Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

Change in the H2 photoproduction capability in a synchronously grown aerobic nitrogen-fixing cyanobacterium,Synechococcus sp. Miami BG 043511

Synchronously growing cells of nitrogen-fixing Synechococcus sp. Miami BG 043511 were harvested periodically and the capability for hydrogen photoproduction in closed vessels was measured under hydrogen production conditions. The capability for hydrogen photoproduction in cells was correlated with that of cellular carbohydrate content. Cells with a high carbohydrate content exhibited a high capacity for hydrogen production and those with low carbohydrate content exhibited low capacity for hydrogen production. Nitrogenase activity at the onset of incubation did not coincide with a capability for the cells to produce hydrogen during the subsequent incubation period. Interestingly, when cells with a high capacity for hydrogen photoaccumulation were incubated, alternate periods of hydrogen and oxygen accumulation were observed at 12 hour intervals. About 0.5 ml of hydrogen per ml of cell suspension was accumulated in flasks during the initial 12-h incubation period. These observations indicate that the use of synchronous culture can be one of the ways of provide materials suitable not only for basic studies but also for applied aspects of hydrogen photoproduction.     

A framework to compare theoretical predictions on trait evolution in temporally varying environments under different life cycles

Predicting the evolution of traits such as dispersal or local adaptation, in a variable environment is an important issue in theoretical evolutionary ecology. With concepts such as hard selection vs. soft selection or fine-grained vs. coarse-grained environmental variability, this issue has attracted much attention, and yet different models seldom agree on qualitative predictions about, e.g. the evolution of generalist or specialist strategies, or the occurrence of stabilizing or disruptive selection on studied traits.Here, I investigate the effect of the order of events in the life cycle on trait evolution in a spatially heterogeneous, temporally varying landscape using a Wright–Fisher island model. I first develop a methodological framework allowing for different life cycles. Then I illustrate the importance of life cycles on selection regimes by looking more closely at the evolution of local adaptation.Model results show that the occurrence of disruptive selection and bi- or tristability mainly depends on the life cycle, the convexity of the trade-off behind local adaptation, the immigration rate, and the autocorrelation in patch state. With the same forces driving the evolution of local adaptation, different life cycles induce different evolutionary outcomes. Model results highlight the importance of accounting for life cycle specificities when attempting to predict the effects of the environment on evolutionarily selected trait values, as well as the need to check the robustness of evolutionary model conclusions against modifications of the life cycle.     

Effect of immersion cycles on growth,phenolics content,and antioxidant properties of Castilleja tenuiflora shoots

Castilleja tenuiflora, a species highly valued for its medicinal properties, is threatened in the wild. We evaluated the effects of six different immersion cycles in a temporary immersion bioreactor on C. tenuiflora shoot growth, proliferation rate, phenolics content, flavonoid content, and antioxidant activity. We also evaluated the regeneration capacity of the shoots. The highest proliferation rate (nine shoots per explant) was obtained using an immersion cycle of 5 min every 12 h, and the longest shoots (38.8?±?1.9 mm) were obtained using an immersion cycle of 5 min every 24 h. Shoots obtained from immersion cycles of 30 min every 24 h or 5 min every 24 h showed 100% rooting efficiency. Shoots obtained from immersion cycles of 30 min every 3 h or 30 min every 12 h accumulated H₂O₂, developed abnormal stomata, and showed symptoms of hyperhydricity. These characteristics were associated with a low survival rate (16–80%) when the plants were transferred to potting mix. The shoots from an immersion cycle of 30 min every 24 h showed the highest total phenolics content, which coincided with the highest antioxidant activity in the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) free-radical scavenging assay (161.74?±?10.06 μmol Trolox/g dry weight (DW)). The shoots from an immersion cycle of 5 min every 24 h showed the highest activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay, and those from an immersion cycle of 5 min every 3 h showed the strongest reducing power. These results show that temporary immersion culture represents a reliable and efficient method for in vitro micropropagation of C. tenuiflora.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Improving the photosynthetic H2-productivity of the green alga Chlorella fusca by physiologically directed O2 avoidance and ammonium stimulation

Low production rates and sensitivity to O₂ are two major obstacles which prevent the technical exploitation of the ability of green algae to produce H₂ from water. Both problems were addressed in the present work. The inhibitory effect of O₂ on the hydrogen photoproduction of the green alga Chlorella fusca could be minimized by using algal cells which had not yet fully restored their oxygen evolving capacities after an artificially induced chloroplast de/regeneration cycle (de-/regreening). The H₂ photoproductivity peaked after 30 h of greening light while the O₂ evolution at this time reached only 59% of its normal capacity. The H₂PP yields could be further increased if NH₄Cl was added to the reaction medium at the beginning of the anaerobic preincubation period. No stimulatory effect was observed when NH₄Cl was added just before illumination, i.e. at the end of the 5-h-preincubation period. It is assumed that NH₄Cl inhibited the photosynthetic reduction of nitrite, which competed with hydrogen photoproduction indirectly by feedback repression of the NO ₂ ^- /NO ₃ ^- -reductive system. The impacts of the given results on an optimized H₂-production in green algae based on photosynthesis are discussed.Abbreviations H₂PP H₂ photoproduction - H₂ase hydrogenase - DA dark adaptation - LRG light regreening - DCMU 3-(3,4-dichlorophenyl)-l, 1-dimethylurea - Dit sodium dithionite - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PS I/II photosystem I/II     

Comparative Amperometric Study of Uptake Hydrogenase and Hydrogen Photoproduction Activities between Heterocystous Cyanobacterium Anabaena cylindrica B629 and Nonheterocystous Cyanobacterium Oscillatoria sp. Strain Miami BG7

Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N₂ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production, photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O₂ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O₂ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

SURVEY OF SELECTED SEA WEEDS FOR SIMULTANEOUS PHOTOPRODUCTION OF HYDROGEN AND OXYGEN1

Ten seaweed species were surveyed for simultaneous photoevolution of hydrogen and oxygen. In an attempt to induce hydrogenase activity (as measured by hydrogen photoproduction) the seaweeds were maintained under anaerobiosis in CO₂-free seawater for varying lengths of time. Although oxygen evolution was observed in every alga studied, hydrogen evolution was not observed. One conclusion of this research is that, in contrast to the microscopic algae, there is not a single example of a macroscopic alga for which the photoevolution of hydrogen has been observed, in spite of the fact that there are now at least nine macroscopic algal species known for which hydrogenase activity has been reported (either by dark hydrogen evolution or light-activated hydrogen uptake). These results are in conflict with the conventional view that algal hydrogenase can catalyze a multiplicity of reactions, one of which is the photoproduction of molecular hydrogen. Two possible explanations for the lack of hydrogen photoproduction in macroscopic algae are presented. It is postulated that electron acceptors other than carbon dioxide can take up reducing equivalents from Photosystem I to the measurable exclusion of hydrogen photoproduction. Alternatively, the hydrogenase system in macroscopic algae may be primarily a hydrogen-uptake system with respect to light-activated reactions. A simple kinetic argument based on recent measurements of the photosynthetic turnover times of simultaneous light-activated hydrogen and oxygen production is presented that supports the second explanation.     

Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa

Photoproduction of hydrogen gas by the green alga Chlorella pyrenoidosa was studied in a large scale culture of 2.1. Hydrogen was produced by adding sodium hydrosulfite directly to an algal suspension after anaerobiosis in darkness for activation of hydrogenase. The hydrogen production rate showed a characteristic course of an initial burst of gas then steady production, and this course appeared most clearly at cell concentrations around 0.6–0.7 kg/m³. In the final third phase, the hydrogen production rate gradually decreased until evolution ceased. The steady hydrogen evolution was inhibited 75% by a herbicide, DCMU, which blocks electron flow through photosystem II, indicating that the electron donor for hydrogen production was mainly water. The average light intensity within the culture vessel was measured with a diffusing sphere photoprobe. The rate of hydrogen evolution increased hyperbolically with the average light intensity. The duration of hydrogen photoproduction was shorter at higher light intensity due to the inhibition of hydrogenase by concomitantly released oxygen. The duration was shorter also at higher concentrations of algal suspension. It was foudd that the optimum concentration of algae, about 0.7 kg/m³ in this system, must be selected to maximize the yield of hydrogen.     

Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures

Plant cell cultures provide a renewable source for synthesis and supply of commercially valuable plant-derived products, particularly for secondary metabolites. However, instability in product yields over multiple passages has hampered the efficient and sustainable use of this technology. Paclitaxel accumulation in Taxus cell suspension culture was quantified over multiple passages and correlated to mean aggregate size, extracellular sugar level, ploidy, and cell cycle distribution. Paclitaxel levels varied approximately 6.9-fold over the 6-month timeframe investigated. Of all of the parameters examined, only mean aggregate size correlated with paclitaxel accumulation, where a significant negative correlation (r = ?0.75, p < 0.01) was observed. These results demonstrate the relevance of measuring, and potentially controlling, aggregate size during long term culture passages, particularly for plant suspensions where industrially relevant secondary metabolites are not pigmented to enable rapid culture selection.     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

Itaconic acid production using an air-lift bioreactor in repeated batch culture of Aspergillus terreus

Repeated itaconic acid production using an air-lift bioreactor was carried out by three methods—two with cell recycling by means of centrifugation and filtration by a stainless steel filter set inside the bioreactor and one by repeated batch culture without cell recycling. In a flask culture, repeated itaconic acid production was stable for 9 cycles (45 d) and the production rate was 0.47 g/l/h. However, in the air-lift bioreactor, it was difficult to produce itaconic acid in the repeated batch culture with cell recycling for a long period due to a decrease in fluidity resulting from an increase in mycelium concentration. In the method without cell recycling, however, repeated itaconic acid production was stable for 4 cycles (21 d) and the production rate was 0.37 g/l/h.     

Effect of sulfide on growth of marine bacteria

Severe hypoxia leads to excess production of hydrogen sulfide in marine environments. In this study, we examined the effect of sulfide on growth of four facultative anaerobic marine bacteria in minimal media under anaerobic conditions. The Gram-negative chemolithoautotrophic Marinobacter sp. tolerated sulfide concentrations up to 0.60 mM, with doubling and lag times increasing as a function of increasing sulfide concentration but with no change in maximum culture yields; growth did not occur at 1.2 mM sulfide. Similar results were obtained for the metabolically diverse Gram-negative denitrifying Pseudomonas stutzeri, except that growth occurred at 1.2 mM and culture yields at 0.60 and 1.2 mM sulfide were approximately 10-fold lower than at sulfide concentrations between 0 and 0.30 mM. Increases in doubling and lag times accompanied by an overall 10-fold decrease in maximum culture yields were found for the Gram-negative chemoheterotrophic Vibrio sp. at all sulfide concentrations tested. In contrast, growth of a Gram-positive chemoheterotrophic Bacillus sp. was resistant to all sulfide concentrations tested (0.15–1.2 mM). Our results highlight the variable responses of marine bacteria to sulfide and provide some insight into shifts that may occur in microbial community structure and diversity as a consequence of changes in sulfide levels that are the result of hypoxia.     

Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light

As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2, and 0.5) in a biological oxygen monitor (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis–irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 μmol m^?2?s^?1. Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 μmol m^?2?s^?1). At lower duty cycles, maintenance respiration reduced net oxygen yield. At higher duty cycles, photon absorption rate exceeded the maximal photon utilization rate, and, as a result, surplus light energy was dissipated which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

Tyrosinase Inhibitory and Anti-oxidative Effects of Lactic Acid Bacteria Isolated from Dairy Cow Feces

Overproduction and accumulation of melanin cause a number of skin diseases. The inhibitors of tyrosinase are important for the treatment of skin diseases associated with hyper-pigmentation after UV exposure and application in cosmetics for whitening and depigmentation. Reactive oxygen species (ROS) including hydrogen peroxide are generated by chemical substances and metabolic intermediates and cause various diseases including cancer and heart diseases. We have isolated four different lactic acid bacteria (LAB) strains from dairy cow feces and investigated the tyrosinase inhibition and anti-oxidative effects of culture filtrates prepared from the isolated bacteria, which are designated as EA3, EB2, PC2, and PD3. To investigate optimal culture conditions isolated LAB strains, the measurements of tyrosinase inhibitory and anti-oxidative activities were performed. The results of tyrosinase inhibitory activities revealed that Enterococcus sp. EA3 showed about 65% at culture conditions (14 h, 30 °C, pH 8, and 0% NaCl), Enterococcus sp. EB2 about 65% at culture conditions (12 h, 30 °C, pH 9, and 0% NaCl), Pediococcus sp. PC2 about 80% at culture conditions (20 h, 30 °C, pH 6, and 0% NaCl), and Pediococcus sp. PD3 about 80% at culture conditions (20 h, 30 °C, pH 8, and 0% NaCl), respectively. In addition, anti-oxidative activities against four different LAB strains showed approximately more than 30% at optimal conditions for the measurements of tyrosinase inhibitory activities. From the results, we have suggested that the isolated four LAB strains could be useful for a potential agent for developing anti-oxidants and tyrosinase inhibitors.     

Hydrogen production by the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis> part I: effects of sulfured nutriments,with thiosulfate as model,on hydrogen production and growth

Background

Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H₂/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen.

Results

Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L^?1 and yeast extract 1 g L^?1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L^?1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol^?1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production.

Conclusions

The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g^?1 of cells), and (2) as both sulfur source and detoxifying agent for H₂ when thiosulfate is present at higher concentrations and, when, simultaneously, the pH₂ is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

     

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.