Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) survive extended periods in winter during which tissue water is frozen. Both low temperature and reduced water activity during freezing present challenges for the structural integrity of cellular lipids. Fatty acids of both phospholipids and triacylglycerols from fat body cells of E. solidaginis were analyzed throughout fall and early winter, a period that encompasses the acquisition of freeze-tolerance, to determine if adaptations to freezing include changes in fatty acid unsaturation. The five most abundant fatty acids from both fractions were (in decreasing order) oleic (40–65%), palmitoleic (18–20%), palmitic (12–17%), linoleic (5–10%), and stearic acids (4 –7%). This represents a typical complement of Dipteran fatty acids, although oleic acid levels were higher in E. solidaginis than those reported from other Dipterans (˜28%; Downer 1985). From September to November, monounsaturates increased from 59 to 70% in phospholipids at the expense of saturated fatty acids (25% –20%) suggesting activation of a Δ⁹-desaturase enzyme. These changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (U/S) from 3.0 to 4.2, although there was no change in the average number of double bonds per fatty acid (unsaturation index, UI ≈ 1.2 in phospholipids and 0.9 in triacylglycerols throughout the season). These changes were temporally correlated to decreasing ambient temperatures and increasing larval and fat body cell freeze-tolerance. Accepted: 31 October 1996     

Physiological characterization of antennular flicking reflexes in the crayfish

The cellular substrates of antennular flicking behavior in the crayfish Procambarus clarkii were investigated. Flicking involves fast downward movements of the external filament of each biramous antennule (1st antenna), and is mediated by phasic contractions of a short muscle, the external filament depressor. Phasic contractions of the external filament depressor depend upon stereotyped impulse bursts in a single motorneuron (P1). These bursts have a characteristic impulse frequency profile that is consistent upon successive occurrences. The temporal characteristics of the impulse burst suggest that the central depolarizations generating each burst may be similar to driver potentials described for motor neurons in crustacean cardiac ganglia. Responses of the external filament to odorants have a long latency and are characterized by repetitive bursts and tonic activity in some external filament depressor fibers. Tonic activity in a slowly contracting muscle, the antennular depressor muscle, is also evoked by chemical stimulation. Flicking is consistently evoked only by mechanical or hydrodynamic stimulation of the cephalothorax, antennae and antennules. The sensitivity and short latency of the hydrodynamic antennule-generated flick reflex is consistent with the sensitivity of rapidly conducting, hydrodynamically activated mechanoreceptor neurons in both antennular filaments. I propose that antennular flicking, which has been shown to enhance the dynamic response characteristics of olfactory receptor neurons on the external antennular filament, has evolved as a response to the turbulence associated with fluid movement, within which chaotic odorant concentration fronts may be imbedded. Accepted: 23 October 1996     

Patch clamp study and kinetic modeling on the most abundant chloride channel on distal fibers of crayfish opener muscle

Whereas the inhibitory innervation of the deep extensor abdominal muscle in crayfish is mediated by a weakly acting common inhibitor, the opener muscle exhibits a stronger inhibition. In the present study the most abundant γ-aminobutyric acid-activated chloride channel on distal fibers of crayfish opener muscle was characterized by measuring the current responses after applying pulses of γ-aminobutyric acid to outside-out patches. The results were compared to those obtained earlier with the chloride channel on the deep extensor abdominal muscle of the same species. The double logarithmic plot of the dose-response relationship had a slope of n _H = 2.2 in contrast to n _H = 5.3 for the channel on the deep extensor abdominal muscle. The rise time of the current response declined to 1 ms at a γ-aminobutyric acid concentration of 50 mmol · l⁻¹. With lower concentrations the rise time increased to a maximal value of 280 ms. No peak of the rise time at low γ-aminobutyric acid concentrations, as observed for the channel on the deep extensor abdominal muscle, was obvious. The open and closed times were similar to those of the channel of the deep extensor abdominal muscle. Different reaction schemes were discussed to describe the kinetics of the chloride channel of the opener muscle. Accepted: 12 August 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     

Fatty acid compositions of arthropod and cephalopod photoreceptors: interspecific,seasonal and developmental studies

Fatty acid compositions of the compound eyes of insects (soldier-bug, Hemiptera, and silk moth, Lepidoptera), crustaceans (crayfish and grapsid crab, Decapoda) and inner and outer segments of visual cells of a squid (Cephalopoda, Mollusca) were analyzed by gaschromatography for interspecific comparison. Fatty acid compositions showed great variation among species. In insect compound eyes, 16:0 and 18:0 were the main saturated fatty acids, and 18:1 was the dominant unsaturated fatty acid. Silk moth eyes contained, in addition, considerable amounts of 18:2 and 20:5. In crustacean compound eyes, the main saturated fatty acids were 16:0 and 18:0, and 14:0 (5.0%) was only detected in grapsid crabs; the main unsaturated fatty acids were 20:4, 20:5 and 22:6. Both whole eyes and rhabdom fraction of crayfish showed similar profiles of fatty acid compositions. Both inner and outer segments of squid retinae were characterized by high amounts of unsaturated fatty acids, especially 22:6. Compound eyes of grapsid crabs were used for the experiments on seasonal changes of fatty acid compositions. UFA/SFA ratios (weight in % of unsaturated fatty acids saturated fatty acids) were lowest (1.0) in July and highest (2.5) in March, and unsaturation indexes (average number of double bonds per molecule) were lowest (1.5) in July and highest (2.3) in March. Fatty acids 18:0 and 20:1 showed a significant correlation with the changes of seasonal temperature. Fatty acid analysis of the developing compound eyes of silk moths during the pupal stage revealed that eicosapentanoic acid (20:5) increased remarkably in parallel with the development of photoreceptive membranes, the rhabdoms. This suggests that eicosapentaenoic acid may play an important role in formation and function of rhabdoms.     

Effects of ultraviolet radiation on early larval stages of the Alpine newt, Triturus alpestris, under natural and laboratory conditions

Although Alpine newts (Triturus alpestris) are found at altitudes up to 2500 m, their larvae proved to be extremely sensitive to UV radiation when exposed in clear tapwater to natural sunlight or to comparable artificial UV-B radiation in the laboratory. The experiments revealed severe skin damages (lysis of epithelial cells) and mortality after a few days of exposure. In their natural habitats above the timberline, however, the larvae are protected by the high concentration of dissolved organic carbon in the water leading to an almost complete absorption of UV radiation within the first few centimeters of the water layer. Furthermore, when exposed to UV radiation larvae show erratic swimming activities that may carry them into protected areas. Only in very shallow natural habitats did we detect sublethal UV-like histological effects. Shallow high mountain ponds with clear water normally lack newt populations, probably as a consequence of both low temperature and the effects of UV radiation. Received: 4 October 1996 / Accepted: 7 February 1997     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

In crayfish photoreceptor cells, Gq-type G-protein plays a central role in the phototransduction pathway, and the translocation of Gqα has been proposed as one of the molecular mechanisms to control photoreceptor sensitivity. We here investigated β subunit of Gq and its localization profiles under various light conditions in the crayfish photoreceptor cells to understand the functional characteristic of visual Gq in the phototransduction pathway. An immunoprecipitation experiment was performed using an anti-Gqα antibody and a thiol-cleavable crosslinker. A 39 kDa protein was co-immunoprecipitated with Gqα, but not by irradiation, in the presence of GTPγS. The partial amino acid sequence of the 39 kDa protein was similar to Gβe in Drosophila photoreceptors, indicating that the crayfish Gβ which combines with Gqα is a Gβe homologue. Immunohistochemical and immunoblot analyses revealed that the amount of the Gβ decreased in the rhabdomeric membranes and increased in the cytoplasm in the light, compared with that in the dark. The profile of the translocation was similar to that reported for Gqα. Since both α and βγ subunits are necessary for G-proteins to be activated by rhodopsin in the rhabdom, the light-modulated translocation of a Gβe homologue possibly controls the amount of Gq which can be activated by light-stimulated rhodopsin. Accepted: 27 June 1998     

Non-spiking local interneurones mediate abdominal extension related descending control of uropod motor neurones in the crayfish terminal abdominal ganglion

The role of non-spiking local interneurones in the synaptic interactions between abdominal extension-evoking descending interneurones and uropod motor neurones in the terminal abdominal ganglion of the crayfish Procambarus clarkii (Girard) was investigated electrophysiologically. Continuous electrical stimulation of the lateral region of the 3rd-4th abdominal connective that included abdominal extension evoking interneurones excited the opener motor neurones and inhibited the closer, reductor motor neurone. Spikes from a single descending interneurone evoked consistent and short latency (0.8–0.9 ms) excitatory postsynaptic potentials (e.p.s.ps) in the opener motor neurones, and evoked rather long-latency (1.5–2.7 ms) inhibitory postsynaptic potentials (i.p.s.ps) in the reductor motor neurone. Many non-spiking interneurones also received depolarizing p.s.ps (0.8–2.5 ms in latency) that were usually faster than i.p.s.ps of the reductor motor neurone if both neurones were recorded sequentially in the same preparation. Non-spiking interneurones received convergent inputs from several descending interneurones and made inverting connection with the reductor motor neurone. Elimination of descending inputs to a particular non-spiking interneurone could reduce the inhibitory response of the reductor motor neurone. These observations strongly suggested that descending inhibitory inputs to the closer, reductor motor neurone were mediated by non-spiking interneurones. Furthermore, some non-spiking interneurones made output connections with the opener motor neurones. The disynaptic pathway through non-spiking interneurones is significant to control and modulate the opening pattern of the uropod during abdominal extension. Accepted: 27 December 1996     

Effect of the removal of cleaner fish on the abundance and species composition of reef fish

The ecological significance of cleaner fish on coral reefs was investigated. I removed all cleaner fish, Labroides dimidiatus, from eight small reefs, measured the subsequent effect on the abundance and species composition of all reef fish after 3 and 6 months, and compared it with eight control reefs with cleaner fish. The removal of cleaner fish had no detectable effect on the total abundance of fish on reefs and the total number of fish species at both times. Multivariate analysis by non-metric multidimensional scaling and ANOSIM pairwise tests based on 191 fish species revealed no effect of cleaners on the community structure of fish. Similar results were obtained using principal components analysis on subsets of the data using the 33 most common fish species and the 15 most abundant species (≥5 individuals per reef ) with both log10 (x + 1) transformed data and with fish numbers standardized for abundance. This study demonstrates that the removal of cleaner fish for 6 months did not result in fish suffering increased mortality nor in fish leaving reefs to seek cleaning elsewhere. Received: 28 October 1996 / Accepted: 7 February 1997     

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons in the crayfish Procambarus clarkii Girard

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons of crayfish have been studied using intradendritic recording and current injection. The input resistance of phasic motoneurons (5.20 ± 0.5 M; mean ± standard error) measured by injecting constant hyperpolarizing current was significantly lower than that of tonic motoneurons (10.3 ± 2.6 M; 0.02 < P < 0.05). The membrane time constant of phasic motoneurons (7.3 ± 0.9 ms) was also significantly shorter than that of tonic motoneurons (24.3 ± 2.5 ms; P < 0.001). Both types of motoneurons behaved linearly during hyperpolarization and sub-threshold depolarization. Nonspiking interneurons showed outward rectification upon depolarization. During hyperpolarization, their membrane behaved linearly and showed significantly higher input resistance (19.5 ± 2.5 M) than phasic and tonic motoneurons (P < 0.001). Their membrane time constant (38.0 ± 5.7 ms) was significantly longer than that of phasic motoneurons (P < 0.001) but not than that of tonic motoneurons (P > 0.05). In response to intracellular injection of sinusoidally oscillating current, phasic motoneurons showed one or two spikes per depolarization period irrespective of oscillating frequency ranging from 1 to 16 Hz. Tonic motoneurons showed larger numbers of spikes per stimulus period at lower frequencies. Nonspiking interneurons also showed phase-locked effects on the motoneuron spike activity. The effective frequency range over which injected oscillating current could modulate motoneuron spike activity was similar for tonic motoneurons and nonspiking interneurons.     

Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

A phospholipase inhibitor, manoalide, and a G-protein activator, Mas-7, both affect the turnover of phototransductive membranes by crab retinas in darkness With a model for the regulation of rhabdomeral membrane turnover

(1) In vitro retinas of a crab, Leptograpsus, were treated with a phospholipase inhibitor, manoalide, or a G-protein activator, Mas-7. Both drugs address early stages of the phototransduction cascade. (2) Manoalide inhibited the light-dependent reduction of rhabdoms during the `day' phase of the light cycle, but did not induce rhabdom overgrowth. Following a period of darkness manoalide failed to affect the diminution of illuminated rhabdoms. (3) The diminution of rhabdoms that follows photoreceptor depolarisation induced by 100 mmol · l<sup>−1</sup> K<sup>+</sup> in darkness was not affected by 2␣μmol · l<sup>−1</sup> manoalide. (4) When retinas in the `night' phase were treated with Mas-7 in darkness, rhabdom diameters were augmented, concurrently with endocytosis of photoreceptor plasma membranes. (5) The results of combining manoalide and Mas-7 with actinomycin D, U-57908 or okadaic acid, drugs used in previous studies to manipulate steps notionally lower in the transduction cascade, lead to a hypothetical model for the regulation of phototransductive membrane turnover by arthropods. Accepted: 3 October 1996     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Sexual dimorphism and species differences in the neurophysiology and morphology of the acoustic communication system of two neotropical hylids

We examined auditory tuning and the morphology of the anatomical structures underlying acoustic communication in female Hyla microcephala and H. ebraccata and compared our findings to data from a previous study (Wilczynski et al. 1993) in which we showed species differences in the traits that in males relate to differences in the species-typical calls. Female species differences in the best excitatory frequency (BEF) of the basilar papilla (BP) were similar to the differences seen in males, and females had a significantly lower BEF in H. ebraccata, but not H. microcephala. In both species, females had lower BP thresholds. Snout-vent length, head width, and tympanic membrane diameters were sexually dimorphic in both species and larger in females, whereas laryngeal components were sexually dimorphic and larger in males. Middle and inner ear volumes were not sexually dimorphic. Despite the significant species differences in laryngeal morphology seen in males, female larynges are not significantly different. Furthermore, the interaction of species and sex differences resulted in significantly different degrees of sex dimorphism in the species, particularly for the larynx, which is more sexually dimorphic in H. microcephala, and measures of body size, which are more dimorphic in H. ebraccata. Accepted: 6 December 1996     

A comparison of the response to induced exercise in red- and white-blooded Antarctic fishes

The physiological responses to forced exercise were studied in yellowbelly and marbled rockcod (Notothenia coriiceps and N. rossii), and the haemoglobinless icefish (Chaenocephalus aceratus), from blood samples obtained via indwelling catheters. The maximal exertion tolerable by N. coriiceps was 3–5 min, although N. rossii was not fully exhausted by this effort, and it proved difficult to elicit sustained maximal activity in C. aceratus. Arterial O₂ tension reflected the relative degree of exhaustion, showing a significant fall in the case of N. coriiceps, little change in N. rossii, and even a rise in C. aceratus as a result of hyperventilation. Such changes in the red-blooded species were not caused by altered O₂ carrying capacity, as there was no change in haematocrit. In Notothenia spp. the decrease in arterial pH was better correlated with a rise in arterial CO₂ tension than with blood lactate concentration, which is reflected in a modest net metabolic acid load. In contrast, the icefish showed an attenuated hypercapnia and a more pronounced lactacidosis, but an insignificant net metabolic acid load. Disturbance in ionoregulation following exercise was limited to an elevated [Cl⁻] in Notothenia, while circulating catecholamine levels remained unusually low in all specimens. The response to stress appears to reflect lifestyle and/or endemic speciation, rather than specific adaptations to the stenothermal environment. Accepted: 9 August 1996     

Post-dispersal seed predation and the establishment of vertebrate dispersed plants in Mediterranean scrublands

The post-dispersal fate of seeds and fruit (diaspores) of three vertebrate-dispersed trees, Crataegus monogyna, Prunus mahaleb and Taxus baccata, was studied in the Andalusian highlands, south-eastern Spain. Exclosures were used to quantify separately the impact of vertebrates and invertebrates on seed removal in relation to diaspore density and microhabitat. The three plant species showed marked differences in the percentage of diaspores removed, ranging from only 5% for C. monogyna to 87% for T. baccata. Although chaffinches (Fringilla coelebs) fed on diaspores, rodents (Apodemus sylvaticus) were the main vertebrate removers of seed and fruit. Two species of ant (Cataglyphis velox and Aphaenogaster iberica) were the only invertebrates observed to remove diaspores. However, the impact of ants was strongly seasonal and they only removed P. mahaleb fruit to any significant extent. While removal of seed by rodents was equivalent to predation, ants were responsible for secondary dispersal. However, their role was limited to infrequent, small-scale redistribution of fruit in the vicinity of parent trees. Rodents and ants differed in their use of different microhabitats. Rodents foraged mostly beneath trees and low shrubs and avoided open areas while the reverse was true of ants. Thus, patterns of post-dispersal seed removal will be contigent on the relative abundance and distribution of ants and rodents. Studies which neglect to quantify separately the impacts of these two guilds of seed removers may fail to elucidate the mechanisms underlying patterns of post-dispersal seed removal. The coincidence of both increased seed deposition by the main avian dispersers (Turdus spp.) and increased seed predation with increasing vegetation height suggested that selection pressures other than post-dispersal seed predation shape the spatial pattern of seed dispersal. Rather than providing a means of escaping post-dispersal seed predators, dispersal appears to direct seeds to microhabitats most suitable for seedling survival. Nevertheless, the reliance of most vertebrate-dispersed trees on regeneration by seed and the absence of persistent soil seed banks imply that post-dispersal seed predators may exert a strong influence on the demography of the plants whose seeds they consume. Even where microsites are limited, the coincidence of the most suitable microhabitats for seedling establishment with those where seed predation is highest provide a means by which selective seed predators can influence community composition. Received: 19 August 1996 / Accepted: 25 January 1997     

The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant

In order to test whether an increased export of carbohydrates by leaves and starch mobilization are critical for floral transition in Arabidopsis thaliana, the Columbia ecotype as well as its starchless mutant pgm and starch-in-excess mutant sex1 were investigated. Induction of flowering was achieved by exposure of plants to either one long day (LD) or one displaced short day (DSD). The following conclusions were drawn: (i) Both the pgm and sex1 mutants have a late-flowering phenotype in days shorter than 16 h. (ii) When inductive treatments cause a large percentage of induced plants, there is always a large, early and transient increase in carbohydrate export from leaves. By contrast, when an inductive treatment results in only a low percentage of induced plants (pgm plants exposed to one DSD), the export of carbohydrates from leaves is not increased, supporting the idea that phloem carbohydrates have a critical function in floral transition. (iii) Starch mobilization is not required to obtain an increased carbohydrate export when induction is by one LD (extended period of photosynthesis), but is absolutely essential when induction is by one DSD (period of photosynthesis unaffected). (iv) Floral induction apparently increases the capability of the leaf phloem-loading system. Received: 27 August 1997 / Accepted: 6 March 1998     

Fatty acid composition of brown adipose tissue in genetically heat-tolerant FOK rats

The phospholipid fatty acid composition of brown adipose tissue (BAT) was examined in inbred heat-tolerant FOK rats and compared with that in conventional Wistar rats not previously exposed to heat. The FOK rats showed higher unsaturation states, as indicated by higher levels of polyunsaturated fatty acids and a higher unsaturation index and polyunsaturated fatty acids/saturated fatty acids ratio. This higher level of unsaturation was characterized by the higher amount of polyunsaturated fatty acids such as linoleic acid, arachidonic acid and docosahexaenoic acid. It may be concluded that the increased docosahexaenoic acid level in BAT phospholipids brings about the hyperplasia of BAT, causing an enhancement of its in vivo thermogernic activity as well as the systemic non-shivering thermogenesis observed in heat-tolerant FOK rats. Received: 17 May 2000 / Revised: 21 August 2000 / Accepted: 21 September 2000