -0 1 4 Heatof Crystallization (or an insect's blood) by 1.86'C. - lo -I- Cryoprotectants may also stabilize pro- teins and cell membranes to prevent a. Supercooled'! injury during freezing and thawing. E1 * Glycerol and sorbitol appear to change the -20 -Y--r IFrozen shape of the ice crystals, effectively "blunt- ing" them and making freezing less dam- aging. Finally, penetrating cryoprotec- -30* * tants, such as glycerol, enter cells and raise the osmotic pressure, thus reducing Time - the amount of cellular dehydration caused by the freeze concentration (Davidson & Figure1. Lee, 1998). Bodytemperature ofan insect during cooling and freezing. Insect freezing atthe temperatureofcrystallization (TJis indicated bythe initial detection ofthe heat Energy Conservation ofcrystallization (adapted from Lee 1989). While subzero temperaturescan pres- .~~~~~~~~~~~-- - ----- ent problems for insects, they are beneficial 30 THEAMERICAN BIOLOGY TEACHER, VOLUME 68,NO. 1,JANUARY 2006 in some ways. During the winter most insects enter a state nological "triggers"have been identified (Figure 2). First, of hibernation or dormancy, referred to as diapause in drying of the plant tissue as the goldenrod plant senesces insects, that is markedby decreased metabolism, slowed or in late summer triggers the production of the cryoprotec- arrested development, and increased tolerance of environ- tant glycerol. A few weeks later, when temperatures fall mental extremes (Danks, 1987; Tauber et al., 1986). The below 5 'C, sorbitol is synthesized from the larva's glyco- primaryfunction of diapause is to conserve energy reserves. gen stores. Thus, the larva monitors two environmental Low winter temperaturespromote conservation by further cues: the moisture level of the gall tissue and the environ- depressing metabolism and energy use. Indeed, it appears mental temperature.This two-step process decreases the that some overwinteringinsects, especially ones that feed chance that it will be "fooled"into premature or late cold- only as larvae, dependupon subzero temperaturesto help hardening. them conserve enough energy to complete their life cycle. The gall fly in particularshows a high mortality and low Gall Ecology: Interactions with fecundity when exposed