Thermal tolerance breadths among groundwater crustaceans living in a thermally constant environment.

The climate variability hypothesis assumes that the thermal tolerance breadth of a species is primarily determined by temperature variations experienced in its environment. If so, aquatic invertebrates living in thermally buffered environments would be expected to exhibit narrow thermal tolerance breadths (stenothermy). We tested this prediction by studying the thermal physiology of three isopods (Asellidae, Proasellus) colonizing groundwater habitats characterized by an annual temperature amplitude of less than 1°C. The species responses to temperature variation were assessed in the laboratory using five physiological variables: survival, locomotor activity, aerobic respiration, immune defense and concentrations of total free amino acids and sugars. The three species exhibited contrasted thermal physiologies, although all variables were not equally informative. In accordance with the climate variability hypothesis, two species were extremely sensitive even to moderate changes in temperature (2°C) below and above their habitat temperature. In contrast, the third species exhibited a surprisingly high thermal tolerance breadth (11°C). Differences in response to temperature variation among Proasellus species indicated that their thermal physiology was not solely shaped by the current temperature seasonality in their natural habitats. More particularly, recent gene flow among populations living in thermally constant yet contrasted habitats might explain the occurrence of eurytherm species in thermally buffered environments.

Cytochrome P450s and cytochrome P450 reductase in the olfactory organ of the cotton leafworm Spodoptera littoralis.

Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules.

Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations.

Fluctuating temperatures are a predominant feature of the natural environment but their effects on ectotherm physiology are not well-understood. The warm periods of fluctuating thermal regimes (FTRs) provide opportunities for repair leading to increased survival, but there are also indications of negative effects of warm exposure. In this study, we examined respiration and oxidative stress in adult Alphitobius diaperinus exposed to FTRs and to constant low temperatures. We hypothesized that cold exposure will cause oxidative stress and that FTRs would reduce the amount of chill injuries, via activation of the antioxidant system. We measured V˙CO2, activities of super oxide dismutase (SOD), amounts of total (GSHt) and oxidized glutathione (GSSG) during cold and warm periods of FTRs. Increased severity of cold exposure caused a decrease in the glutathione pool. SOD levels increased during the recovery period in the more severe FTR. The antioxidant response was sufficient to counter the reactive oxygen species production, as the GSH:GSSG ratio increased. We conclude that cold stress causes oxidative damage in these beetles, and that a warm recovery period activates the antioxidant system allowing repair of cold-induced damage, leading to the increased survival previously noted in beetles exposed to fluctuating versus constant temperatures.

Effects of cold-exposure and subsequent recovery on cellular proliferation with influence of 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2).

In developing insects, the peak level of 20-hydroxyecdysone (20E) initiates a decrease in cyclin expression, which subsequently triggers an arrest of cellular proliferation and the start of differentiation, finally culminating in the moult. We investigated the impact of cold-exposure (4 degrees C) and recovery (26 degrees C) on the cell cycle activity of the Plodia interpunctella Lepidoptera cell line IAL-PID2 and on the expression of B-type cyclin (PcycB), ecdysone receptor (B1-isoform; PiEcR-B1), and Hsc70 (PiHsc70) mRNA. Cold-exposure significantly reduced expression of these mRNAs, while their levels increased to above control values during subsequent recovery at the normal growth temperature. When cold-exposed cells were returned to 26 degrees C, cell cycle activity restarted, but apoptosis was strongly increased. The presence of 20E appeared to increase this apoptotic phenomenon. This result is consistent with the described protective role of 20E against a variety of stressors and with the capacity of 20E to induce cell death in different situations. Here, we illustrate for the first time a connection between 20E treatment and Hsc70 expression during cold-exposure and subsequent recovery in insect cells. Combined with the 20E-induced apoptotic response, our results suggest that regulation of Hsc70 expression by 20E could act in synergy with the control of apoptotic cell death in order to optimize the survival of specific cell populations after a period of cold-exposure.

Ageing and thermal performance in the sub-Antarctic wingless fly Anatalanta aptera (Diptera: Sphaeroceridae): older is better.

Senescence is a progressive biological process expressed in behavioural, morphological, physiological, biochemical and cellular age-related changes. Age-associated alterations in activity are regularly found in insects when examining whole-organism senescence over the adult lifespan. In addition, overall stress resistance usually decreases with senescence. In the present study, we measured the critical thermal minimum (CT(min)) and the subsequent recovery period over the lifespan of the sub-Antarctic wingless fly, Anatalanta aptera. Experiments were conducted on males and females in seven age groups: newly emerged, 1.5-, 5-, 7-, 13-, 15- and 18-month-old adults. Surprisingly, CT(min) decreased significantly with ageing in A. aptera, from -3.8 +/- 0.5 degrees C just after the emergence to -5.6 +/- 0.7 degrees C in the 18-month-old flies. The subsequent recovery period remained similar between the seven groups tested. Our unexpected results contradict the previous data collected in other insects. We have demonstrated for the first time that ageing may improve rather than impair locomotor activity during unfavourable thermal conditions. It raises questions and challenges the literature dealing with ageing. These fascinating results also question the underpinning mechanisms involved in the improvement of the thermal performance with ageing in A. aptera.

Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes.

Environmental stress deleteriously affects every aspect of an ectotherm's biological function. Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and physiological mechanisms. However, the physiological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied. We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols. The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with constant cold exposure and untreated controls. The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins. Some FAAs have been shown to have cryoprotective properties in insects, but the relationship between FAAs, cold tolerance and survival has not yet been well defined. Instead of considering FAAs only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation. Under FTRs, polyol accumulation probably contributes to the increased duration of survival in A. diaperinus.