Photoperiodic Time Research Articles

Distinct Physiological Mechanisms Induce Latitudinal and Sexual Differences in the Photoperiodic Induction of Diapause in a Fly

Many temperate insects enter diapause (dormancy) for overwintering in response to short days (long nights). A latitudinal cline in the critical day lengths for the photoperiodic induction of diapause has been reported in various insect species. However, the physiological mechanisms underlying this cline have remained elusive. We approached this issue in the flesh fly Sarcophaga similis, in which the photoperiodic time measurement system meets the "external coincidence model." In this model, measuring day lengths depends on whether the photoinducible phase (φi), determined by a circadian clock, is exposed to light or not. First, we detected a clear latitudinal cline in the critical day lengths of flies collected from 4 localities at different latitudes. The phase positions of the φi, which can be verified by night interruption photoperiods, also showed a clear latitudinal cline. This result supports the hypothesis that the latitudinal cline in the critical day length is produced by the difference in the phase positions of the φi among different strains. A sexual difference in the critical day length for photoperiodic induction has also been detected in various species. In this study, a sexual difference in the critical day length was observed in the southern strains but there was no sexual difference in the phase positions of the φi. This result indicates that both sexes measure photoperiods in the same manner. Males are less sensitive than females to the light pulse given at the φi, suggesting a quantitative difference in the photoperiodic time measurement and counter systems. This study clearly reveals that distinct mechanisms induce latitudinal and sexual differences in the critical day length for the photoperiodic induction of diapause in a fly.

Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.

Seasonality of reproduction in Asian elephantsElephas maximusand African elephantsLoxodonta africana: underlying photoperiodic cueing?

AbstractAnimals in seasonal environments often rely on photoperiodicity to time their reproduction. Elephants have a gestation length of approximately two years and a historical geographic distribution including higher latitudes than at present, so the evolution of a seasonal breeding pattern cued by photoperiodicity and timed to the long‐day period is a theoretical option in both species.We reviewed literature on reproductive patterns in free‐ranging, semi‐captive and captive Asian and African elephants, photoperiodic cueing, seasonal variation in body condition, and other factors influencing their reproduction, as well as data from zoological collections on the timing of births.Most of the free‐ranging and all the semi‐captive and captive elephant populations showed a moderate yet distinct seasonal breeding pattern.Peak breeding activity of free‐ranging Asian elephants took place in either the dry or the wet season, with no preference for short‐day or long‐day breeding at low latitudes (close to the equator) but a preference for long‐day breeding at higher latitudes. Semi‐captive Asian elephants mainly bred in the dry season when body condition was lowest and day‐lengths were increasing. Peak conception often occurred in the wet season in free‐ranging African elephants when body condition was highest, with no evident preference for short‐day or long‐day breeding at low latitudes but preference for long‐day breeding at higher latitudes.Asian and African elephants in zoos at latitudes from 43 to 53°N tended to conceive more often during spring and summer, i.e. when day‐lengths were increasing. Body condition was not reported to vary significantly throughout the year and was rather high compared to in the wild.We hypothesise that elephants are ‘long‐day breeders’ in which the photoperiodic timing of conception can be influenced by many additional factors. Strategies to encourage natural conception in captive populations should include measures aimed at increasing breeding incentives in the northern hemisphere spring.

Molecular characterization of a CONSTANS gene from Sapium sebiferum (L.) Rxob

Sapium sebiferum (L.) Roxb [S. sebiferum L.] is not only one of the most important economic woody oil trees, but is also a significant traditional herbal medicine in China. The CONSTANS (CO) gene is a key regulator of the long day-dependent flowering pathway in Arabidopsis and other plants. To gain insight into the role of CO in woody oil trees, SsCO from S. sebiferum L. was isolated and characterized in this study. The corresponding SsCO protein, with 340 amino acid residues, included two putative zinc finger motifs B-Box1 and B-Box2 in the N-terminal region and a conserved CCT domain in the C-terminal region. SsCO expression was high in flowers and exhibited distinct circadian regulation. In addition, SsCO had a transcriptional activation effect in yeast strains. Moreover, heterologous expression of SsCO complemented the late-flowering phenotype of the Arabidopsis CO mutant co-1. These results indicate that SsCO is a transcription factor and may regulate the photoperiodic flowering time and SsCO is regulated by circadian rhythms in Sapium sebiferum L.

Effects of environmental variables on the calling behaviour of Northern Leopard Frogs (Lithobatespipiens) in Alberta, Canada

Breeding behaviour of Temperate Zone amphibians is influenced by environmental variables, but the initiation of calling (phenology) and influences on calling activity may vary with species and region. We investigated the influence of the timing of ice melt, water temperature, and photoperiod on the breeding phenology of the Northern Leopard Frog (Lithobates pipiens (Schreber, 1782)) in southern Alberta, Canada, using automated recording units. We also examined the influence of wind speed, relative humidity, water temperature, and time of day on calling activity. The initiation of calling varied by 13 days at our three sites, suggesting that calling was influenced more by water temperature and timing of ice melt than photoperiod. Calling was first observed 8–11 days after ice melt at water temperatures of 7.5–8 °C at our sites. No calling was detected at water temperature <5 °C. We recorded nocturnal and diurnal calling at all sites; >50% of calling was diurnal, even on days with warm overnight temperatures. Calling activity was influenced by time of day, water temperature, wind, and relative humidity. Our results suggest that date of initiation of calling varies considerably among breeding ponds and that the time of day of peak calling varies with both site and water temperature.

Keeping time without a spine: what can the insect clock teach us about seasonal adaptation?

Seasonal change in daylength (photoperiod) is widely used by insects to regulate temporal patterns of development and behaviour, including the timing of diapause (dormancy) and migration. Flexibility of the photoperiodic response is critical for rapid shifts to new hosts, survival in the face of global climate change and to reproductive isolation. At the same time, the daily circadian clock is also essential for development, diapause and multiple behaviours, including correct flight orientation during long-distance migration. Although studied for decades, how these two critical biological timing mechanisms are integrated is poorly understood, in part because the core circadian clock genes are all transcription factors or regulators that are able to exert multiple effects throughout the genome. In this chapter, we discuss clocks in the wild from the perspective of diverse insect groups across eco-geographic contexts from the Antarctic to the tropical regions of Earth. Application of the expanding tool box of molecular techniques will lead us to distinguish universal from unique mechanisms underlying the evolution of circadian and photoperiodic timing, and their interaction across taxonomic and ecological contexts represented by insects.This article is part of the themed issue 'Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals'.

Photoperiodic time measurement and photoentrainment of a circadian locomotor activity rhythm in subtropical tree sparrows.

Experiments were performed on the subtropical tree sparrow (Passer montanus) to investigate the involvement of an endogenous circadian rhythm in photoperiodic time measurement during the initiation of gonadal growth and functions and also to study the photic entrainment of the circadian activity rhythm. Groups of photosensitive birds of both sexes were exposed to different "night-interruption" light-dark regimes of 24 h duration viz., G1 - 6L/5D/1L/12D, G2 - 6L/6D/1L/11D, G3 - 6L/7D/1L/10D, G4 - 6L/8D/1L/9D, G5 - 6L/10D/1L/7D, G6 - 6L/12D/1L/5D, G7 - 6L/14D/1L/3D and G8 - 6L/16D/1L/1D for 30 days in which 17 h long nights were interrupted by 1 h light pulse falling at different ZTs (i.e., 11, 12, 13, 14, 16, 18, 20 and 22). A control group, for each sex, was maintained under 7L/17D. The birds of all groups behaved as though they had been exposed to a stimulatory photoperiod except those of G1, G8 and the control. Furthermore, the serum levels of testosterone in males and estradiol-17β in females ran almost parallel to changes in the gonadal size. Birds, when exposed to 12L/12D, showed entrainment of their locomotor activity rhythm with the activity confined mainly during the light phase. However, various characteristics of the activity rhythm differed significantly upon transfer of birds to various night interruption regimes. These results suggest the involvement of the endogenous circadian rhythm in photoperiodic time measurement during the induction of gonadal growth and functions in the tree sparrow. Also, there is photic entrainment of the locomotor activity rhythm that shows variations in various circadian characteristics under different night interruption cycles suggesting that the photoinducible phase is differentially sensitive to light.

Neural control of daily and seasonal timing of songbird migration

Bird migration is one of most salient annual events in nature. It involves predictable seasonal movements between breeding and non-breeding habitats. Both circadian and circannual clocks are entrained by photoperiodic cues and time daily and seasonal changes in migratory physiology and behavior. This mini-review provides an update on daily and seasonal rhythms of migratory behavior, and examines the neuroendocrine and molecular pathways involved in the timing of migration in songbirds. Recent findings have identified key neural substrates, and suggest the involvement of multiple neuroendocrine regulatory systems in controlling seasonal states in migrants. We propose that four distinct neural substrates are involved in the timing of migration and include (1) pineal gland and suprachiasmatic nucleus (mSCN); (2) a cluster of hypothalamic nuclei, the mediobasal hypothalamus (MBH); (3) dorsomedial hypothalamic nucleus (DMH); and (4) tanycytes along ependymal layer of the 3rd ventricle (3V). Cluster N, a nucleus in the telencephalon involved in the integration of geomagnetic cues, likely maintains functional connectivity with brain regions involved in timing songbird migration. These nuclei form an interconnected network that coordinates daily timing (pineal gland/mSCN), annual photoperiodic response (MBH, 3V), energetic state (MBH, DMH, 3V), and magnetic compass information (i.e., cluster N) for migration in songbirds.

MicroRNA319-regulated TCPs interact with FBHs and PFT1 to activate CO transcription and control flowering time in Arabidopsis.

The transcription factor CONSTANS (CO) is a central component that promotes Arabidopsis flowering under long-day conditions (LDs). Here, we show that the microRNA319-regulated TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors promote photoperiodic flowering through binding to the CO promoter and activating its transcription. Meanwhile, these TCPs directly interact with the flowering activators FLOWERING BHLH (FBHs), but not the flowering repressors CYCLING DOF FACTORs (CDFs), to additively activate CO expression. Furthermore, both the TCPs and FBHs physically interact with the flowering time regulator PHYTOCHROME AND FLOWERING TIME 1 (PFT1) to facilitate CO transcription. Our findings provide evidence that a set of transcriptional activators act directly and additively at the CO promoter to promote CO transcription, and establish a molecular mechanism underlying the regulation of photoperiodic flowering time in Arabidopsis.

Role of Pineal and Melatonin in the Avian Circadian and Photoperiodic Systems

The pineal gland is an important component of the multioscillatory avian circadian timekeeping system. The other principal clock components reside in the retinae of the eyes and the hypothalamus. The best known output signal from the pineal gland is melatonin, which is a lipophilic molecule. The presence of melatonin is however not limited to the organisms having a pineal gland. Melatonin is present from plants to protozoa to humans. Melatonin seems to have been evolutionarily conserved as an adaptive molecule of darkness of the daily day-night environment. In birds, the major physiological roles of pineal melatonin are in its involvement in the daily and seasonal timekeeping as well as photoperiodic time measurement. Birds use daily rhythm in melatonin secretion to decode the time-of-day as well as the time-of-year information. Besides, melatonin performs other physiological roles, namely in the immune function, free radical scavenging, etc. Avian pineal (melatonin) directly regulates several circadian behaviors, but intriguingly not the circadian rhythm-mediated photoperiodic induction of gonadal development. Melatonin, however, may act as an endocrine modulator of seasonal reproduction. In this article, we describe briefly the avian timekeeping system and then discuss the potential roles of pineal gland and melatonin in daily and seasonal timing of physiology in birds, particularly in songbirds.

The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

AbstractMany insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth‐instar nymphal stage, which is the most photoperiod‐sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod‐dependent. The RNA interference‐mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.

Distribution of PERIOD-immunoreactive neurons and temporal change of the immunoreactivity under long-day and short-day conditions in the larval brain of the flesh fly Sarcophaga similis

ABSTRACTThe flesh fly Sarcophaga similis show a clear photoperiodic response; they develop into adults under long days, whereas they arrest their development at the pupal stage under short days. Although the involvement of a circadian clock in photoperiodic time measurement is suggested in this species, the anatomical location of the clock neurons responsible for the time measurement has been unknown. We detected two PERIOD-immunoreactive cell clusters in the larval brain; one cluster was located at the dorsoanterior region and the other at the medial region. We further investigated their temporal changes in PERIOD-immunoreactivity and compared their patterns under different photoperiods.

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length

Seasonal reproduction in many organisms requires detection of day length. This is achieved by integrating information on the light environment with an internal photoperiodic time‐keeping mechanism. Arabidopsis thaliana promotes flowering in response to long days (LDs), and CONSTANS (CO) transcription factor represents a photoperiodic timer whose stability is higher when plants are exposed to light under LDs. Here, we show that PSEUDO RESPONSE REGULATOR (PRR) proteins directly mediate this stabilization. PRRs interact with and stabilize CO at specific times during the day, thereby mediating its accumulation under LDs. PRR‐mediated stabilization increases binding of CO to the promoter of FLOWERING LOCUS T (FT), leading to enhanced FT transcription and early flowering under these conditions. PRRs were previously reported to contribute to timekeeping by regulating CO transcription through their roles in the circadian clock. We propose an additional role for PRRs in which they act upon CO protein to promote flowering, directly coupling information on light exposure to the timekeeper and allowing recognition of LDs.

Clock gene polymorphism, migratory behaviour and geographic distribution: a comparative study of trans-Saharan migratory birds.

Migratory behaviour is controlled by endogenous circannual rhythms that are synchronized by external cues, such as photoperiod. Investigations on the genetic basis of circannual rhythmicity in vertebrates have highlighted that variation at candidate 'circadian clock' genes may play a major role in regulating photoperiodic responses and timing of life cycle events, such as reproduction and migration. In this comparative study of 23 trans-Saharan migratory bird species, we investigated the relationships between species-level genetic variation at two candidate genes, Clock and Adcyap1, and species' traits related to migration and geographic distribution, including timing of spring migration across the Mediterranean Sea, migration distance and breeding latitude. Consistently with previous evidence showing latitudinal clines in 'circadian clock' genotype frequencies, Clock allele size increased with breeding latitude across species. However, early- and late-migrating species had similar Clock allele size. Species migrating over longer distances, showing delayed spring migration and smaller phenotypic variance in spring migration timing, had significantly reduced Clock (but not Adcyap1) gene diversity. Phylogenetic confirmatory path analysis suggested that migration date and distance were the most important variables directly affecting Clock gene diversity. Hence, our study supports the hypothesis that Clock allele size increases poleward as a consequence of adaptation to the photoperiodic regime of the breeding areas. Moreover, we show that long-distance migration is associated with lower Clock diversity, coherently with strong stabilizing selection acting on timing of life cycle events in long-distance migratory species, likely resulting from the time constraints imposed by late spring migration.

Rainfall, leafing phenology and sunrise time as potential Zeitgeber for the bimodal, dry season laying pattern of an African rain forest tit (Parus fasciiventer)

Recent studies have documented a mismatch between the phenology of leaf production, prey availability and the nestling food requirements of north temperate songbirds, attributed to climate change effects. Although tropical forest species have often been regarded as relatively aseasonal breeders, similar disruptive effects can be expected at equatorial latitudes, where comparatively little is known of the links between weather, leafing phenology, food availability and bird breeding activity, particularly in complex rain forest habitats. During a 19-year study at 1°S in Bwindi Impenetrable Forest, Uganda, Stripe-breasted Tits Parus fasciiventer showed a strongly bimodal laying pattern, breeding mainly in the two dry seasons, with 50 % of breeding activity occurring in January–February and 19 % in June–July. Individual females bred in both dry seasons, laying their first and last clutches up to 28 weeks apart. Breeding activity was linked to leaf production, which peaked mainly in November–December, following the September–November wet season. Increased leaf production is likely to have stimulated a rise in caterpillar numbers during December–February, coinciding with peak food demands by nestling tits. Laying was thus positively correlated with increased leaf production in the preceding calendar month, but was also linked to day length and a change in sunset time. To investigate possible links between egg laying and photic cues I compared the median date of first clutches laid by marked females in each half of the breeding year (October–March and April–September), with annual changes in photoperiod (varying by 7 min p.a.) and sunrise time (varying bimodally, by 31 min p.a.). The two median laying dates fell 138–139 days after the last date on which sunrise had occurred at 07:05 in August and January, suggesting the potential for sunrise time to act as a cue, or Zeitgeber, for breeding in tropical birds. Further work is required to establish whether the relationship is causative or coincidental.

Drosophila ezoana uses an hour-glass or highly damped circadian clock for measuring night length and inducing diapause.

Insects inhabiting the temperate zones measure seasonal changes in day or night length to enter the overwintering diapause. Diapause induction occurs after the duration of the night exceeds a critical night length (CNL). Our understanding of the time measurement mechanisms is continuously evolving subsequent to Bünning's proposal that circadian systems play the clock role in photoperiodic time measurement (Bünning, 1936). Initially, the photoperiodic clocks were considered to be either based on circadian oscillators or on simple hour‐glasses, depending on ‘positive’ or ‘negative’ responses in Nanda–Hamner and Bünsow experiments (Nanda & Hammer, 1958; Bünsow, 1960). However, there are also species whose responses can be regarded as neither ‘positive’, nor as ‘negative’, such as the Northern Drosophila species Drosophila ezoana, which is investigated in the present study. In addition, modelling efforts show that the ‘positive’ and ‘negative’ Nanda–Hamner responses can also be provoked by circadian oscillators that are damped to different degrees: animals with highly sustained circadian clocks will respond ‘positive’ and those with heavily damped circadian clocks will respond ‘negative’. In the present study, an experimental assay is proposed that characterizes the photoperiodic oscillators by determining the effects of non‐24‐h light/dark cycles (T‐cycles) on critical night length. It is predicted that there is (i) a change in the critical night length as a function of T‐cycle period in sustained‐oscillator‐based clocks and (ii) a fixed night‐length measurement (i.e. no change in critical night length) in damped‐oscillator‐based clocks. Drosophila ezoana flies show a critical night length of approximately 7 h irrespective of T‐cycle period, suggesting a damped‐oscillator‐based photoperiodic clock. The conclusion is strengthened by activity recordings revealing that the activity rhythm of D. ezoana flies also dampens in constant darkness.

Physiological and molecular mechanisms underlying photoperiodism in the spider mite: comparisons with insects.

Photoperiodism is an adaptive, seasonal timing system that enables organisms to coordinate their development and physiology to annual changes in the environment using day length (photoperiod) as a cue. This review summarizes our knowledge of the physiological mechanisms underlying photoperiodism in spider mites. In particular, the two-spotted spider mite Tetranychus urticae is focussed, which has long been used as a model species for studying photoperiodism. Photoperiodism is established by several physiological modules, such as the photoreceptor, photoperiodic time measurement system, counter system, and endocrine effector. It is now clear that retinal photoreception through the ocelli is indispensable for the function of photoperiodism, at least in T. urticae. Visual pigment, which comprised opsin protein and a vitamin A-based pigment, is involved in photoreception. The physiological basis of the photoperiodic time measurement system is still under debate, and we have controversial evidence for the hourglass-based time measurement and the oscillator-based time measurement. Less attention has been centred on the counter system in insects and mites. Mite reproduction is possibly regulated by the ecdysteroid, ponasterone A. Prior physiological knowledge has laid the foundation for the next steps essential for the elucidation of the molecular mechanisms driving photoperiodism.

Enhanced GABAergic inhibition in the premammillary nucleus of photorefractory turkey hens via GABAA receptor upregulation

The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABAA receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABAA receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABAA receptor α1-3, β2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABAA receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABAA receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABAA receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of photorefractoriness and the termination of breeding activities in the turkey, a temperate zone bird.

The temporal ‘structure’ and function of the insect photoperiodic clock: a tribute to Colin S. Pittendrigh

AbstractIn 1936, Erwin Bünning suggested that photoperiodic time measurement was a function of the circadian system. Colin Pittendrigh became an ardent supporter of Bünning's hypothesis, drawing parallels between photoperiodism and his own group's investigations of adult eclosion rhythmicity in the fruit fly Drosophila pseudoobscura. They developed several more modern versions of Bünning's general hypothesis based on the entrainment of circadian oscillations to the light cycle, including ‘external coincidence’, which is a derivation of Bünning's original model, and ‘internal coincidence’, which relied upon seasonal changes in the mutual phase relationship of oscillators within a multi‐oscillator circadian system. This review considers the experimental evidence for the central role of the circadian system in photoperiodic timing and, in some species, for both external and internal coincidence. Pittendrigh, however, pursued the idea of internal coincidence further with his analysis of the pacemaker–slave organization of eclosion rhythmicity in D. pseudoobscura and proposed a similar theoretical model for photoperiodism comprising a group of slave oscillators driven by a light‐sensitive pacemaker. In this model, the phase relationships of the slaves to the pacemaker were affected by (i) the relative periods of the pacemaker and slave(s); (ii) the strength(s) of the coupling between the two; and (iii) the dampening coefficients of the various slaves. Manipulation of these variables showed that the slaves adopted different internal phase relationships (both to each other and to the pacemaker) under the influence of changes in daily photophase, the period of the Zeitgeber and phase shifts of the entraining light cycle.

Bioprocess Development for Chlorella vulgaris Cultivation and Biosynthesis of Anti-phytopathogens Silver Nanoparticles

Bioprocess Development for Chlorella vulgaris Cultivation and Biosynthesis of Anti-phytopathogens Silver Nanoparticles In this study, growth media formulations and environmental conditions were varied to optimize the growth media composition and conditions for maximized algal biomass production. The production both of Chlorella vulgaris in different photobioreactor configurations (250 ml flask, 5L flask and 7.5L stirred tank reactor) was investigated, using different organic and inorganic media formulations. The effect of initial cell concentrations, agitation speed, airflow rate and photoperiodic time were studied using inexpensive poultry manure medium, the results indicate that 0.5 × 107 (n/ml), 200 rpm, 1 bar and 16:8hr respectively which better for biomass productivity at growth periods. The highest overall specific growth rate (μ) and doubling time (td) were found when growing C. vulgaris in batch operation in a 7.5 L stirred tank bioreactor, μ=0.08 d-1 and 3.5 days respectively which also reached the maximum biomass concentration of 0.59 g/L. Biosynthesized silver nanoparticles (AgNPs) are being widely using in the field of agriculture. Biosynthesis of silver nanoparticles was carried out by using the algal cells for the reduction of aqueous silver ions in short period. A Plackett-Burman experimental design was applied to screen seven parameters for biosynthesis of AgNPs, the most important parameters were pH, temperature and container head space; the results indicate that pH 6, 40ºC and 75% container head space which better for biosynthesis of AgNPs which also reached the maximum final product concentration of 5.59 mg/L. The AgNP formation was confirmed with the help of SEM, XRD and UV-Vis spectroscopy. These AgNPs were tested for antibacterial and antifungal activities. The tested strains are; Erwinia carotovora and Alternaria alternata were used. The results indicate that the AgNPs may have an important advantage over conventional antibiotics.