Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

Field measurements of the CO2 evolution rate under different crops during an irrigation cycle in a mountain oasis of Oman

For millennia oasis agriculture has been the backbone of rural livelihood in the desertic Sultanate of Oman. However, little is known about the functioning of these oasis systems, in particular with respect to the C turnover. The objective was to determine the effects of crop, i.e. alfalfa, wheat and bare fallow on the CO2 evolution rate during an irrigation cycle in relation to changes in soil water content and soil temperature. The gravimetric soil water content decreased from initially 24% to approximately 16% within 7 days after irrigation. The mean CO2 evolution rates increased significantly in the order fallow (27.4 mg C m^−2 h^−1) < wheat (45.5 mg C m^−2 h^−1) < alfalfa (97.5 mg C m^−2 h^−1). It can be calculated from these data that the CO2 evolution rate of the alfalfa root system was nearly four times higher than the corresponding rate in the wheat root system. The decline in CO2 evolution rate, especially during the first 4 days after irrigation, was significantly related to the decline in the gravimetric water content, with r = 0.70. CO2 evolution rate and soil temperature at 5 cm depth were negatively correlated (r = -0.56,n = 261) due to increasing soil temperature with decreasing gravimetric water content.

Effects of fertilizer type and rate on partitioning of soil organic matter into pools of different stability

Type and rate of fertilizers influence the level of soil organic carbon (Corg) and total nitrogen (Nt) markedly, but the effect on C and N partitioning into different pools is open to question. The objectives of the present work were to: (i) quantify the impact of fertilizer type and rate on labile, intermediate and passive C and N pools by using a combination of biological, chemical and mathematical methods; (ii) explain previously reported differences in the soil organic matter (SOM) levels between soils receiving farmyard manure with or without biodynamic preparations by using Corg time series and information on SOM partitioning; and (iii) quantify the long-term and short-term dynamics of SOM in density fractions and microbial biomass as affected by fertilizer type and rate and determine the incorporation of crop residues into labile SOM fractions. Samples were taken from a sandy Cambisol from the long-term fertilization trial in Darmstadt, Germany, founded in 1980. The nine treatments (four field replicates) were: straw incorporation plus application of mineral fertilizer (MSI) and application of rotted farmyard manure with (DYN) or without (FYM) addition of biodynamic preparations, each at high (140 – 150 kg N ha-1 year-1; MSIH, DYNH, FYMH), medium (100 kg N ha-1 year-1; MSIM, DYNM, FYMM) and low (50 – 60 kg N ha-1 year-1; MSIL, DYNL, FYML) rates. The main findings were: (i) The stocks of Corg (t ha-1) were affected by fertilizer type and rate and increased in the order MSIL (23.6), MSIM (23.7), MSIH (24.2) < FYML (25.3) < FYMM (28.1), FYMH (28.1). Stocks of Nt were affected in the same way (C/N ratio: 11). Storage of C and N in the modelled labile pools (turnover times: 462 and 153 days for C and N, respectively) were not influenced by the type of fertilizer (FYM and MSI) but depended significantly (p ≤ 0.05) on the application rate and ranged from 1.8 to 3.2 t C ha 1 (7 – 13% of Corg) and from 90 to 140 kg N ha-1 (4-5% of Nt). In the calculated intermediate pool (C/N ratio 7), stocks of C were markedly higher in FYM treatments (15-18 t ha-1) compared to MSI treatments (12-14 t ha-1). This showed that differences in SOM stocks in the sandy Cambisol induced by fertilizer rate may be short-lived in case of changing management, but differences induced by fertilizer type may persist for decades. (ii) Crop yields, estimated C inputs (1.5 t ha-1 year-1) with crop residue, microbial bio¬mass C (Cmic, 118 – 150 mg kg-1), microbial biomass N (17 – 20 mg kg-1) and labile C and N pools did not differ significantly between FYM and DYN treatments. However, labile C increased linearly with application rate (R2 = 0.53) from 7 to 11% of Corg. This also applied for labile N (3.5 to 4.9% of Nt). The higher contents of Corg in DYN treatments existed since 1982, when the first sampling was conducted for all individual treatments. Contents of Corg between DYN and FYM treatments con-verged slightly since then. Furthermore, at least 30% of the difference in Corg was located in the passive pool where a treatment effect could be excluded. Therefore, the reported differences in Corg contents existed most likely since the beginning of the experiment and, as a single factor of biodynamic agriculture, application of bio-dynamic preparations had no effect on SOM stocks. (iii) Stocks of SOM, light fraction organic C (LFOC, ρ ≤ 2.0 g cm-3), light fraction organic N and Cmic decreased in the order FYMH > FYML > MSIH, MSIL for all sampling dates in 2008 (March, May, September, December). However, statistical significance of treatment effects differed between the dates, probably due to dif-ferences in the spatial variation throughout the year. The high proportion of LFOC on total Corg stocks (45 – 55%) highlighted the importance of selective preservation of OM as a stabilization mechanism in this sandy Cambisol. The apparent turnover time of LFOC was between 21 and 32 years, which agreed very well with studies with substantially longer vegetation change compared to our study. Overall, both approaches; (I) the combination of incubation, chemical fractionation and simple modelling and (II) the density fractionation; provided complementary information on the partitioning of SOM into pools of different stability. The density fractionation showed that differences in Corg stocks between FYM and MSI treatments were mainly located in the light fraction, i.e. induced by higher recalcitrance of the organic input in the FYM treatments. Moreover, the use of the combination of biological, chemical and mathematical methods indicated that effects of fertilizer rate on total Corg and Nt stocks may be short-lived, but that the effect of fertilizer type may persist for longer time spans in the sandy Cambisol.

Photoperiod-dependent plasticity of circadian pacemaker center in the brain of the Madeira cockroach Rhyparobia maderae

To various degrees, insects in nature adapt to and live with two fundamental environmental rhythms around them: (1) the daily rhythm of light and dark, and (2) the yearly seasonal rhythm of the changing photoperiod (length of light per day). It is hypothesized that two biological clocks evolved in organisms on earth which allow them to harmonize successfully with the two environmental rhythms: (1) the circadian clock, which orchestrates circadian rhythms in physiology and behavior, and (2) the photoperiodic clock, which allows for physiological adaptations to changes in photoperiod during the course of the year (insect photoperiodism). The circadian rhythm is endogenous and continues in constant conditions, while photoperiodism requires specific light inputs of a minimal duration. Output pathways from both clocks control neurosecretory cells which regulate growth and reproduction. This dissertation focuses on the question whether different photoperiods change the network and physiology of the circadian clock of an originally equatorial cockroach species. It is assumed that photoperiod-dependent plasticity of the cockroach circadian clock allows for adaptations in physiology and behavior without the need for a separate photoperiodic clock circuit. The Madeira cockroach Rhyparobia maderae is a well established circadian clock model system. Lesion and transplantation studies identified the accessory medulla (aMe), a small neuropil with about 250 neurons, as the cockroach circadian pacemaker. Among them, the pigment-dispersing factor immunoreactive (PDF-ir) neurons anterior to the aMe (aPDFMes) play a key role as inputs to and outputs of the circadian clock system. The aim of my doctoral thesis was to examine whether and how different photoperiods modify the circadian clock system. With immunocytochemical studies, three-dimensional (3D) reconstruction, standardization and Ca2+-imaging technique, my studies revealed that raising cockroaches in different photoperiods changed the neuronal network of the circadian clock (Wei and Stengl, 2011). In addition, different photoperiods affected the physiology of single, isolated circadian pacemaker neurons. This thesis provides new evidence for the involvement of the circadian clock in insect photoperiodism. The data suggest that the circadian pacemaker system of the Madeira cockroach has the plasticity and potential to allow for physiological adaptations to different photoperiods. Therefore, it may express also properties of a photoperiodic clock.