Cold hardiness in Entomobrya nivalis (Collembola, Entomobryidae): annual cycle of polyols and antifreeze proteins, and antifreeze triggering by temperature and photoperiod

In the Swiss Prealps Entomobrya nivalis hibernates in an inactive state, hidden under bark flakes on spruce. For freeze avoidance it relies on thermal hysteresis proteins (THPs) and polyols (mainly ribitol, with small amounts arabitol and threitol). Polyols are present only during the inactive state, THPs additionally protect during the transition phase in spring and autumn, when animals are still active but frosts may occur. Peak values were recorded in February/March for THPs (3.5 °C hysteresis between melting and freezing point) and for polyols (26 μg mg⁻¹ FW; hemolymph osmolality 680 mosmol l⁻¹). E. nivalis is able to control its hemolymph osmolality independently of body water content. Mean osmolality in summer was 350– 440 mosmol l⁻¹, in winter it was elevated to 650 mosmol l⁻¹, due to a synthesis mainly of ribitol. Body water content varied between 1.8 and 3.3 mg H₂O mg⁻¹ DW, depending on humidity conditions. Experiments on triggering of antifreeze synthesis showed the action of temperature and photoperiod as cues, but there was also evidence for an endogenous rhythm. No clear correlation between antifreeze concentration and supercooling ability could be established, suggesting that gut content or other parameters also play an inportant role. Accepted: 18 November 1995     

Ultrastructure of the neurosecretory system of the Colorado potato beetle,Leptinotarsa decemlineata (Say)

Summary The ultrastructure of seven types of neurosecretory cells (NSC) in the medial and lateral groups of the protocerebrum is described. The differences among cell types established earlier by light microscopy parallel differences in size and appearance of the neurosecretory particles observed in electron micrographs. No relationship was found between the affinity for Gomori's paraldehyde fuchsin stain and the nature of the particles.The secretions of the A-, A₁-, and C-types of NSC of the medial group are characterized by electron-dense neurosecretory granules of 1250 Å dia., medium-dense granules of 2100 Å, and electron-lucent vesicles of 1700 Å, respectively. The L-type NSCof the lateral group contain smaller (1300 Å) or larger (1700 Å) neurosecretory granules. The medial B- and E-types of NSC and the lateral L_B-type contain granulated vesicles (1200 Å) of the same appearance. These cell types differ in other respects and most likely have separate functions.The author wishes to thank the Laboratory of Virology of the Agricultural University for the use of the electron microscope, Mr. J. Groenewegen and Miss J. van Rinsum for technical assistance, and Professor J. Lattin for correcting the English text. Part of the work has been done while the author was in the service of the Netherlands Organization for the Advancement of Pure Research (ZWO, grant 942-48), and the National Council for Agricultural Research (TNO).     

Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold

The effect of exposure to low temperatures (5 °C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 °C compared to that of animals kept at 22 °C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 °C for the same length of time. Mean serum complement activity after 1 month at 5 °C was significantly reduced in comparison to animals held at 22 °C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 °C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 °C) in frogs returned to 22 °C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 °C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 °C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 °C failed to cause death or observable symptoms of disease in frogs that were returned to 22 °C. Accepted: 20 November 1996     

Quantifying the effects of distance and conspecifics on colonization: experiments and models using the loosestrife leaf beetle, Galerucella calmariensis

The ability of an insect to disperse to new habitat patches is difficult to quantify, but key to the establishment and persistence of populations. In this study, we examined dispersal of the phytophagous chrysomelid beetle, Galerucella calmariensis, which is currently being introduced into North America for the biological control of purple loosestrife (Lythrum salicaria), an aggressive wetland weed. We used a mark, release, and recapture approach to determine how rates of colonization of host patches by this beetle are influenced by the distance of the patch from the source of dispersers, and by the presence of conspecifics at the patch. We released color-coded beetles at six distances from a long, linear patch of purple loosestrife that was divided into segments with and without conspecifics. We observed initial flight directions as beetles left the release points and collected all beetles that settled at the target patch. We found a bias in initial flight toward the target for distances up to 50 m. Over the 7 days of the experiment, beetles arrived at the target from all release points, including the farthest release point, 847 m away. G. calmariensis was strongly attracted to conspecifics when settling after dispersal; 86% of the 582 recovered beetles came from the segments inhabited by conspecifics. The probability of an individual arriving at the patch declined steeply with release distance. This relationship fits a model in which beetles move in a random direction and stop if they intercept the target patch, and where beetles are lost at a constant rate with distance travelled. The dispersal and patch-colonizing behavior of G. calmariensis is likely to have important consequences for the biological control program against purple loosestrife. Received: 23 January 1996 / Accepted:30 September 1996     

Geographic variation in winter hardiness of a common agricultural pest,Leptinotarsa decemlineata,the Colorado potato beetle

Successful latitudinal expansions into temperate climates depend largely upon the evolution of novel adaptive traits or the presence of pre-adaptive or exapted mechanisms for survival in seasonal climates. Geographic comparisons of ancestral (pre-expansion) and derived (post-expansion) populations provide a useful framework for understanding the evolutionary conditions that facilitate geographic expansions. Using a common agricultural pest, the Colorado Potato Beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) as a model, we conducted a regional comparison of cold hardiness and overwintering success among ancestral (southern Mexico) and derived (Vermont and Kansas, USA) L. decemlineata populations. In order to determine if ancestral and derived beetle populations vary physiologically for cold hardiness, we compared supercooling points (SCPs) of three geographic populations of L. decemlineata. We also tested if ancestral and derived beetle populations differed in their overwintering behavior and success by performing an overwintering field experiment. Ancestral and derived populations did not express different physiological responses (i.e. SCPs) to freezing temperatures. However, ancestral and derived populations responded differently to the onset of winter conditions and displayed dissimilar overwintering behaviors. The majority of ancestral beetles failed to initiate diapause and dug upward within experimental mesocosms. Differences in overwintering behavior also resulted in significant variation in overwintering success as derived populations displayed higher overwintering survivorship when compared with ancestral populations. Given our results, it is evident that research exploring the interaction of the ecological factors and evolutionary processes is necessary to fully realize the dynamics of biological invasions.     

Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say)

 A modified gene of Bacillus thuringiensis var. Tolworthi (Bt), encoding a coleopteran insect-specific CryIIIB toxin, was transferred via Agrobacterium tumefaciens to the female parent of the eggplant commercial F₁ hybrid ‘Rimina’. One-hundred and fifty eight transgenic plants were regenerated and tested by PCR and NPTII expression assays. The presence of the CryIIIB toxin in leaf extracts was demonstrated in 57 out of 93 transgenic plants tested by DAS-ELISA assay. High Bt-expressing plants contained a 74-kDa protein cross-reacting with serum anti-CryIIIB toxin. Twenty three out of 44 S. melongena plants tested by insect bioassay showed significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus, transgenic Bt eggplants represent a very effective means of CPB pest control. Received: 25 November 1996/Accepted: 31 January 1997     

Responses to object approach by a wide field visual neurone, the LGMD2 of the locust: Characterization and image cues

The LGMD2 belongs to a group of giant movement-detecting neurones which have fan-shaped arbors in the lobula of the locust optic lobe and respond to movements of objects. One of these neurones, the LGMD1, has been shown to respond directionally to movements of objects in depth, generating vigorous, maintained spike discharges during object approach. Here we compare the responses of the LGMD2 neurone with those of the LGMD1 to simulated movements of objects in depth and examine different image cues which could allow the LGMD2 to distinguish approaching from receding objects. In the absence of stimulation, the LGMD2 has a resting discharge of 10–40 spikes s⁻¹ compared with <1 spike s⁻¹ for the LGMD1. The most powerful excitatory stimulus for the LGMD2 is a dark object approaching the eye. Responses to approaching objects are suppressed by wide field movements of the background. Unlike the LGMD1, the LGMD2 is not excited by the approach of light objects; it specifically responds to movement of edges in the light to dark direction. Both neurones rely on the same monocular image cues to distinguish approaching from receding objects: an increase in the velocity with which edges of images travel over the eye; and an increase in the extent of edges in the image during approach. Accepted: 23 October 1996     

Influence of stand structure on carbon-13 of vegetation, soils, and canopy air within deciduous and evergreen forests in Utah, United States

Carbon isotope ratios (δ¹³C) were studied in evergreen and deciduous forest ecosystems in semi-arid Utah (Pinus contorta, Populus tremuloides, Acer negundo and Acer grandidentatum). Measurements were taken in four to five stands of each forest ecosystem differing in overstory leaf area index (LAI) during two consecutive growing seasons. The δ¹³C_leaf (and carbon isotope discrimination) of understory vegetation in the evergreen stands (LAI 1.5–2.2) did not differ among canopies with increasing LAI, whereas understory in the deciduous stands (LAI 1.5–4.5) exhibited strongly decreasing δ¹³C_leaf values (increasing carbon isotope discrimination) with increasing LAI. The δ¹³C values of needles and leaves at the top of the canopy were relatively constant over the entire LAI range, indicating no change in intrinsic water-use efficiency with overstory LAI. In all canopies, δ¹³C_leaf decreased with decreasing height above the forest floor, primarily due to physiological changes affecting c _i/c _a (> 60%) and to a minor extent due to δ¹³C of canopy air (< 40%). This intra-canopy depletion of δ¹³C_leaf was lowest in the open stand (1‰) and greatest in the denser stands (4.5‰). Although overstory δ¹³C_leaf did not change with canopy LAI, δ¹³C of soil organic carbon increased with increasing LAI in Pinus contorta and Populus tremuloides ecosystems. In addition, δ¹³C of decomposing organic carbon became increasingly enriched over time (by 1.7–2.9‰) for all deciduous and evergreen dry temperate forests. The δ¹³C_canopy of CO₂ in canopy air varied temporally and spatially in all forest stands. Vertical canopy gradients of δ¹³C_canopy, and [CO₂]_canopy were larger in the deciduous Populus tremuloides than in the evergreen Pinu contorta stands of similar LAI. In a very wet and cool year, ecosystem discrimination (Δ_e) was similar for both deciduous Populus tremulodies (18.0 ± 0.7‰) and evergreen Pinus contorta (18.3 ± 0.9‰) stands. Gradients of δ¹³C_canopy and [CO₂]_canopy were larger in denser Acer spp. stands than those in the open stand. However, ¹³C enrichment above and photosynthetic draw-down of [CO₂]_canopy below tropospheric baseline values were larger in the open than in the dense stands, due to the presence of a vigorous understory vegetation. Seasonal patterns of the relationship δ¹³C_canopy versus 1/[CO₂]_canopy were strongly influenced by precipitation and air temperature during the growing season. Estimates of Δ_e for Acer spp. did not show a significant effect of stand structure, and averaged 16.8 ± 0.5‰ in 1933 and 17.4 ± 0.7‰ in 1994. However, Δ_e varied seasonally with small fluctuations for the open stand (2‰), but more pronounced changes for the dense stand (5‰). Received: 15 April 1996 / Accepted: 19 October 1996     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996     

Dynamic responses of series force receptors innervating the opener muscle apodeme in the blue crab, Callinectes sapidus

Receptors monitoring muscle force innervate the opener muscle apodeme in the walking legs of the blue crab, Callinectes sapidus. Biocytin backfills reveal 9–15 bipolar neurons with somata as large as 60 μm positioned at the distal end of the apodeme. Sensory endings insert into the apodeme and are in series with the opener muscle. The axons of these neurons form the opener apodeme sensory nerve that merges with the most distal branch of the opener motor nerve. Recordings reveal that the receptors are not spontaneously active nor do they respond to passive muscle stretch. Isometric muscle contraction evoked by stimulating the opener excitor motor neuron is the adequate stimulus for receptor firing. Most significant is the finding that during contraction, over a wide range of forces, the firing rate of individual receptors closely parallels the rate of change of isometric force. The peak instantaneous frequency typically occurs at the force derivative maximum, but not at maximum force development. Thus, receptors of the opener apodeme sensory nerve more closely monitor changes in isometric force rather than the total force achieved. Accepted: 20 September 1996     

Effects of four Bacillus spp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say)

Four species of Bacillus were isolated from soil in an effort to find safe, effective and alternative biological control agents against plant pests. These bacteria were identified as Bacillus pumilus, Bacillus sphaericus, Bacillus megaterium and Bacillus cereus on the basis of fatty acid methyl ester analysis and carbon utilization profiles by using Microbial Identification and Biolog Microplate Systems. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that B. pumilus caused 95.7 and 26.7% mortality and B. sphaericus caused 74.5 and 23.3% mortality of Leptinotarsa decemlineata larvae and adults, respectively. B. cereus and B. megaterium showed 51.1 and 29.7%, respectively, of L. decemlineata larvae. This study presents at least two Turkish isolates from the genus Bacillus showing high insecticidal activity against L. decemlineata.     

The effect of mermithid parasitism on predation of nymphal Baetis bicaudatus (Ephemeroptera) by invertebrates

We investigated how infection by the mermithid nematode Gasteromermis sp. affected predation on its nymphal mayfly host, Baetisbicaudatus, by two invertebrate predators – the stonefly nymphs of Kogotusmodestus and the caddisfly larvae of Rhyacophilahyalinata. Predation trials and behavioral observations were conducted in stream-side, flow-through experimental chambers. When parasitized and unparasitized prey were offered in equal numbers, K. modestus consumed significantly more parasitized than unparasitized nymphs. R. hyalinata consumed equal numbers of both prey types. Behavioral observations of foraging K.␣modestus on parasitized and unparasitized prey suggested that the increased consumption of parasitized nymphs was due to differences in the behavior of infected mayflies in response to the predator. Specifically, parasitized nymphs drifted less often to escape an approaching predator (non-contact encounters) compared to unparasitized nymphs, which increased the number of contact encounters and attacks that occurred between K.␣modestus and parasitized prey. Because all hosts are castrated, these behavioral alterations affect only the fitness of the parasite, which is killed along with its host by invertebrate predation. We present a number of hypotheses to explain why the parasite causes increased predation on its host. These include the large size of the parasite affecting the sensory abilities of the host, the larger energetic costs of escape behavior for parasitized individuals, and natural selection from fish predation against drifting behavior by parasitized individuals. Received: 27 May 1996 / Accepted: 30 September 1996     

Young chickens learn to localize the centre of a spatial environment

Young chickens were trained to find food by ground-scratching in the centre of a closed uniform arena and were then tested in arenas of similar areas but of different shapes. Chickens showed localized searching behaviour in the square-shaped arena, and maintained this behaviour when placed in a circular or triangular (both equilateral and isosceles) arena. With a rectangular-shaped arena, obtained by doubling the original square-shaped one, chickens showed more dispersed searching along the major axis, but searching tended to be concentrated around the centres of the composing squares and around the centre of the rectangle itself. When trained in a square- or triangle-shaped arena and then tested in an arena of the same shape but a larger area, chickens displayed searching behaviour at two different distances from the wall of the arena, one corresponding to the correct distance (i.e. centre) in the smaller (training) arena, the other to the actual centre of the test arena. On the other hand, in a circular arena, chickens searched mainly at a distance midway between the radius of the small (training) and of the large (testing) circular arena. These results suggest that, during training, chickens encoded information on both the absolute and the relative distance of the food from the walls of the arena, the latter information being more accurate when the arena displayed identifiable features such as corners. Accepted: 28 December 1996     

Rhythmicity of torpor in a marsupial hibernator, the mountain pygmy-possum (Burramysparvus), under natural and laboratory conditions

Circadian rhythms have been observed in most mammals, but their importance and function remain controversial with respect to daily cycles during hibernation. We investigated the timing of arousals from and entries into hibernation for both free-living and captive mountain pygmy-possums (Burramys parvus). Under both natural and laboratory conditions most arousals and entries were entrained with the light-dark cycle. Entries occurred mainly during the night and arousals preferably around dusk, which coincides with the onset of the normal activity phase for the nocturnal pygmy-possums. This entrainment prevailed throughout the hibernation season although only the laboratory animals were constantly subjected to photoperiodic stimuli, whereas under natural conditions hibernacula are shielded from photic cues and diurnal temperature fluctuations. Nevertheless, possums left their hibernacula frequently throughout winter and were occasionally trapped close to the snow surface suggesting that during the periods of post-arousal normothermia they can be exposed to environmental stimuli. It thus appears that the synchronisation with the photocycle was governed by a temperature-compensated circadian clock which was reset periodically during short activity periods. For the mountain pygmy-possum, entrainment with the photocycle probably has two functions: 1. Entrainment ensures that foraging bouts during the hibernation season remain synchronised with the dark phase. 2. Information about the prevailing climatic conditions sampled during short activity periods enables them to time final spring emergence from hibernation when snow melt begins and ensures that the breeding season can commence as early as possible. Accepted: 26 August 1998     

Effects of aerobic exercise on the torque-velocity relationship in cycling

The kinetics of the torque-velocity (T-ω) relationship after aerobic exercise was studied to assess the effect of fatigue on the contractile properties of muscle. A group of 13 subjects exercised until fatigued on a cycle ergometer, at an intensity which corresponded to 60% of their maximal aerobic power for 50 min (MAP60%); ten subjects exercised until fatigued at 80% of their maximal aerobic power for 15 min (MAP80%). Of the subjects 7 exercised at both intensities with at least a 1-week interval between sessions. Pedalling rate was set at 60 rpm. The T-ω relationship was determined from the velocity data collected during all-out sprints against a 19 N · m braking torque on the same ergometer, according to a method proposed previously. Maximal theoretical velocity (ω₀) and maximal theoretical torque (T ₀) were estimated by extrapolation of the linear T-ω relationship. Maximal power (P _max) was calculated from the values of T ₀ and ω₀ (P _max = 0.25 ω₀ T ₀). The T-ω relationships were determined before, immediately after and 5 and 10 min after the aerobic exercise. The kinetics of ω₀, T ₀ and P _max was assumed to express the effects of fatigue on the muscle contractile properties (maximal shortening velocity, maximal muscle strength and maximal power). Immediately after exercise at MAP60% a 7.8% decrease in T ₀ and 8.8% decrease in P _max was seen while the decrease in ω₀ was nonsignificant, which suggested that P _max decreased in the main because of a loss in maximal muscle strength. In contrast, MAP80% induced a 8.1% decrease in ω₀ and 12.8% decrease in P _max while the decrease in T ₀ was nonsignificant, which suggested that the main cause of the decrease in P _max was probably a slowing of maximal shortening velocity. The short recovery time of the T-ω relationship suggests that the causes of the decrease of torque and velocity are processes which recover rapidly. Accepted: 25 November 1996