Ecophysiological studies on citrate synthase: (II) enzyme regulation of selected crustaceans with regard to life-style and the climatic zone

Citrate synthase is a regulatory enzyme of the energy metabolism pathway controlling the citric acid cycle. It was studied in order to determine modes of enzyme regulation with regard to the life-style of the investigated species. Citrate synthase from crustaceans with different life-styles were compared: the pelagic euphausiids Euphausia superba from the Antarctic and Meganyctiphanes norvegica from the Scandinavian Kattegat and the Mediterranean were compared to the benthic isopods Serolis polita from the Antarctic and Idotea baltica from the Baltic. Citrate synthase was partly purified chromatographically and the influence of adenosine 5′-triphosphate on enzyme activity was examined. Mechanisms of inhibition and inhibitor constants were determined. Two different mechanisms of enzyme regulation by ATP were found. Citrate synthase from isopods was only competitively inhibited, while citrate synthase from euphausiids showed not only competitive inhibition but also activation by low concentrations of ATP. This activation is equivalent to the reversed methanism of uncompetitive inhibition. The ecophysiological relevances of the coupling of these mechanisms are discussed. The degree of competitive inhibition was different in the two groups of investigated crustaceans. Inhibitor constants were similar within the euphausiids but not in isopods, which showed higher or lower inhibition depending on the climatic zone: the colder the ambient temperature the lower the ATP inhibition. A possible mechanism of temperature adaptation through effects of varying inhibition constants is concluded.     

Effect of modulation of enzyme inactivation on temperature optimization for reactor operation with chitin-immobilized lactase

Temperature is a critical variable to be optimized in any enzymatic process, producing opposite effects on enzyme activity and inactivation rate. Temperature functions for all kinetic and inactivation parameters were validated for chitin-immobilized yeast lactase (CIL). Enzyme inactivation was described by a two-stage series mechanism. The effect of galactose and lactose on inactivation was determined in terms of modulation factors that were positive for galactose and negative for lactose. Modulation factors were mild functions of temperature in the first stage and strong functions in the second stage of enzyme inactivation, where galactose positive modulation factors increase while lactose negative modulation factors decrease with temperature. Temperature-explicit functions for kinetic and inactivation parameters were incorporated into a scheme to optimize temperature in the simulation of a continuous packed-bed reactor operation with chitin-immobilized lactase, based on an annual cost objective function. Optimum temperature was 20°C at enzyme replacement of 25% residual activity, and increased only slightly at higher replacement frequencies. The effect of modulation factors on reactor design and temperature optimization is presented and discussed. Software for temperature optimization that allows the introduction of variations in all parameters and operational criteria to perform sensitivity analysis was developed.     

Correlation of seasonal acclimatization in metabolic enzyme activity with preferred body temperature in the Eastern red spotted newt (Notophthalmus viridescens viridescens)

Eastern red spotted newts, as aquatic adults, are active year round. They are small and easy to handle, and thus lent themselves to a laboratory study of seasonal changes in preferred body temperature and biochemical acclimatization. We collected newts in summer (n=20), late fall (n=10) and winter (n=5). Ten each of the summer and late fall newts were subjected to an aquatic thermal gradient. Summer newts maintained higher cloacal temperatures than late fall newts (26.8+/-0.5 degrees C and 17.2+/-0.4 degrees C, respectively). In addition, the activity of three muscle metabolic enzymes (cytochrome c oxidase (CCO), citrate synthase (CS) and lactate dehydrogenase (LDH)) was studied in all newts collected. Newts compensated for lower late fall and winter temperatures by increasing the activity of CCO during those seasons over that in summer newts at all assay temperatures (8, 16 and 26 degrees C). The activity of CS was greater in winter over summer newts at 8 and 16 degrees C. No seasonal differences in LDH activity were demonstrated. These data in newts indicate that this amphibian modifies some muscle metabolic enzymes in relation to seasonal changes and can modify its behavioral in a way that correlates with those biochemical changes.     

海南山蛭对土壤湿度和气温的适应

在室内以土壤湿度梯度作为海南山蛭Haemadipsa hainana的栖息试验表明,海南山蛭栖息于含水量的15.1％－16.77％水分的土壤表面;野外调查表明,海南岛橡胶林内海南山蛭栖息于16.54％－17.66％含水量的土壤表面;热带雨林者栖息于19.92%-25.73%含水量的土壤,室内实验表明,海南山蛭爬向30－35℃温度范围,占试验蛭数79.15％,在8－9℃不活动,10℃能山蛭活动,随温度升高活动蛭数增加,而15℃试验蛭答部活动,活动蛭数与温度关系为Y－18.6Z-172.3,R-0.976,P=0.01。在橡胶林内8－9℃山蛭不活动;10℃开始有山蛭活动,且随温度升高活动蛭数增加,当17℃时所有山蛭活动,活动蛭数与温度关系Y＝4.73X-3.97,R=0.93,P=0.1。首次使用土壤湿度梯度测定山蛭对土壤湿度的要求,这是一个新的有普遍意义的方法,并讨论了海南山蛭对土壤湿度气温的适应。     

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea,Decapoda, Penaeidae): effects of development and temperature

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 °C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII–XIV (PL XII–XIV). PK activity reached a pronounced peak in PL V–VI, followed by a further decrease in PL XII–XIV. Temperature reduction produced variation in oxygen consumption rates (QO₂), ammonia–N excretion and in enzyme activities. Ammonia–N excretion was higher at 20 °C in mysis III (M III), PL V–VI and PL XII–XIV, resulting in substantially lower O:N ratios in these stages. QO₂ was increased in protozoea II (PZ II) and mysis I (M I) at 26 °C, while no difference in QO₂ was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V–VI and PL XII–XIV at 20 °C compared with 26 °C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85–0.92 for CS and 1.1–1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 °C for both enzymes. Weight-specific CS activity was positively correlated with QO₂ at 20 and 26 °C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.     

Chitobiase of planktonic crustaceans from South Atlantic coast (Southern Brazil): Characterization and influence of abiotic parameters on enzyme activity

Chitobiase is one of the enzymes involved in chitin degradation in nature. It is produced and released by a variety of organisms from bacteria to fish. In crustaceans, it is associated with digestive function and acts on the epidermis during the molting process. In the present study, the influence of water pH, temperature and salinity on maximum chitobiase activity (MCA), as well as the enzyme affinity (Km) for a substrate, the methylumbelliferyl N-acetyl-ß-d-glucosaminide (MUFNAG) was evaluated in the copepod Acartia tonsa. Km values for chitobiases of other crustaceans from the Patos Lagoon estuary and Cassino Beach (Southern Brazil) were also determined. For A. tonsa, MCA was observed at pH 5-6 and 30-35 °C. The range of pH was quite similar to that reported for other aquatic organisms. However, the range of temperature was lower than that previously reported. For salinity, no previous studies have considered the influence of this parameter on MCA. For A. tonsa, MCA was observed in freshwater, showing a significant linear decrease with increasing salinity. Considering that maximum copepod survival and growth rates are observed between 15 and 25 ppt, these findings suggest that the observed enzyme activity in this range of salinity (68 to 47% of that measured in freshwater) is not a limiting factor for A. tonsa growth. However, the extremely decreased enzyme activity observed in salinity 30 ppt (33% of that measured in freshwater) suggests that chitobiase activity might be one of the limiting factor for copepod growth at 30 ppt salinity or higher. Km values (μM) determined for organisms evaluated in the present study (copepod A. tonsa = 20.77; mysid Metamysidopsis elongata atlantica = 14.67; nauplii barnacle Balanus improvisus = 18.19; decapod zoea = 14.30; decapod megalopa = 24.77) were lower than those reported for other crustaceans from Northern Hemisphere. Also, they were much lower than those of organisms from different taxonomic groups like bacteria and fungi, but much higher than in protozoans and dinoflagelates. These findings suggest that chitobiase might be differentially evolved in each specific group of organism, and even within different ontogenetic stages of the same species, for a better adaptation to cope with its respective environmental needs.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Histological studies on Halicryptus spinulosus (Priapulida) with regard to environmental hydrogen sulfide resistance

The priapulid Halicryptus spinulosus has an outstanding resistance to anoxia and hydrogen sulfide, which enables the animal to survive in deteriorating environments. Whole-body staining procedures, as well as light and scanning electron microscopy were used to study structures and mechanisms possibly involved in sulfide detoxification.The cuticle of the trunk is covered by a coat of mucus and bacteria. Within this coat considerable amounts of finely dispersed iron are precipitated, probably as a Fe²⁺-compound. It is suggested that the iron functions as a rechargeable buffer against hydrogen sulfide, protecting both the bacteria and the priapulid host. Although this chemical shield may not alone account for long-term protection, it allows the animal to gain time for metabolic adaptations.Contribution No. 289 of the Alfred-Wegener-Institut für Polar- and Meeresforschung (AWI Bremerhaven).     

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

Life history characteristics of the western flower thrips,Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), under fluctuating conditions of temperature or relative humidity

Frankliniella occidentalis (Pergande) is a major insect pest of greenhouse crops such as leaf vegetables, flowers and vegetable fruits worldwide. The life history characteristics of F. occidentalis were investigated at control temperature and humidity (27.3 ± 0.54 °C, 79.9 ± 2.79% RH) (mean ± SD), a 10 °C-range fluctuation in temperature (27.1 ± 5.28 °C, 81.5 ± 4.03% RH), a 20 °C-range fluctuation in temperature (26.5 ± 10.09 °C, 80.4 ± 5.76% RH), a 20%-range fluctuation in humidity (26.8 ± 0.37 °C, 80.7 ± 9.55% RH) and a 30%-range fluctuation in humidity (27.3 ± 0.41 °C, 76.3 ± 15.28% RH). Overall, the life history traits of F. occidentalis were more negatively affected by fluctuating environmental conditions. The impact of temperature fluctuation was more severe than that of humidity fluctuation. Additionally, the degree of impact increased as the fluctuation range of the temperature increased, while the reverse trend was observed with humidity fluctuations. With the 20 °C-range fluctuation in temperature, F. occidentalis died at the 1st instar larval stage. The offspring’s sex ratio was significantly higher at the 20%- and 30%-range fluctuations in humidity (0.47 and 0.49, respectively) compared to the control (0.35) and at the 10 °C-range fluctuation in temperature (0.33). From the fertility life table analysis, the intrinsic rate of increase (r) was higher at the 30%-range fluctuation in humidity and control conditions as 0.218 and 0.205, respectively. At the 10 °C-range fluctuation in temperature conditions, r was significantly lower as 0.169. High fluctuations in temperature and low fluctuations in humidity appear to be the best conditions for controlling F. occidentalis populations in greenhouses.     

Effect of acclimation temperature and substrate type on selected temperature,movement and activity of juvenile spiny softshell turtles (Apalone spinifera) in an aquatic thermal gradient

In some turtle species, temperature selection may be influenced by environmental conditions, including acclimation temperature and substrate quality. These factors may be particularly important for softshell turtles that are highly aquatic and often thermoregulate by burying in the substrate in shallow water microhabitats. We tested for effects of acclimation temperature (22 °C or 27 °C) and substrate type (sand or gravel) on the selected temperature and movement patterns of 20 juvenile spiny softhshell turtles (Apalone spinifera; Reptilia: Trionychidae) in an aquatic thermal gradient of 14–34 °C. Among 7–11 month old juvenile softshell turtles, acclimation temperature and substrate type did not influence temperature selection, nor alter activity and movement patterns. During thermal gradient tests, both 22- and 27 °C-acclimated turtles selected the warmest temperature (34 °C) available most frequently, regardless of substrate type (sand or gravel). Similarly, acclimation temperature and substrate type did not influence movement patterns of turtles, nor the number of chambers used in the gradient tests. These results suggest that juvenile Apalone spinifera are capable of detecting small temperature increments and prefer warm temperatures that may positively influence growth and metabolism, and that thermal factors more significantly influence aquatic thermoregulation in this species than does substrate type.     

Female mate choice of male signals is unlikely to promote ecological adaptation in Enchenopa treehoppers (Hemiptera: Membracidae)

A key question in speciation research is how ecological and sexual divergence arise and interact. We tested the hypothesis that mate choice causes local adaptation and ecological divergence using the rationale that the performance~signal trait relationship should parallel the attractiveness~signal trait relationship. We used female fecundity as a measure of ecological performance. We used a species in the Enchenopa binotata treehopper complex, wherein speciation involves adaptation to novel environments and divergence in sexual communication. We used a full‐sibling, split‐family rearing design to estimate genetic correlations (r_G) between fecundity and signal traits, and compared those relationships against population‐level mate preferences for the signal traits. Animal model estimates for r_G between female fecundity and male signal traits overlapped zero—rejecting the hypothesis—but could reflect sample size limitations. The magnitude of r_G correlated with the strength of the mate preferences for the corresponding signal traits, especially for signal frequency, which has the strongest mate preference and the most divergence in the complex. However, signal frequencies favored by the population‐level mate preference are not associated with high fecundity. Therefore, mate preferences do not appear to have been selected to favor high‐performance genotypes. Our findings suggest that ecological and sexual divergence may arise separately, but reinforce each other, during speciation.     

Review of methodological problems of 'Computer cladistics' exemplified with a case study on isopod phylogeny (Crustacea: Isopoda)

Using a computerized phylogenetic analysis of the Isopoda (Crustacea: Peracarida) as source of typical errors and misunderstandings, problems that may occur in computer cladistics are reviewed. It is concluded that in addition to the errors that are possible in a conventional Hennigian analysis some specific methodological problems exist in computer cladistics. It is recommended that the OTU be replaced by the groundpattern concept. Tree statistics are not useful for comparing different competing hypotheses. Arguments ought to concentrate on the hypo-thetico-deductive steps of the analysis, i.e. on character analysis. The use of computers does not add objectivity to character analysis. Single outgroup taxa should not be used in assessing the character states of ingroups. Concerning isopod phylogeny, it is argued here that the tail fan of the Isopoda can probably be derived from the eumalacostracan groundpattern and did not evolve de novo within the Isopoda.     

Local adaptation with high gene flow: temperature parameters drive adaptation to altitude in the common frog (Rana temporaria)

Both environmental and genetic influences can result in phenotypic variation. Quantifying the relative contributions of local adaptation and phenotypic plasticity to phenotypes is key to understanding the effect of environmental variation on populations. Identifying the selective pressures that drive divergence is an important, but often lacking, next step. High gene flow between high‐ and low‐altitude common frog (Rana temporaria) breeding sites has previously been demonstrated in Scotland. The aim of this study was to assess whether local adaptation occurs in the face of high gene flow and to identify potential environmental selection pressures that drive adaptation. Phenotypic variation in larval traits was quantified in R. temporaria from paired high‐ and low‐altitude sites using three common temperature treatments. Local adaptation was assessed using Q_ST–F_ST analyses, and quantitative phenotypic divergence was related to environmental parameters using Mantel tests. Although evidence of local adaptation was found for all traits measured, only variation in larval period and growth rate was consistent with adaptation to altitude. Moreover, this was only evident in the three mountains with the highest high‐altitude sites. This variation was correlated with mean summer and winter temperatures, suggesting that temperature parameters are potentially strong selective pressures maintaining local adaptation, despite high gene flow.     

延腹小蜂属榕小蜂对榕果果内空腔大小差异的行为和形态适应

大多数榕小蜂的雄蜂终生都在密闭的榕果中生活,榕果间果内空腔的大小存在很大的差异,这种差异可能限制对小蜂的活动。已有研究表明榕果的大小会影响果内榕小蜂雄蜂的打斗行为,那么这种差异是否会影响雄蜂的果内爬行行为? 雄蜂是否会产生适应性的形态特征？针对这些问题,在2008年6月到2009年10月期间,我们在野外采集榕果,并带回实验室中, 对榕果内生活的延腹小蜂属Philotrypesis 5种雄蜂的果内爬行行为和足的形态特征进行了研究。结果表明,根据Philotrypesis spp.雄蜂后足的跗节形态特征的不同,其后足可分成2种形态型--Ⅰ型：后足跗节基部两节的突出部分排列紧密, 后足中跗节不发达; Ⅱ型：后足跗节基部两节的突出部分排列疏松,二者之间有较宽的分隔,后足中跗节发达。GLM分析结果表明各种榕树上生活的Philotrypesis雄蜂前足腿节长宽比存在极显著差异（F_{8, 81}=94.86, P<0.001）。在大果中生活的Philotrypesis雄蜂都具有较细长的前足腿节,在小果中生活的Philotrypesis雄蜂除了M7#c-ben外,都具有较粗壮的前足腿节。当雄蜂在榕果内寻偶时,主要依靠粗壮的前足或灵活的后足在果内爬行。结果提示, 榕果空腔大小可能限制了Philotrypesis雄蜂果内爬行行为, 进而影响了雄蜂足形态的适应性进化。     

Effects of temperature on the development and reproduction of sugarcane top borer Scirpophaga excerptalis (Lepidoptera: Pyralidae)

To determine the effects of temperature on the development and reproduction of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae), the durations of different developmental stages and reproductive capacity of S. excerptalis were measured at 20, 23, 26, 29 and 32℃. The results showed that the average durations of various developmental stages of S. excerptalis shortened with increasing temperatures from 20 to 32℃. The durations to complete one life cycle were 113.17 days at 20℃, and 39.50 days at 32℃. The developmental rate of each stage and generation was positively correlated with the temperature, which was consistent with the Logistic regression model. The temperature thresholds for egg, larval, pupal and preoviposition stages and the total cycle were 13.73, 14.73, 13.91, 13.66 and 14.10℃, respectively. The effective accumulative temperature was 112.62, 370.01, 188.17, 23.82 and 718.07 degree-days (DD), respectively. The adult longevity shortened with increasing temperatures, and the highest number of eggs laid per female was 204.74 eggs at 29℃. The survival rates of eggs, larvae and the whole generation were the highest at 26℃, which were 87.25%, 56.67% and 37.21%, respectively while the highest survival rate of the pupa was 76.69% at 29℃. There was no significant difference for the total cycle at 23-29℃. The results indicated that temperature was the key factor affecting the development and reproduction of S. excerptalis, and the optimum temperature for development and reproduction ranged from 23 to 29℃.