Circadian regulation of bioluminescence in the prey-luring glowworm, Arachnocampa flava

The glowworms of New Zealand and Australia are bioluminescent fly larvae that generate light to attract prey into their webs. Some species inhabit the constant darkness of caves as well as the dim, natural photophase of rain-forests. Given the diversity of light regimens experienced by glowworms in their natural environment, true circadian rhythmicity of light output could be present. Consequently the light emission characteristics of the Australian subtropical species Arachnocampa flava, both in their natural rainforest habitat and in artificial conditions in the laboratory, were established. Larvae were taken from rainforest and kept alive in individual containers. When placed in constant darkness (DD) in the laboratory they maintained free-running, cyclical light output for at least 28 days, indicating that light output is regulated by an endogenous rhythm. The characteristics of the light emission changed in DD: individuals showed an increase in the time spent glowing per day and a reduction in the maximum light output. Most individuals show a free-running period greater than 24 h. Manipulation of the photophase and exposure to skeleton photoperiods showed that light acts as both a masking and an entraining agent and suggests that the underlying circadian rhythm is sinusoidal in the absence of light-based masking. Manipulation of thermoperiod in DD showed that temperature cycles are an alternative entraining agent. Exposure to a period of daily feeding in DD failed to entrain the rhythm in the laboratory. The endogenous regulation of luminescence poses questions about periodicity and synchronization of bioluminescence in cave glowworms.     

Presence of circadian rhythms in the locomotor activity of a cave-dwelling millipede Glyphiulus cavernicolus sulu (Cambalidae, Spirostreptida)

The locomotor activity of the millipede Glyphiulus cavernicolus (Spirostreptida), which occupies the deeper recesses of a cave, was monitored in light-dark (LD) cycles (12h light and 12h darkness), constant darkness (DD), and constant light (LL) conditions. These millipedes live inside the cave and are apparently never exposed to any periodic factors of the environment such as light-dark, temperature, and humidity cycles. The activity of a considerable fraction of these millipedes was found to show circadian rhythm, which entrained to a 12:12 LD cycle with maximum activity during the dark phase of the LD cycle. Under constant darkness (DD), 56.5% of the millipedes (n = 23) showed circadian rhythms, with average free-running period of 25.7h ± 3.3h (mean ± SD, range 22.3h to 35.0h). The remaining 43.5% of the millipedes, however, did not show any clear-cut rhythm. Under DD conditions following an exposure to LD cycles, 66.7% (n = 9) showed faint circadian rhythm, with average free-running period of 24.0h ± 0.8h (mean ± SD, range 22.9h to 25.2h). Under constant light (LL) conditions, only 2 millipedes of 11 showed free-running rhythms, with average period length of 33.3h ± 1.3h. The results suggest that these cave-dwelling millipedes still possess the capacity to measure time and respond to light and dark situations. (Chronobiology International, 17(6), 757-765, 2000)     

Distribution and phylogenetic relationships of Australian glow-worms Arachnocampa (Diptera, Keroplatidae)

Glow-worms are bioluminescent fly larvae (Order Diptera, genus Arachnocampa) found only in Australia and New Zealand. Their core habitat is rainforest gullies and wet caves. Eight species are present in Australia; five of them have been recently described. The geographic distribution of species in Australia encompasses the montane regions of the eastern Australian coastline from the Wet Tropics region of northern Queensland to the cool temperate and montane rainforests of southern Australia and Tasmania. Phylogenetic trees based upon partial sequences of the mitochondrial genes cytochrome oxidase II and 16S mtDNA show that populations tend to be clustered into allopatric geographic groups showing overall concordance with the known species distributions. The deepest division is between the cool-adapted southern subgenus, Lucifera, and the more widespread subgenus, Campara. Lucifera comprises the sister groups, A. tasmaniensis, from Tasmania and the newly described species, A. buffaloensis, found in a high-altitude cave at Mt Buffalo in the Australian Alps in Victoria. The remaining Australian glow-worms in subgenus Campara are distributed in a swathe of geographic clusters that extend from the Wet Tropics in northern Queensland to the temperate forests of southern Victoria. Samples from caves and rainforests within any one geographic location tended to cluster together within a clade. We suggest that the morphological differences between hypogean (cave) and epigean (surface) glow-worm larvae are facultative adaptations to local microclimatic conditions rather than due to the presence of cryptic species in caves.     

Cold resistance in two species of cave-dwelling beetles (Coleoptera: Cholevidae)

Supercooling points (SCPs), lower lethal temperatures (LLTs), and the effect of short-term exposures (1 min) to low temperatures were examined in the adults of two stenothermal leptodirin species, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae). Specimens were collected from two caves in the Venetian Prealps (NE-Italy). Inter-species comparison highlighted lower values of SCP in N. mancinii (−7.1±0.9 °C) than in N. pasai (−6.4±0.3 °C), with no significant intersexual differences in both species. N. pasai (LLT₅₀±SE=−16.96±2.30 °C; LLT₁₀₀=−25.41 °C) tolerated short exposures to subzero temperatures better than N. mancinii (LLT₅₀±SE=−4.89±1.08 °C; LLT₁₀₀=−11.72 °C). According to the mortality and cumulative proportion of individual freezing curves (CPIF), SCPs and LLT₁₀₀, N. pasai may be defined as “strongly freeze tolerant”, N. mancinii as “moderately freezing tolerant”. Overall, these results may justify the different in-cave habitat selection showed by the two species (N. pasai was abundant close to the entrance where the temperature is variable whereas N. mancinii was confined to the internal part of the cave where the temperature is constant throughout the year), and suggest hypotheses on the effects of such habitat selection on freeze tolerance strategy adopted. Finally, they give new insights into possible responses to climate changes in cave dwelling species.     

New cave-dwelling armored spiders (Araneae,Tetrablemmidae) from Southwest China

A new genus and five new species belonging to the family Tetrablemmidae are described from caves in Southwest China, i.e., Sinammaoxycera gen. & sp. n., Singaporemma banxiaoensis sp. n., Singaporemma wulongensis sp. n., Tetrablemma ziyaoensis sp. n. andTetrablemma menglaensis sp. n. The following new combination is proposed: Sinamma sanya (Lin & Li, 2010), comb. n. ex. Shearella Lehtinen, 1981. The relationships of the Sinamma gen. n. with other genera are discussed. Diagnoses and illustrations for all new taxa are given.     

Shifting the phase of the circadian rhythm in bioluminescence in gonyaulax with vanillic Acid

Exposure for 4 hours to vanillic acid (4-hydroxy 3-methoxy benzoic acid) caused large delay phase shifts (5 to 6 hours) in the circadian rhythm of bioluminescence in Gonyaulax polyedra, when assayed at either 10 to 14 circadian time or 22 to 02 circadian time in constant light and temperature, provided that the pH of the medium was 7.1 or lower. Corresponding changes in the pH with acetic acid did not shift phase. Vanillic acid caused detectable depolarization of the membranes of Gonyaulax, as demonstrated with the cyanine dye fluorescence technique.     

RAP, an integrated program for monitoring bioluminescence and analyzing circadian rhythms in real time

To process the large number of circadian rhythmic bioluminescence data that we generated with specially developed, high-throughput monitoring apparatuses, we developed an integrated rhythm-analyzing program (RAP) with a user-friendly graphical interface. RAP does all of the following in real time: (i) displays the bioluminescence time course, (ii) records time-series data, (iii) analyzes the data, (iv) displays the analyzed results, (v) statistically evaluates the analyzed results, and (vi) provides printouts. Because RAP can import files, it can analyze not only bioluminescence data but also other data such as those obtained by DNA array experiments and by Northern and Western blot analyses. The program is a powerful tool for large-scale investigations of biological rhythmic phenomena in general.     

Propagation of bioluminescence in Euphysa japonica hydromedusae, (Tubulariidae)

The jellyfish Euphysa emits light when touched, stimulated electrically or shaken in the water. Light is emitted over the whole subumbrellar surface. The source of light is the subumbrellar endodermal epithelium. The response accompanies muscular involution (crumpling) and is spread by all-or-none action potentials propagated in the epithelium itself. Light emission is calcium dependent, but propagation of the triggering events is not. Octanol blocks propagation but light is still emitted in the immediate vicinity of the stimulating electrode. Light emission consists of discrete flashes which sum and facilitate with repeated stimulation. Although the triggering impulses propagate over the entire epithelium, the threshold for light emission varies regionally. The response more closely resembles that described for the siphonophore Hippopodius than those of leptomedusae such as Aequorea. Light emission is best viewed as a startle response.     

Synchronized maturation and breeding in natural populations ofXiphophorus variatus (Poeciliidae)

Synopsis Reproductive events tend to be synchronized in natural populations ofXiphophorus variatus. Males physically resembling one another tend to mature at the same rate and time. This coordinate development has also been seen in the laboratory. We hypothesize that juveniles resembling each other experience fewer aggressive responses from males than do juveniles without look-alikes, because of habituation of aggression. This would decrease social inhibition and permit their synchronous development.Females have an ovarian cycle. In the laboratory, crowding stress causes a suspension of breeding while a return to uncrowded conditions permits its resumption. Females released simultaneously from crowding tend to be synchronized in subsequent broods, although the synchrony deteriorates with time. Synchronizing events are shown to occur in natural populations.The consequences of reproductive synchrony in males and females are briefly discussed.     

N-terminal acetyltransferase 3 gene is essential for robust circadian rhythm of bioluminescence reporter in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model species of algae for studies on the circadian clock. Previously, we isolated a series of mutants showing defects in the circadian rhythm of a luciferase reporter introduced into the chloroplast genome, and identified the genes responsible for the defective circadian rhythm. However, we were unable to identify the gene responsible for the defective circadian rhythm of the rhythm of chloroplast 97 (roc97) mutant because of a large genomic deletion. Here, we identified the gene responsible for the roc97 mutation through a genetic complementation study. This gene encodes a protein that is homologous to the subunit of N-terminal acetyltransferase (NAT) which catalyzes N-terminal acetylation of proteins. Our results provide the first example of involvement of the protein N-terminal acetyltransferase in the circadian rhythm.     

Energy, water balance and the roost microenvironment in three Australian cave-dwelling bats (Microchiroptera)

The ghost bat, Macroderma gigas, and the orange leaf-nosed bat, Rhinonycteris aurantius, occupy similar ranges across northern Australia and are often found in the same roost caves. Both species are considered rare and vulnerable to further population decline. A third small species, the large bent-wing bat, Miniopterus schreibersii, has a similar body mass to R. aurantius, but has one of the largest ranges of any Australian mammal. In the present study we examine the effect and sensitivity of the animals' roosting microclimates on their energy and water balance. M. schreibersii exhibits a basal metabolic rate about 40% greater than other bats of similar body mass, whereas the other two species are close to predicted levels. R. aurantius shows a decrease in body temperatures below thermoneutrality. R. aurantius has levels of pulmocutaneous water loss among the highest seen for a mammal, and calculations based on nasal tip temperatures suggest that most of this loss is across the skin. Calculated ambient temperatures at which metabolic water production is equal to pulmocutaneous water loss in dry air are −14.7 °C for R. aurantius, 9.8 °C for M. schreibersii and −0.3 °C for M. gigas. Exposing the animals to relative humidities of between 80% and 90% shifted these calculated temperatures to 5.6 °C, 25.2 °C, and 2.9 °C, respectively. For each species the ratio of metabolic water production to evaporative water loss has been treated as a joint function of humidity and ambient temperature. The resulting surface plot shows that under known roosting conditions in caves R. aurantius and M. schreibersii remain in positive water balance, whereas M. gigas does not. Accepted: 20 May 2000     

The methamphetamine-sensitive circadian oscillator (MASCO) in mice

The suprachiasmatic nucleus (SCN) orchestrates synchrony among many peripheral oscillators and is required for circadian rhythms of locomotor activity and many physiological processes. However, the unique effects of methamphetamine (MAP) on circadian behavior suggest the presence of an SCN-independent, methamphetamine-sensitive circadian oscillator (MASCO). Substantial data collected using rat models show that chronic methamphetamine dramatically lengthens circadian period of locomotor activity rhythms and induces rhythms in animals lacking an SCN. However, the anatomical substrate and the molecular components of the MASCO are unknown. The response to MAP is less well studied in mice, a model that would provide the genetic tools to probe the molecular components of this extra-SCN oscillator. The authors tested the effects of chronic MAP on 2 strains of intact and SCN-lesioned mice in constant dark and constant light. Furthermore, they applied various MAP availability schedules to SCN-lesioned mice to confirm the circadian nature of the underlying oscillator. The results indicate that this oscillator has circadian properties. In intact mice, the MASCO interacts with the SCN in a manner that is strain, sex, and dose dependent. In SCN-lesioned mice, it induces robust free-running locomotor rhythmicity, which persists for up to 14 cycles after methamphetamine is withdrawn. In the future, localization of the MASCO and characterization of its underlying molecular mechanism, as well as its interactions with other oscillators in the body, will be essential to a complete understanding of the organization of the mammalian circadian system.     

Evolution and speciation of cave-dwelling Fulgoroidea in the Canary Islands (Homoptera: Gixiidae and Meenoplidae)

Ten new cavernicolous Fulgoroidea species of the families Cixiidae and Meenoplidae are described from the Canary Islands: Cixius palmeros, C. pinarcoladus, C. ratonicus, C. tacandus, Memoplus claustrophilus from La Palma, C. ariadne, C. nycticolus, M. charon from El Hierro, and Tachycixius crypticus, T. retrusus from Tenerife. Notes on their ecology and distribution are given, and where possible, phylogenetic affinities to existing epigean species of the corresponding families are discussed. Despite the present-day relict status of the majority of cavernicolous species, it cannot be concluded with certainty whether their evolution has followed allopatric or parapatric modes of speciation.     

A new (and different) circadian pacemaker

Comment on: Mohawk JA, Baer ML, Menaker M. The methamphetamine-sensitive circadian oscillator does not employ canonical clock genes. Proc Natl Acad Sci U S A 2009; 106:3519-24.     

Persistent cell-autonomous circadian oscillations in fibroblasts revealed by six-week single-cell imaging of PER2::LUC bioluminescence

Biological oscillators naturally exhibit stochastic fluctuations in period and amplitude due to the random nature of molecular reactions. Accurately measuring the precision of noisy oscillators and the heterogeneity in period and strength of rhythmicity across a population of cells requires single-cell recordings of sufficient length to fully represent the variability of oscillations. We found persistent, independent circadian oscillations of clock gene expression in 6-week-long bioluminescence recordings of 80 primary fibroblast cells dissociated from PER2::LUC mice and kept in vitro for 6 months. Due to the stochastic nature of rhythmicity, the proportion of cells appearing rhythmic increases with the length of interval examined, with 100% of cells found to be rhythmic when using 3-week windows. Mean period and amplitude are remarkably stable throughout the 6-week recordings, with precision improving over time. For individual cells, precision of period and amplitude are correlated with cell size and rhythm amplitude, but not with period, and period exhibits much less cycle-to-cycle variability (CV 7.3%) than does amplitude (CV 37%). The time series are long enough to distinguish stochastic fluctuations within each cell from differences among cells, and we conclude that the cells do exhibit significant heterogeneity in period and strength of rhythmicity, which we measure using a novel statistical metric. Furthermore, stochastic modeling suggests that these single-cell clocks operate near a Hopf bifurcation, such that intrinsic noise enhances the oscillations by minimizing period variability and sustaining amplitude.     

A new genus and species for the first recorded cave-dwelling Cavernicola (Platyhelminthes) from South America

Species diversity of Brazilian cave fauna has been seriously underestimated. A karst area located in Felipe Guerra, northeastern Brazil, which is a hotspot of subterranean diversity in Brazil, has revealed more than 20 troglobitic species, most of them still undescribed. Based on recent samplings in this karst area, we document the occurrence of the suborder Cavernicola (Platyhelminthes) in South American hypogean environments for the first time and describe a new genus and species for this suborder. Hausera Leal-Zanchet & Souza, gen. n. has features concordant with those defined for the family Dimarcusidae. The new genus is characterized by two unique features, viz. an intestine extending dorsally to the brain and ovovitelline ducts located dorsally to the nerve cords, which is complemented by a combination of other characters. The type-specimens of Hauserahauseri Leal-Zanchet & Souza, sp. n. are typical stygobionts, unpigmented and eyeless, and they may constitute an oceanic relict as is the case of other stygobiotic invertebrates found in this karst area in northeastern Brazil.     

Reassessing the role of a 3'-UTR-binding translational inhibitor in regulation of circadian bioluminescence rhythm in the dinoflagellate Gonyaulax

The nightly bioluminescence of the dinoflagellate Gonyaulax is a circadian rhythm caused by the presence in cells of specialized bioluminescent organelles, termed scintillons, containing the reaction catalyst luciferase, the substrate luciferin and a luciferin-binding protein (LBP). LBP levels increase at the start of the night phase because of increased protein synthesis rates in vivo, and this regulation has been ascribed to circadian binding of an inhibitory protein factor binding to the 3' untranslated region (UTR) of lbp mRNA at times when LBP is not normally synthesized. To purify and characterize the binding factor, the electrophoretic mobility shift assays and UV crosslinking experiments used to first characterize the factor were repeated. However, neither these protocols nor binding to biotinylated RNA probes confirmed the presence of a specific circadian RNA-binding protein. Furthermore, neither RNA probe screening of a cDNA library expressed in bacteria nor three-hybrid assays in yeast were successful in isolating a cDNA encoding a protein able to bind specifically to the lbp 3'UTR. Taken together, these results suggest that alternative mechanisms for regulating lbp translation should now be examined.