Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria)

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K⁺], causing dissipation of K⁺ equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K⁺] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K⁺- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K⁺] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K⁺] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K⁺ is decoupled. Chill coma recovery time, after 2 h at −4 °C, differed significantly between fasted animals and those fed on high K⁺ diet. This difference was not associated with an increased disturbance of haemolymph [K⁺] in the fed animals, instead it was associated with a slowed recovery of muscle [K⁺], muslce water, haemolymph [Na⁺] and K⁺equilibrium potential in the fed animals.     

<Emphasis Type="Italic">Myrmica</Emphasis> host-ants limit the density of the ant-predatory large blue <Emphasis Type="Italic">Maculinea nausithous</Emphasis>

Butterflies of the highly endangered genus Maculinea are parasites of red Myrmica ants. Prior to the adoption by Myrmica worker ants Maculinea caterpillars feed on a specific host plant. This field study aims to answer the question whether the density and distribution of the host plant Sanguisorba officinalis or the density of the host ant M. rubra limit the density of M. nausithous egg, larval and adult stage. We found that the density of M. nausithous egg stage and adult stage increased with the density of the host ant. The density of M. nausithous caterpillars was not associated with ant density or plant density. This study suggests that the density of M. nausithous is limited by the density of the host ant M. rubra. We conclude that habitat management for M. nausithous should focus on the maintenance of habitats that hold both resources, but that enable high densities of M. rubra. In addition, it is discussed why high densities of host ants might be more important in predatory than in cuckoo-feeding Maculinea.     

Influence of developmental temperature on cold shock and chill coma recovery in Drosophila ananassae: Acclimation and latitudinal variations among Indian populations

Exposure of various Drosophila species to mild increase or decrease in temperature has consistently been shown to result in increased resistance to subsequent temperature extremes. We investigated cold tolerance in 45 Indian natural populations of Drosophila ananassae collected from all over India by monitoring the time taken by adults to recover from chill-coma after a treatment for 16 h at 4 °C. Significant latitudinal and altitudinal differentiation was observed for chill coma recovery in D. ananassae. Chill-coma recovery was closely associated with local climatic factors like average annual temperature and relative humidity of origin of populations.     

The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster

Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO₂ concentrations (4 h at 18,000 ppm) and chronic cold (72 h at 4 °C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO₂ and cold. We conclude that although caution must be used in choosing an immobilisation method, CO₂ appears to have less long-term impact than cold.     

Dissecting chill coma recovery as a measure of cold resistance: evidence for a biphasic response in Drosophila melanogaster

Cold resistance in insects has traditionally been measured in terms of survival following a stress, but alternative methods are increasingly being used because of their relevance to the ecology of organisms and their utility in characterizing variation among species, populations and individuals. One such method capable of discriminating among Drosophila species and conspecific Drosophila populations from different environments is adult chill coma recovery time, the time taken for adults to become active again after being knocked down by a cold stress. Here we characterized the chill coma response of D.melanogaster in detail. Adults were exposed to a range of temperatures and stressful periods prior to measuring recovery. Recovery from chill coma in D.melanogaster was biphasic; as flies were stressed under cooler temperatures, recovery times leveled off and then decreased before sharply increasing again as mortality starts to occur. This biphasic response has previously been observed in D.subobscura where it has a somewhat different shape. A second mechanism therefore acts at relatively lower temperatures to ameliorate the effects of the cold stress. When D.melanogaster were reared at 19 and 25 °C for two generations, the shape of the curve relating temperature to recovery time was similar, but flies from the warmer temperature had longer recovery times and showed responses that leveled off and then decreased at relatively higher temperatures. As exposure time to cold stress was increased, recovery times also increased except at mild stress levels. Chill coma recovery in D.melanogaster is a complex trait and likely to reflect multiple underlying components.     

Ion and water balance in Gryllus crickets during the first twelve hours of cold exposure

Insects lose ion and water balance during chilling, but the mechanisms underlying this phenomenon are based on patterns of ion and water balance observed in the later stages of cold exposure (12 or more hours). Here we quantified the distribution of ions and water in the hemolymph, muscle, and gut in adult Gryllus field crickets during the first 12 h of cold exposure to test mechanistic hypotheses about why homeostasis is lost in the cold, and how chill-tolerant insects might maintain homeostasis to lower temperatures. Unlike in later chill coma, hemolymph [Na⁺] and Na⁺ content in the first few hours of chilling actually increased. Patterns of Na⁺ balance suggest that Na⁺ migrates from the tissues to the gut lumen via the hemolymph. Imbalance of [K⁺] progressed gradually over 12 h and could not explain chill coma onset (a finding consistent with recent studies), nor did it predict survival or injury following 48 h of chilling. Gryllus veletis avoided shifts in muscle and hemolymph ion content better than Gryllus pennsylvanicus (which is less chill-tolerant), however neither species defended water, [Na⁺], or [K⁺] balance during the first 12 h of chilling. Gryllus veletis better maintained balance of Na⁺ content and may therefore have greater tissue resistance to ion leak during cold exposure, which could partially explain faster chill coma recovery for that species.     

Selection for rapid and slow recovery from chill‐ and heat‐coma in Drosophila melanogaster

To assess the trade‐offs associated with cold and heat tolerance, selection experiments were conducted on the rate of recovery from chill‐ and heat‐coma using Drosophila melanogaster. Flies were treated with cold and heat to induce coma, and those that showed rapid or slow recovery from coma were selected. The lines selected for rapid (or slow) recovery from chill‐coma also showed rapid (slow) recovery from heat‐coma, although such a correlation was not observed in the lines selected for the rate of recovery from heat‐coma. On the other hand, survival after cold was enhanced in both lines selected for rapid and slow recovery from chill‐coma, and survival after heat was enhanced in both lines selected for rapid and slow recovery from heat‐coma. It was assumed that cold and heat treatments to induce coma caused some damages to flies and those that were tolerant to cold or heat were unintentionally selected in the present coma‐based selection. Only a weak trade‐off was observed between survival‐based cold and heat tolerance. On the other hand, developmental time was prolonged and desiccation resistance, walking speed, and longevity were reduced in the lines selected for rapid and slow recovery from chill‐ and/or heat‐coma, suggesting that these resistance and life‐history traits are under trade‐offs with cold and/or heat tolerance. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 72–80.     

Choice behaviour of Myrmica rubra workers between ant larvae and larvae of their Phengaris (Maculinea) nausithous nest parasites

Larvae of Phengaris (Maculinea) butterflies are adopted by Myrmica workers and are obligate myrmecophiles. Brood recognition by Myrmica rubra workers was tested for concolonial larvae (M. rubra) versus allocolonial larvae (M. rubra and P. nausithous) to assay the mimetic efficiency of P. nausithous. In addition, we tested M. rubra ant colonies from different populations with and without the presence of Phengaris, to test for potential local adaptation in adoption behaviour. We show that M. rubra can distinguish between nest-mate and foreign larvae as well as between P. nausithous and their own larvae. Workers from the allopatric population inspected and rejected more P. nausithous larvae than workers from the sympatric population. This might reflect a local host adaptation in which the social parasite more efficiently mimes its sympatric host ants than allopatric ones.     

Functional Characterization of the Frost Gene in Drosophila melanogaster: Importance for Recovery from Chill Coma

Background

Almost all animals, including insects, need to adapt to temperature fluctuations. The molecular basis of thermal adaptation is not well understood, although a number of candidate genes have been proposed. However, a functional link between candidate genes and thermal tolerance has rarely been established. The gene Frost (Fst) was first discovered when Drosophila flies were exposed to cold stress, but the biological function(s) of Fst has so far not been characterized. Because Fst is up-regulated after a cold stress, we tested whether it was essential for chill-coma recovery.

Methodology/Principal Findings

A marked increase in Fst expression was detected (by RT-PCR) during recovery from cold stress, peaking at 42-fold after 2 h. The GAL4/UAS system was used to knock down expression of Fst and recovery ability was assessed in transgenic adults following 12 h of chill coma at 0°C. The ability to recover from cold stress (short-, medium- and long-term) was significantly altered in the transgenic adults that had Fst silenced. These findings show that Fst plays an essential role in the recovery from chill coma in both males and females.

Conclusions/Significance

The Frost gene is essential for cold tolerance in Drosophila melanogaster and may play an important role in thermal adaptation.     

Development of parasitic <Emphasis Type="Italic">Maculinea teleius</Emphasis> (Lepidoptera,Lycaenidae) larvae in laboratory nests of four <Emphasis Type="Italic">Myrmica</Emphasis> ant host species

Maculinea butterflies are social parasites of Myrmica ants. Methods to study the strength of host ant specificity in the Maculinea–Myrmica association include research on chemical and acoustic mimicry as well as experiments on ant adoption and rearing behaviour of Maculinea larvae. Here we present results of laboratory experiments on adoption, survival, development and integration of M. teleius larvae within the nests of different Myrmica host species, with the objective of quantifying the degree of specialization of this Maculinea species. In the laboratory, a total of 94 nests of four Myrmica species: M. scabrinodis, M. rubra, M. ruginodis and M. rugulosa were used. Nests of M. rubra and M. rugulosa adopted M. teleius larvae more readily and quickly than M. ruginodis colonies. No significant differences were found in the survival rates of M. teleius larvae reared by different ant species. Early larval growth of M. teleius larvae differed slightly among nests of four Myrmica host species. Larvae reared by colonies of M. rugulosa which were the heaviest at the beginning of larval development had the lowest mean larval body mass after 18 weeks compared to those reared by other Myrmica species. None of the M. teleius larvae was carried by M. scabrinodis or M. rubra workers after ant nests were destroyed, which suggests a lack of integration with host colonies. Results indicate that Myrmica species coming from the same site differ in their ability to adopt and rear M. teleius larvae but there was no obvious adaptation of this butterfly species to one of the host ant species. This may explain why, under natural conditions, all four ants can be used as hosts of this butterfly species. Slight advantages of particular Myrmica species as hosts at certain points in butterfly larval development can be explained by the ant species biology and colony structure rather than by specialization of M. teleius.     

Effects of photoperiodically induced reproductive diapause and cold hardening on the cold tolerance of Drosophila montana

Coping with seasonal and daily variation in environmental conditions requires that organisms are able to adjust their reproduction and stress tolerance according to environmental conditions. Females of Drosophila montana populations have adapted to survive over the dark and cold winters at high latitudes and altitudes by spending this season in photoperiodically controlled reproductive diapause and reproducing only in spring/summer. The present study showed that flies of a northern population of this species are quite tolerant of low temperatures and show high seasonal and short-term plasticity in this trait. Culturing the flies in short day length (nearly all females in reproductive diapause), as well as allowing the flies to get cold hardened before the cold treatment, increased the cold tolerance of both sexes both in chill coma recovery time test and in mortality assay. Chill coma recovery time test performed for the females of two additional D. montana populations cultured in a day length where about half of the females enter diapause, also showed that diapause can increase female cold tolerance even without a change in day length. Direct linkage between diapause and cold tolerance was found in only two strains representing a high-altitude population of the species, but the phenomenon will certainly be worth of studying in northern and southern populations of the species with larger data sets.     

Cold tolerance in flour beetle species differing in body size and selection temperature

Cold tolerance is an important trait directly related to survival and hence fitness. In the present study, the link is addressed between cold tolerance and body size, which is associated with many key fitness traits, at both the intra‐ and interspecific levels. Specifically, chill coma recovery time, as a metric of cold tolerance, is examined in five related flour beetle species (four of them belonging to the genus Tribolium), two additional Tribolium castaneum Herbst populations selected for higher temperatures, and a mutant showing reduced body size. Recovery times are negatively correlated with the species average body size but not within each species. Females usually recover faster than males, although this difference is significant in only a single species, and is unrelated to body size. Repeating the experimental procedure with the same individuals, after 2 days in isolation with a limited amount of food, results in longer recovery times. Therefore, even if cold acclimation takes place, its influence appears to be diminished by the deleterious effects associated with the experimental procedure. Hence, the findings provide evidence for an association between body size and cold tolerance in the genus Tribolium, with larger species recovering faster from chill than smaller species. By contrast, the smalleyed flour beetle Palorus ratzeburgii Wissmann does not follow this pattern. Additionally, a population of T. castaneum selected for the highest temperature takes longer to recover from chill coma, indicating a trade‐off between cold and heat adaptations and not to a cross‐protection effect, as sometimes demonstrated.     

Myrmica ants host highly diverse parasitic communities: from social parasites to microbes

Myrmica ants have been model species for studies in a variety of disciplines, including insect physiology, chemical communication, ant social dynamics, ant population, community ecology, and ant interactions with other organisms. Species belonging to the genus Myrmica can be found in virtually every habitat within the temperate regions of the northern hemisphere and their biology and systematics have been thoroughly studied. These ants serve as hosts to highly diverse parasitic organisms from socially parasitic butterfly caterpillars to microbes, and many Myrmica species even evolved into parasitizing species of their own genus. These parasites have various impacts both on the individuals and on the social structure of their hosts, ranging from morphological malformations to reduction in colony fitness. A comprehensive review of the parasitic organisms supported by Myrmica and the effects of these organisms on individuals and on whole ant colonies has not yet been compiled. Here, we provide a review of the interactions of these organisms with Myrmica ants by discussing host and parasite functional, behavioral or physiological adaptations. In addition, for all “symbiont groups” of Myrmica ants described in this paper, we examine the present limitations of the knowledge at present of their impact on individuals and host colony fitness. In conclusion, we argue that Myrmica ants serve as remarkable resource for the evolution of a wide variety of associated organisms.     

Dufour gland secretions of Myrmica rugulosa and Myrmica schencki workers

The chemical composition and behavioural activities of the secretions of the Dufour glands of Myrmica rugulosa and M. schencki have been studied, as part of an extended study on Myrmica ants. Chemically, the Dufour gland of M. rugulosa is filled with a mixture of hydrocarbons dominated by straight chain alkanes and alkenes with 13 to 19 carbon atoms, as found in M. rubra. Significant quantities of (Z,E)-α-farnesene and its homologues, homofarnesene and bishomofarnesene, are also present. In M. schencki, the major compounds present are homofarnesene and bishomofarnesene. In both species, the very volatile portion of the Dufour gland secretion is identical to that analysed in M. rubra. From an ethological point of view, this very volatile part is efficient in attracting workers at a distance (6 to 8 cm) and in decreasing their wandering movements. No specificity was observed when performing cross-tests with Dufour glands freshly isolated from workers of other Myrmica species, but obvious specificities were detected when testing the less volatile part of the Dufour glands' contents, known to be used for marking newly discovered areas.     

Expression of stress proteins of HSP70 family in response to cold in Myrmica ants from various geographic populations

Expression of HSP70 is induced by stress factors, including sublethal chilling. However, the role of HSP70 for overcoming the consequences of cold stress is not clear. If it is positive, the level of HSP70 expression might be higher in populations from cold climates. Using the immunoblotting technique we investigated dynamics of HSP70 expression in response to cold stress in two Myrmica species (M. rubra and M. ruginodis) from three localities of different latitudes (50, 60 and 67°N). The results showed that in the more thermophilic species Myrmica rubra, expression of HSP70 after cold stress was higher. Within both species, HSP70 expression did not show a direct relationship with latitude. The southernmost population of M. rubra and northernmost population of M. ruginodis displayed the fastest and the most intense response. However, two other populations of M. rubra were similar in timing and intensity of the response, while in M. ruginodis the intermediate population showed the slowest and weakest response. The data suggest that expression of HSP70 may play essential role for adaptation to cold only in the northernmost population of M. ruginodis     

Not only the butterflies: managing ants on road verges to benefit Phengaris (Maculinea) butterflies

Obligate myrmecophilic butterfly species, such as Phengaris (Maculinea) teleius and P. nausithous, have narrow habitat requirements. Living as a caterpillar in the nests of the ant species Myrmica scabrinodis and M. rubra, respectively, they can only survive on sites with both host ants and the host plant Great Burnet Sanguisorba officinalis. After having been reintroduced into a nature reserve in the Netherlands in 1990, both butterfly species expanded their distribution to linear landscape elements such as road verges and ditch edges outside this reserve. As additional habitat of both butterfly species, vegetation management of these landscape elements became important. Our results show that a management beneficial for Phengaris butterflies should aim to increase the nest density of Myrmica species, at the same time reducing the density of nests of the competitor Lasius niger or at least keeping them at a low density. Unfavourable grassland management under which L. niger thrives, includes not mowing or flail-cutting the grass, or depositing dredgings along the side of the ditch. Management favourable for the two Myrmica species differs, demanding some flexibility if both species are to benefit. M. scabrinodis is best supported with early mowing of the road verge vegetation or late mowing in the nature reserve, both of which result in an open vegetation and warm microclimate. In contrast, the nest sites of M. rubra should be left undisturbed during the summer, and mown in late autumn. Mowing of butterfly habitat should be avoided between mid-June and mid-September as this would remove the flowerheads of the Sanguisorba plants, on which the butterflies lay their eggs.     

Cryoprotection in dampwood termites (Termopsidae, Isoptera)

In contrast to the majority of the Order, the dampwood termites of the family Termopsidae found in colder regions can experience frost and snow, either in cool temperate areas at high latitudes (45°), or alpine areas at high elevations (>1000 m). This suggests that dampwood termites are adapted to cold climates. We investigated this hypothesis in two dampwood termites, Porotermes adamsoni Froggatt and Stolotermes victoriensis Hill. We measured nest temperatures and atmospheric temperatures of their alpine habitat during winter, and measured survival and recovery at subzero temperatures. We also determined the minimum temperature at which these species remain active and the LT50 values. We used a novel gas chromatographic strategy to examine eight metabolites from individuals of both species collected in winter and summer to identify possible cryoprotectants. Both P. adamsoni and S. victoriensis had significantly higher levels of trehalose, a known cryoprotectant, in winter than in summer; in addition S. victoriensis also had higher levels of unsaturated fatty acid ligands in winter than in summer, consistent with patterns observed for cold adaptation in other organisms. These results are the first to reveal that dampwood termites are adapted to cold climates and use trehalose and unsaturated lipids as cryoprotectants.     

Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster

Chill susceptible insects like Drosophila lose the ability to regulate water and ion homeostasis at low temperatures. This loss of hemolymph ion and water balance drives a hyperkalemic state that depolarizes cells, causing cellular injury and death. The ability to maintain ion homeostasis at low temperatures and/or recover ion homeostasis upon rewarming is closely related to insect cold tolerance. We thus hypothesized that changes to organismal ion balance, which can be achieved in Drosophila through dietary salt loading, could alter whole animal cold tolerance phenotypes. We put Drosophila melanogaster in the presence of diets highly enriched in NaCl, KCl, xylitol (an osmotic control) or sucrose (a dietary supplement known to impact cold tolerance) for 24 h and confirmed that they consumed the novel food. Independently of their osmotic effects, NaCl, KCl, and sucrose supplementation all improved the ability of flies to maintain K⁺ balance in the cold, which allowed for faster recovery from chill coma after 6 h at 0 °C. These supplements, however, also slightly increased the CT_min and had little impact on survival rates following chronic cold stress (24 h at 0 °C), suggesting that the effect of diet on cold tolerance depends on the measure of cold tolerance assessed. In contrast to prolonged salt stress, brief feeding (1.5 h) on diets high in salt slowed coma recovery, suggesting that the long-term effects of NaCl and KCl on chilling tolerance result from phenotypic plasticity, induced in response to a salty diet, rather than simply the presence of the diet in the gut lumen.     

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster

When ectotherms are exposed to low temperatures, they enter a cold‐induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill‐coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold‐induced perturbations. The metabolites of cold‐hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.