Relative susceptibility of Tribolium confusum life stages exposed to elevated temperatures

Methyl bromide, a space fumigant used in food-processing facilities, may be phased out in the United States by 2005. The use of elevated temperatures or heat treatment is gaining popularity as a methyl bromide alternative. During heat treatment, the temperature of the whole food-processing facility, or a portion of it, is raised and held between 50 and 60 degrees C for 24-36 h to kill stored-product insects. We determined time-mortality responses of the confused flour beetle, Tribolium confusum (Jacquelin du Val), eggs, young larvae, old larvae, pupae, and adults exposed to six constant temperatures between 46 and 60 degrees C. Responses of all five insect stages also were measured using exposure times of 160, 40, and 12 min at 46, 50, and 60 degrees C, respectively. Time-mortality responses of all T. confusum life stages increased with an increase in exposure time and temperature. Both time-mortality and fixed time responses showed eggs and young larvae to be most susceptible at elevated temperatures and old larvae to be least susceptible. Our results suggest that old larvae should be used as test insects to gauge heat treatment effectiveness, because heat treatment aimed at controlling old larvae should be able to control all other T. confusum life stages. Besides providing baseline data for successful use of heat treatments, time-mortality data collected at the six temperatures can be used for developing thermal death kinetic models for this species to predict mortality during actual facility heat treatments.     

Time-mortality relationships for Tribolium castaneum (Coleoptera: Tenebrionidae) life stages exposed to elevated temperatures

The use of elevated temperatures (> or = 40-60 degrees C) or heat treatments for managing insects in food-processing facilities is a viable alternative to space fumigation with methyl bromide. Quantitative data are lacking on the responses of life stages of the red flour beetle, Tribolium castaneum (Herbst), an important pest of food-processing facilities worldwide, to elevated temperatures used during heat treatments. We determined time-mortality relationships for eggs, young (neonate) larvae, old larvae, pupae, and adults of T. castaneum, exposed to constant temperatures of 42, 46, 50, 54, 58, and 60 degrees C. Generally, mortality of each stage increased with an increase in temperature and exposure time. Young larvae were the most heat-tolerant stage, especially at temperatures > or = 50 degrees C. Exposure for a minimum of 7.2 h at > or = 50 degrees C was required to kill 99% of young larvae, whereas the other stages required < or = 1.8 h. Heat treatments that control young larvae should control all other stages of T. castaneum, and young larvae should be used as test insects to evaluate efficacy against T. castaneum during an actual facility heat treatment. These results provide the basis for successful use of elevated temperatures for management of T. castaneum life stages associated with food-processing facilities.     

Reproductive performance of Tribolium castaneum (Coleoptera: Tenebrionidae) exposed to the minimum heat treatment temperature as pupae and adults

Managing stored-product insect pests by heating the ambient air of a food-processing facility to high temperatures (50-60 degrees C), also referred to as heat treatment, is an effective technology that has been used since the early 1900s. The minimum temperature during heat treatment for effective disinfestation is 50 degrees C. The effect of sublethal exposures to 50 degrees C on the reproductive performance of stored-product insects associated with food-processing facilities is unknown. The red flour beetle, Tribolium castaneum (Herbst), is a pest commonly found in food-processing facilities worldwide. The adverse effects on fecundity, egg-to-adult survival, and progeny production of T. castaneum exposed as 1-d-old pupae and 2-wk-old adults to 50 degrees C for 60 and 39 min, respectively, were determined in the laboratory. Pupae and adults exposed for the same time periods at 28 degrees C served as the control treatment. Four possible reciprocal crosses were carried out among adults from the heat-treated (50 degrees C) and control (28 degrees C) treatments. The number of eggs produced during the first 2 wk of adult life, survival of these eggs to adulthood, and adult progeny production after 2 and 8 wk of oviposition in treatments representing all four reciprocal crosses were determined. Fecundity, egg-to-adult survival, and adult progeny production decreased by 17-63, 52-63, and 66-78%, respectively, when males, females, and both males and females were exposed to 50 degrees C. These effects were relatively more pronounced in treatments in which pupae were exposed to the high temperature compared to adults, and in exposed females than in males. The impaired reproductive performance in T. castaneum pupae and adults surviving sublethal exposures to the minimum heat treatment temperature is valuable for understanding population rebound following a heat treatment intervention.     

Thermal death kinetics of red flour beetle (Coleoptera: Tenebrionidae)

While developing radio frequency heat treatments for dried fruits and nuts, we used a heating block system developed by Washington State University to identify the most heat-tolerant life stage of red flour beetle, Tribolium castaneum (Herbst), and to determine its thermal death kinetics. Using a heating rate of 15 degrees C/min to approximate the rapid heating of radio frequency treatments, the relative heat tolerance of red flour beetle stages was found to be older larvae > pupae and adults > eggs and younger larvae. Lethal exposure times for temperatures of 48, 50, and 52 degrees C for the most heat-tolerant larval stage were estimated using a 0.5th order kinetic model. Exposures needed for 95% mortality at 48 degrees C were too long to be practical (67 min), but increasing treatment temperatures to 50 and 52 degrees C resulted in more useful exposure times of 8 and 1.3 min, respectively. Red flour beetle was more sensitive to changes in treatment temperature than previously studied moth species, resulting in red flour beetle being the most heat-tolerant species at 48 degrees C, but navel orangeworm, Amyelois transitella (Walker), being most heat tolerant at 50 and 52 degrees C. Consequently, efficacious treatments for navel orangeworm at 50-52 degrees C also would control red flour beetle.     

Susceptibility of Lasioderma serricorne (Coleoptera: Anobiidae) life stages to elevated temperatures used during structural heat treatments

Heat treatment of food-processing facilities involves using elevated temperatures (50-60 degrees C for 24-36 h) for management of stored-product insects. Heat treatment is a viable alternative to the fumigant methyl bromide, which is phased out in the United States as of 2005 because of its adverse effects on the stratospheric ozone. Very little is known about responses of the cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: Anobiidae), a pest associated with food-processing facilities, to elevated temperatures. Responses of L. serricorne life stages to elevated temperatures were evaluated to identify the most heat-tolerant stage. Exposure of eggs, young larvae, old larvae, and adults during heat treatment of a food-processing facility did not clearly show a life stage to be heat tolerant. In the laboratory, exposure of eggs, young larvae, old larvae, pupae, and adults at fixed times to 46, 50, and 54 degrees C and 22% RH indicated eggs to be the most heat-tolerant stage. Time-mortality responses at each of these three temperatures showed that the time for 99% mortality (LT99) based on egg hatchability and egg-to-adult emergence was not significantly different from one another at each temperature. Egg hatchability alone can be used to determine susceptibility to elevated temperatures between 46 and 54 degrees C. The LT99 based on egg hatchability and egg-to-adult emergence at 46 degrees C was 605 and 598 min, respectively, and it decreased to 190 and 166 min at 50 degrees C and 39 and 38 min at 54 degrees C. An exponential decay equation best described LT99 as a function of temperature for pooled data based on egg hatchability and egg-to-adult emergence. Our results suggest that during structural heat treatments eggs should be used in bioassays for gauging heat treatment effectiveness, because treatments aimed at controlling eggs should be able to control all other L. serricorne life stages.     

Susceptibility of Tribolium castaneum life stages exposed to elevated temperatures during heat treatments of a pilot flour mill: influence of sanitation, temperatures attained among mills floors, and costs

The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), was investigated in a pilot flour mill subjected to three, 24-h heat treatments by using forced-air gas heaters fueled by propane. Two sanitation levels, dusting of wheat flour and 2-cm-deep flour, were created in 25 plastic bioassay boxes, each holding eggs, young larvae, old larvae, pupae, and adults of T. castaneum plus two temperature sensors. Data loggers (48) were placed on the five mill floors to record air temperatures. The time required to reach 50 degrees C, time above 50 degrees C, and the maximum temperature among mill floors and in bioassay boxes were measured. The maximum temperature in bioassay boxes and in the mill was lower on the first floor than on other floors. This trend was apparent in time required to reach 50 degrees C and time above 50 degrees C, especially in compartments with 2-cm-deep flour. The mean +/- SE mortality of T. castaneum life stages on the first floor was 55.5 +/- 12.9-98.6 +/- 0.8%; it was 93.2 +/- 6.7-100 +/- 0.0% on other floors. Adults were the least susceptible stage. Mortality of T. castaneum stages in compartments with 2-cm-deep flour was generally lower than those with flour dust. Costs for the three heat treatments ranged from US$27,438 to $28,838. An effective heat treatment can be conducted within 24 h, provided temperatures on mill floors reach 50 degrees C in 8-12 h and are held above 50 degrees C for at least 10-14 h, with maximum temperatures held between 50 and 60 degrees C.     

Temperature-dependent post-diapause development and prediction of spring emergence of the pine needle gall midge (Dipt., Cecidomyiidae)

Abstract:  We determined the influence of temperature on post-diapause development of overwintered Thecodiplosis japonensis Uchida et Inouye (Dipt., Cecidomyiidae) under various treatments (12, 15, 18, 21, 24, 27 and 30°C) in an effort to predict its spring emergence. Survival and developmental period for the overwintered larvae and pupae were significantly influenced by temperature. Linear and nonlinear regression models quantitatively described temperature-dependent development and survival of T. japonensis . The survival models exhibited right-skewed bell shape patterns for all stages, indicating a more detrimental impact on survival at high temperatures. Theoretical optimum temperatures with highest survival were 22.3, 24.0 and 24.0°C for the overwintered larvae, pupae and total post-diapause development (the larvae to adults) respectively. Pupal mortality was higher at all temperatures than larval mortality and the suitable range of temperature for pupae was narrower than that of larvae. The nonlinear Briere model estimated that optimum temperatures with the fastest development were 29.1°C for larvae, 27.6°C for pupae and 27.0°C for larvae to adults. In a linear model, the lower threshold temperatures were 5.1, 7.1 and 5.9°C for larvae, pupae, and larvae to adults respectively. A predictive degree-day model was developed using trap catches of T. japonensis adult emergence during 1991–1995. The model accounted for 84.6% of year-to-year variation in adult emergence and predicted accurately the median emergence time in 1996.     

Environmental factors affecting reproductive incompatibility in flour beetles, genus Tribolium

This paper reports the effect of high temperatures on the reproductive compatibility of geographic strains of flour beetles, Tribolium confusum. Some geographic strains are reproductively incompatible with other strains owing to a maternally inherited microorganism: Males infected with the microorganism are reproductively incompatible with uninfected females. All other crosses are fertile. Previous reports indicated that infected strains can be cured of the microorganisms by culturing the beetles on medium containing tetracycline and that cured males become reproductively compatible. This study reports that raising larvae at high temperatures (37 degrees C) promotes curing. The proportion of individuals cured is a function of sex and the period of exposure to high temperature.     

Susceptibility of last instar red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae) to hydroprene.

Last instar larvae of the red flour beetle, Tribolium castaneum (Herbst), and the confused flour beetle, Tribolium confusum Jacquelin du Val, were either exposed for 8-144 h on concrete treated with 1.9 x 10(-3) mg(AI)/per cm2 hydroprene, or continually exposed on concrete treated with 9.8 x 10(-4) to 1.9 x 10(-3) mg[AI]/per cm2 hydroprene. In both tests, larvae were exposed and held at 27 or 32 degrees C and 40, 57, or 75% RH. When larvae were exposed with no food to hydroprene for different time intervals, then transferred to untreated concrete containing flour, consistent effects were produced only at 144 h. At this exposure interval, the percentage of beetles arrested in the larval stage after 3-4 wk was generally greater at 75% RH compared with 40 and 57% RH, but there were no differences between species or temperature. The percentages of dead adult red flour beetles and live adults with morphological deformities were also greatest at 75% RH, and defects were more prevalent in red flour beetles than in confused flour beetles. When larvae were continually exposed to different concentrations of hydroprene on concrete that contained flour, the percentage of arrested larvae, dead adults, and live adults of both species generally increased with concentration. There were more deleterious effects at 75% RH compared with either 40 or 57% RH, and effects were more pronounced in the red flour beetle compared with the confused flour beetle. In both experiments, temperature effects were variable and inconclusive. Results indicate that continual exposure of last instar red flour beetle and confused flour beetle to hydroprene can limit population development, but exposure intervals of >6 d may be required for maximum effectiveness.     

Residual efficacy of methoprene for control of Tribolium castaneum (Coleoptera: Tenebrionidae) larvae at different temperatures on varnished wood, concrete, and wheat

The residual efficacy of the juvenile hormone analog methoprene (Diacon II) was evaluated in bioassays using larvae of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) exposed on unsealed concrete or varnished wood treated with a liquid formulation and held at different temperatures. When these two types of surfaces were stored at 20, 30 or 35 degrees C for 0-24 wk, the percentage of adult emergence on concrete increased with time. In contrast, there was no adult emergence from larvae exposed to varnished wood at 24 wk after treatment at any of these temperatures. The presence of flour reduced residual efficacy of methoprene on concrete, but not on varnished wood, with no differences between cleaning frequencies. Methoprene was also stable for 48 h on concrete held at 65 degrees C and wheat, Triticum aestivum L., held at 46 degrees C. Results show that methoprene is stable at a range of temperatures commonly encountered in indoor food storage facilities and at high temperatures attained during insecticidal heat treatments of structures. The residual persistence of methoprene applied to different surface substrates may be affected more by the substrate than by temperature.     

Effects of constant and varying temperatures on the development of blue swimmer crab (Portunus pelagicus) larvae: Laboratory observations and field predictions for temperate coastal waters

Temperature is widely held to be a critical factor for the development of marine invertebrate larvae. We investigated three specific aspects of this relationship for the blue swimmer crab, Portunus pelagicus, in a temperate gulf: (1) the effects of different but temporally constant temperatures on the survival and developmental period of larvae reared in the laboratory, (2) the effects of varying temperatures on the survival and developmental period of larvae reared in the laboratory, and (3) prediction of larval developmental periods under seasonal temperature changes found in the field. Temperature had a marked effect on larval survival. At constant temperatures of 22.5 and 25 °C larval survival was far greater than at lower temperatures down to 17 °C, and developmental period of the larval period was inversely related to (constant) temperature. However, larvae in temperate coastal waters will usually be exposed to seasonally varying, rather than constant, temperatures. To account for this, a larval developmental period model was created and then verified in the laboratory by rearing larvae under varying temperatures. Results of this work demonstrated that developmental periods were markedly different under constant versus varying temperature regimes. Using different temperature simulations for a temperate gulf (Gulf St Vincent, South Australia), the developmental period model predicted that in years of ‘average’ seasonal temperature changes, total larval developmental period could range from 26 to 45 days depending on the day of hatching. In such cases, peak postlarval settlement was predicted to occur between mid January and mid March. Results from this study also predict that larval survival (and thus postlarval settlement) will be maximised in years with abnormally warm summers. Whilst the developmental period model was used to make predictions of developmental period for P. pelagicus in a temperate gulf, it could readily be adapted to predict developmental periods in other coastal environments.     

SELECTION FOR HEAT-SHOCK RESISTANCE IN LARVAL AND IN ADULT DROSOPHILA BUZZATII: COMPARING DIRECT AND INDIRECT RESPONSES

Direct and correlated responses in selection for heat-shock resistance in adult and in larval Drosophila buzzatii were studied. Two lines were artificially selected for higher survival to heat stress as adults, and two other lines were reared under a fluctuating thermal environment as larvae, 35°C for 6 h and 25°C for 18 h, to “naturally” select for higher resistance as larvae. The latter two lines were duplicated after nine generations to yield additional lines to be “naturally” selected as larvae at a higher temperature, 38.2°C for 6 h. Control lines were maintained separately for the adult and larval selection lines. A significant direct response to selection was found for the adult selection lines. However, larvae of these adult selection lines were no more heat resistant than were larvae of the control lines. One of the two larval selection lines increased significantly in heat resistance as larvae. However, adult heat resistance was similar for lines selected as larvae and the corresponding control lines maintained at 25°C. Changes in developmental time accompanied changes in survival after stress in both sets of lines selected for increased heat resistance.     

Thermal susceptibility of Streptococcus faecium strains isolated from frankfurters

The heat resistance of nine strains of Streptococcus faecium isolated from frankfurters was determined at 63 and 68 degrees C in brain heart infusion broth. Exponential-phase cultures (approximately 10(7) colonies/mL) were used as inoculants. The heat resistance of S. faecium DP2181, a moderately resistant isolate, was further examined in broth (55, 63, and 68 degrees C) and frankfurter emulsion (63 and 68 degrees C). The decimal reduction times (D values) were determined by regression. In broth, both time-temperature combinations resulted in a 3-4 log decline in bacterial numbers for the nine S. faecium strains tested. For S. faecium DP2181, the survivor curves deviated from the logarithmic order of death at all three heating temperatures. An initial slow period of death was evident at 55 degrees C and a resistant tail of organisms was observed at 55, 63, and 68 degrees C. The D55D63, and, D68 values for the logarithmic portion of the corresponding survivor curves were 105.6, 9.36, and 3.34 min, respectively. The survival of DP2181 was enhanced by the frankfurter emulsion. The results indicate that populations of S. faecium existed that were very heat resistant and could survive normal frankfurter processing if initially present in high numbers.     

Thermal requirements of Galleria mellonella L. (Lepidoptera: Pyralidae) immature stages

The rearing of Galleria mellonella L. in laboratory is important for multiplication of entomopathogenic nematodes, mandatory for biological control studies. The objective of this study was to evaluate the effect of three thermal profiles on development stages of this insect, allowing synchronization of cycle production. Two distinct rearing phases were done: firstly, using nucleous of incubation for development of eggs and, secondly, using circular-aluminum manifolds for development of larvae and pupae. The time necessary for development of the immature stages decreased with higher temperatures. Incubation periods lasted 13.4 days at 22 degrees C, 8.3 at 27 degrees C and 6.8 days at 32 degrees C, while periods for larvae development lasted 40.4, 27.2, and 23.4 days, respectively, for the same temperatures. Development to pupal stage was observed 18.2, 15.0, and 12.2 days, respectively, for the same temperatures. Larval survival was higher at 32 degrees C, however embryonic stages and pupae survival were higher at 27 degrees C. and 22 degrees C, respectively. The threshold temperature was 11.209167 degrees C for the embryonic development stage, 7.695869 degrees C for larval stage, and 1.943050 degrees C for pupal stage of G. mellonella. Thermal constants were 138.380533 DG (degree day) for egg, 554.968830 DG for larvae, and 369.054080 DG for pupae.     

Impact of varying levels of sanitation on mortality of Tribolium castaneum eggs and adults during heat treatment of a pilot flour mill

The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), an economically important pest in flour mills, was investigated in a pilot flour mill subjected to two, 24-h heat treatments. One hundred eggs or 100 adults of T. castaneum were exposed inside each 20-cm diameter by 15-cm high PVC rings holding 0.1-, 0.2-, 1.0-, 3.0-, 6.0-, and 10.0-cm-deep wheat flour to simulate different sanitation levels that may exist in a flour mill. These rings were placed on the first and third floors of a pilot flour mill. On the first floor, temperatures inside rings with eggs reached 50 degrees C in 7-11 h only in 0.1- and 0.2-cm-deep flour treatments. In all other treatments the maximum temperatures attained generally were below 50 degrees C and inversely related to flour depth. Adults of T. castaneum on this floor were less susceptible than eggs. The egg mortality decreased linearly with an increase in flour depth, whereas that of adults decreased exponentially. All eggs and adults in rings on the third floor were killed irrespective of flour depth, because temperatures inside rings reached 50 degrees C in 15-17 h and were held above 50 degrees C for 6-8 h with the maximum temperatures ranging between 55.0 and 57.0 degrees C. Although the protective effects of flour on survival of T. castaneum eggs and adults were evident only if temperatures did not reach 50 degrees C, removal of flour accumulations is essential to improve heat treatment effectiveness.     

Influence of temperature on the zoeal development and elemental composition of the cancrid crab, Cancer setosus Molina, 1782 from Pacific South America

Temperature changes during ENSO cause mass mortalities of adult Cancer setosus, but the effects on early life stages are unknown. The influence of temperature on survival, development and biochemical composition was studied in larvae of the hairy crab, C. setosus, from a population off the northern Chilean coast. In rearing experiments conducted at four different temperatures (12, 16, 20, 22 °C), zoeal development was only completed at 16 and 20 °C, after 78 and 36 days, respectively. Instar duration was negatively correlated with temperature. A multiple linear model relating larval body mass (in carbon) to temperature and developmental time suggests that successful larval development is possible within a narrow temperature range only. The biochemical composition, measured as carbon, hydrogen, and nitrogen (C, H, N) content, show in general the typical oscillating changes during the moult cycle of brachyuran crab larvae. However, at high (22 °C) and low (16 °C) temperatures, CHN values show deviations from the typical pattern, indicating threshold temperatures for larval activity and survival. These findings indicate that the larval development of C. setosus is compromised under conditions of El Niño, with temperatures exceeding the upper thermal temperature tolerance threshold of larvae. Effects of El Niño on early life history stages and recruitment rates should be increasingly taken into account in fisheries management strategies.     

Critical thermal limits, temperature tolerance and water balance of a sub-Antarctic kelp fly, Paractora dreuxi (Diptera: Helcomyzidae)

Paractora dreuxi displays distinct ontogenetic differences in thermal tolerance and water balance. Larvae are moderately freeze tolerant. Mean larval onset of chill coma was -5.1 degrees C, and onset of heat stupor was 35.5 degrees C. Larval supercooling point (SCP) was -3.3 degrees C with 100% recovery, although mortality was high below -4 degrees C. Starvation caused SCP depression in the larvae. Adults were significantly less tolerant, with critical thermal limits of -2.7 and 30.2 degrees C, no survival below the SCP (-9.6 degrees C), and no change in SCP with starvation. Moderate freeze tolerance in the larvae supports the contention that this strategy is common in insects from southern, oceanic islands. Fly larvae survived desiccation in dry air for 30 h, and are thus less desiccation tolerant than most other sub-Antarctic insect larvae. Water loss rates of the adults were significantly lower than those of the larvae. Lipid metabolism did not contribute significantly to water replacement in larvae, which replaced lost body water by drinking fresh water, but not sea water. Kelp fly larvae had excellent haemolymph osmoregulatory abilities. Current climate change has led to increased temperatures and decreased rainfall on Marion Island. These changes are likely to have significant effects on P. dreuxi, and pronounced physiological regulation in larvae suggests that they will be most susceptible to such change.     

Susceptibility of Plodia interpunctella (Lepidoptera: Pyralidae) developmental stages to high temperatures used during structural heat treatments

Heating the ambient air of a whole, or a portion of a food-processing facility to 50 to 60 degrees C and maintaining these elevated temperatures for 24 to 36 h, is an old technology, referred to as heat treatment. There is renewed interest in adopting heat treatments around the world as a viable insect control alternative to fumigation with methyl bromide. There is limited published information on responses of the Indian meal moth, Plodia interpunctella (Hübner), exposed to elevated temperatures typically used during heat treatments. Time-mortality relationships were determined for eggs, fifth-instars (wandering-phase larvae), pupae, and adults of P. interpunctella exposed to five constant temperatures between 44 and 52 degrees C. Mortality of each stage increased with increasing temperature and exposure time. In general, fifth-instars were the most heat-tolerant stage at all temperatures tested. Exposure for a minimum of 34 min at 50 degrees C was required to kill 99% of the fifth-instars. It is proposed that heat treatments aimed at controlling fifth-instars should be able to control all other stages of P. interpunctella.     

Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle

The sub-Antarctic beetle Hydromedion sparsutum (Coleoptera, Perimylopidae) is common locally on the island of South Georgia where sub-zero temperatures can be experienced in any month of the year. Larvae were known to be weakly freeze tolerant in summer with a mean supercooling point (SCP) around -4 degrees C and a lower lethal temperature of -10 degrees C (15min exposure). This study investigated the effects of successive freezing exposures on the SCP and subsequent survival of summer acclimatised larvae. The mean SCP of field fresh larvae was -4.2+/-0.2 degrees C with a range from -1.0 to -6.1 degrees C. When larvae were cooled to -6.5 degrees C on 10 occasions at intervals of 30min and one and four days, survival was 44, 70 and 68%, respectively. The 'end of experiment' SCP of larvae surviving 10 exposures at -6.5 degrees C showed distinct changes and patterns from the original field population depending on the interval between exposure. In the 30min interval group, most larvae froze between -6 and -8 degrees C, a depression of up to 6 degrees C from the original sample; all larvae were dead when cooling was continued below the SCP to -12 degrees C. In the one and four day interval groups, most larvae froze above -6 degrees C, showing no change as a result of the 10 exposures at -6.5 degrees C. As with the 30min interval group, some larvae froze below -6 degrees C, but with a wider range, and again, all were dead when cooled to -12 degrees C. However, in the one and four day interval groups, some larvae remained unfrozen when cooled to -12 degrees C, a depression of their individual SCP of at least 6 degrees C, and were alive 24h after cooling. In a further experiment, larvae were cooled to their individual SCP temperature at daily intervals on 10 occasions to ensure that every larva froze every day. Most larvae which showed a depression of their SCP of 2-4 degrees C from their day one value became moribund or died after six or seven freezing events. Survival was highest in larvae with SCPs of -2 to -3 degrees C on day one and which froze at this level on all 10 occasions. The results indicate that in larvae in which the SCP is lowered following sub-zero exposure, the depression of the SCP is greatest in individuals that do not actually freeze. Further, the data suggest that after successive frost exposures in early winter the larval population may become segregated into two sub-populations with different overwintering strategies. One group consists of larvae that freeze consistently in the temperature range from -1 to -3 degrees C and can survive multiple freeze-thaw cycles. A second group with lower initial SCPs (around -6 degrees C), or which fall to this level or lower (down to -12 degrees C) after freezing on one or more occasions, are less likely to freeze through extended supercooling, but more likely to die if freezing occurs.     

The relative importance of temperature and diet to larval development and adult size of the winter stonefly, Soyedina carolinensis (Piecoptera: Nemouridae)

SUMMARY. 1. Soyedina carolinensis Claassen, a leaf shredding stonefly, was reared in a series of three laboratory experiments from early instar to adult on different species of deciduous leaves and at various constant and fluctuating temperature regimes.
2. Experiment 1, which involved rearing larvae on fourteen different leaf diets at ambient stream temperatures, showed that diet significantly affected larval growth and adult size but did not affect overall developmental time.
3. Experiment 2, which involved rearing larvae on five different leaf diets at each of three fluctuating temperature regimes (viz ambient White Clay Creek (WCC), ambient WCC+3°C, and ambient WCC+6°C), showed that: (i) adding 6°C to the normal temperature regime of WCC was lethal to 99% of the larvae regardless of diet; and (ii) warming WCC by 3°C did not affect developmental time but did significantly reduce adult size relative to adults reared at WCC temperatures on certain diets.
4. Experiment 3, which involved rearing larvae on five different leaf diets at each of five constant temperatures (viz 5, 10, 15, 20, 25°C), showed that: (i) temperature significantly affected the mortality, growth, and development time of larvae whereas diet only affected larval growth and mortality; (ii) temperatures at or near 10°C yielded maximum larval growth and survival for most diets; (iii) at 5°C, larval mortality was high and growth was low resulting in a few small adults for most diets; (iv) larval mortality was at or near 100% at 15°C regardless of diet; and (v) no larvae survived at 20 and 25°C.