Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Inhibition of hydrocarbon biosynthesis in the housefly by 2-Octadecynoate

The elongation of [9,10-³H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ²⁼) inhibited the in vivo incorporation of [1-¹⁴C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-¹⁴C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-³H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-¹⁴C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.     

DOPA and tyrosine decarboxylase activity in tissues of Periplanet a americana in relation to cuticle formation and ecdysis

DOPA decarboxylase activity in haemolymph and integument was low in last instar and early pharate adult Periplaneta americana, but began to increase shortly before ecdysis. Decarboxylation rates of l-DOPA, about 10 times the larval level by the start of ecdysis, reached a peak about 6 hr afterward, coinciding with the main period of cuticular sclerotization. Activity decreased rapidly during the next 18 hr, then decreased gradually for several days. Haemolymph DOPA decarboxylase activity was about four times greater than the integument, based on tissue dry weights. The fat body and gut tissues had low DOPA decarboxylase activity in all ages tested, and this did not increase at ecdysis. Tyrosine decarboxylase activity was significant only in the haemolymph and at consistently low levels.DOPA decarboxylase, therefore, apparently plays a major rôle in production of catecholamine derivatives for cuticular sclerotization in P. americana, while tyrosine decarboxylation is minor. Both haemolymph and integument appear to be important sites of dopamine biosynthesis.     

Production of farnesoic acid and methyl farnesoate by mandibular organs of the crayfish,Procambarus clarkii

The mandibular organs (MO) of crustaceans secrete methyl farnesoate (MF) and farnesoic acid (FA). To better understand the secretory activity of MO, the kinetics of production and release of both compounds were determined in vitro by following incorporation of [2-¹⁴C]acetate and l-[³H-methyl]methionine into MF and [2-¹⁴C]acetate into FA by MO of Procambarus clarkii. MO released more FA than MF but contained more MF. In medium lacking unlabeled acetate, the percentage incorporation of [¹⁴C]acetate into MF, relative to [³H]methionine, was between 21 and 40%, suggesting that there may be an alternative source of C₂ units.MO produce similar amounts of MF at concentrations of acetate from 0.08 to 10 mM. However, the addition of exogenous unlabelled FA to incubation media did not stimulate the biosynthesis of MF, raising the possibility that unlike JH biosynthesis in insects, the last step in MF production may be rate-limiting. Nonetheless, exogenous FA significantly reduced the incorporation of [¹⁴C]acetate into MF, suggesting that the glands use exogenous FA to synthesize MF. The absence of stimulation of FA production by exogenous FA indicates that there is no feedback effect of this product on the early steps in the biosynthetic pathway.     

Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig

Corpora allata of adult female Euborellia annulipes, incubated in medium containing ³H-methionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 ± 1.6% of the total juvenile hormone III synthesized was released and 96.5% ± 0.3 in medium supplemented with 60 μM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 ± 0.004 mm), hormone production was only 0.59 ± 0.13 fmol hr⁻/corpus allatum; production increased to 1.52 ± 0.25 fmol hr⁻¹/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 ± 0.13 fmol hr⁻¹/corpus allatum. The addition of 60 μM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of l-methionine in the medium also enhanced hormone biosynthesis. Maximal hormone production was 32.8 ± 10.9 fmol hr⁻¹/corpus allatum, at an l-methionine concentration of 51 μM.     

Enhancement of erythritol production by Trichosporonoides oedocephalis ATCC 16958 through regulating key enzyme activity and the NADPH/NADP ratio with metal ion supplementation

Erythritol, a well-known natural sweetener, is mainly produced by microbial fermentation. Various metal ions (Al³⁺, Cu²⁺, Mn²⁺, and Ni²⁺) were added to the culture medium of Trichosporonoides oedocephalis ATCC 16958 at 30?mg/L in shake flask cultures. Compared with controls, Cu²⁺ increased the erythritol content by 86% and decreased the glycerol by-product by 31%. After 48 hr of shake flask culture, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that expression levels of erythrose reductase (ER) in the presence of 30?mg/L CuSO₄?·?5H₂O were higher than those obtained after treatment with other examined metal ions. Furthermore, after 108 hr of batch culture in a 5-L bioreactor, supplementation with 30?mg/L of CuSO₄?·?5H₂O increased the specific erythritol content by 27%. Further studies demonstrated that ER activity under 30?mg/L CuSO₄?·?5H₂O supplementation in a fermentor was overtly increased compared with the control after 60 hr, while glycerol-3-phosphate dehydrogenase activity was clearly reduced in most of the fermentation process. Furthermore, the NADPH/NADP ratio was slightly lower in T. oedocephalis cells treated with Cu²⁺ compared with control cells. These results provide further insights into Cu²⁺ effects on erythritol biosynthesis in T. oedocephalis and should improve the industrial production of erythritol by biological processes.     

The allatostatic effect of 20-hydroxyecdysone on the adult viviparous cockroach, Diploptera punctata

The effect of 20-hydroxyecdysone upon the activity of corpora allata (CA) from female Diploptera punctata has been investigated. This ecdysteroid inhibits juvenile hormone (JH) biosynthesis by the CA, whether they have been implanted into a male, or remained in situ within the female. In the female, this inhibition is reflected in reduced oöcyte growth and vitellin content. The allatostatic effect of 20-hydroxyecdysone becomes apparent in vivo within 24 hr. However, no inhibition was observed when the CA were maintained in vitro for 42 hr in medium containing up to 1·10^?5 M 20-hydroxyecdysone. This suggests that the effect of the hormone upon the CA is indirect. These experiments raise the possibility that ecdysteroids play an allatostatic role during the normal gonotrophic cycle in Diploptera.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

The horizontal and vertical distribution of flying insects near artificial windbreaks

Lath fences 3 ft and 8 ft high affected the horizontal distribution of flying insects similarly: most insects accumulated at a distance equal to 2–3 times the height of each fence to leeward, where catches, measured at 14 and 38 in. (≡ to 0·4 times the height of the fence above the ground) were 30–40 % greater than in exposed positions. Behind an 8 ft fence accumulations extended vertically to 12–16 ft (≡ to 1·5-2·0 times the height of the fence). Vertical profiles of weak-flying insects, in winds > 3 m.p.h. differed in sheltered and exposed positions; in shelter the boundary layer was deeper and insects were more abundant nearer the ground than elsewhere. For strong flyers, and insects which flew only in winds < 2 m.p.h., vertical profiles in sheltered and exposed positions were indistinguishable.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae

The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pieris brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13·0 per cent and in the fat body of 4-day pupae 6·3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). The label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-1-¹⁴C 39·0 per cent of label was in PTC, 52·8 per cent in PTE, and 2·0 per cent in sphingomyelin. In late fifth instar 45·0 per cent was in PTC, 45·5 per cent in PTE, and 6·5 per cent in sphingomyelin, while in 4-day female pupae 45·2 per cent was in PTC, 41·3 per cent in PTE, and 13·5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51·8 per cent being in PTC and 48·2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55·8 and 43·2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58·6 per cent), PTE (24·4 per cent), and sphingomyelin (17·0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the rôle of the investigated fatty acids in the biosynthesis of PL are discussed.     

Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment,especially for anaerobic treatment

In recent years considerable effort has been made in the Netherlands toward the development of a more sophisticated anaerobic treatment process, suitable for treating low a strength wastes and for applications at liquid detention times of 3–4 hr. The efforts have resulted in new type of upflow anaerobic sludge blanket (UASB) process, which in recent 6 m³ pilot-plant experiments has shown to be capable of handling organic space loads of 15–40 kg chemical oxygen demand (COD)·m^?3/day at 3–8 hr liquid detention times. In the first 200 m³ full-scale plant of the UASB concept, organic space loadings of up to 16 kg COD·m^?3/day could be treated satisfactorily at a detention times of 4 hr, using sugar beet waste as feed. The main results obtained with the process in the laboratory as well as in 6 m³ pilot plant and 200 m³ full-scale experiments are presented and evaluated in this paper. Special attention is given to the main operating characteristics of the UASB reactor concept. Moreover, some preliminary results are presented of laboratory experiments concerning the use of the USB reactor concept for denitrification as well as for the acid formation step in anaerobic treatment. For both purposes the process looks feasible because very satisfactory results with respect to denitrification and acid formation can be achieved at very high hydraulic loads (12 day^?1) and high organic loading rates, i.e., 20 kg COD·m^?3/day in the denitrification and 60–80 kg COD·m^?3/day in the acid formation experiments.     

THE CELL POPULATION KINETICS AND RESPONSE TO AN S-PHASE SPECIFIC AGENT OF THREE TRANSPLANTABLE COLON TUMOR LINES

The relative cell population kinetics of three transplantable murine colon tumor lines (Colon 26, 36 and 38) with different histological and metastatic characteristics were studied in relation to the response of each line to an S-phase specific agent. The mean doubling times for the three lines between 0·1 and 1·0 g are similar (4·2 days) but marked differences are apparent in times to tumor appearance (0·1 g) and in median days to death. The length of the cell cycle is about one day and the length of the S-phase 10–11 hr for Colon 36 and 38. The length of the cell cycle in Colon 26 is difficult to estimate by conventional methods but probably exceeds 24 hr and the S-phase is 10–11 hr; [³H]TdR pulse labeling indices for Colon 36 and 38 decrease with time and tumor size from about 0·45 in 0·1 to 0·2 g tumors to about 0·33 at 3 g. The decrease in the [³H]TdR labeling index for Colon 26 is more pronounced (from about 0·38 at 0·1 g to 0·21 at 1·0 g). The shapes of the PLM curves and the [³H]TdR labeling index data are consistent with the observed sensitivity to an S-phase specific agent (Palmo-AraC, NSC 135962) in Colon 36 and the minimal response observed in Colon 26. Colon 38 is intermediate between Colon 36 and Colon 26 in kinetic properties and in response to the S-phase agent.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Neurophysiological changes associated with paralysis arising from body stress in the cockroach, Periplaneta americana

Two hours after physical stress, Periplaneta americana could be separated into three behavioural categories: normal to hyperactive; torpid with ataxia; and paralysed. At 2 hr, 68 per cent were either torpid or paralysed, at 20 hr, 83 per cent were paralysed. Weight loss was a distinct physiological symptom of stress paralysis: The calculated mean loss was 9·5 mg/hr for torpid insects and 12·4 mg/hr for paralysed cockroaches, losses were four to six times larger than those occurring in starved cockroaches. However, the haemolymph osrnolarities of the three categories showed no appreciable differences. Only starved and paralysed cockroaches showed a noticeable reduction in muscle fibre membrane potentials of the flexor tibia—a mean value below 40 mV for starved insects and a mean value below 50 mV for paralysed insects. Both of these categories consistently showed a lower amplitude for junctional potentials, but paralysed cockroaches showed a much higher incidence of complete failure to neural stimulation. Most muscle fibres of completely paralysed insects lost their sensitivity to direct extracellular stimulation while the loss in sensitivity was less evident in starved cockroaches. Axonal conduction on the crural nerve was not changed by stress, and the spontaneous efferent activity of completely paralysed insects was similar to the pattern of activity for normal cockroaches. Stress seemed to alter the volume and content of the intermyofibral spaces of muscles.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.     

Intrathyroidal iodine metabolism in the rat. The influence of diet and the administration of thyroid-stimulating hormone

1. Ratios of mono[¹³¹I]iodotyrosine and di[¹³¹I]iodotyrosine (R values) and the incorporation of ¹³¹I into iodothyronines have been estimated in rat thyroid glands from 30min. to 38hr. after the administration of [¹³¹I]iodide. 2. In rats receiving a powdered low-iodine diet the R values were close to unity and did not change with time after the administration of [¹³¹I]iodide. In rats receiving a commercial pellet diet the R values fell from a mean of 0·8 at 30min. after [¹³¹I]iodide administration to 0·49 at 38hr. 3. Administration of 0·5–2·0i.u. of thyroid-stimulating hormone before giving the injection of [¹³¹I]iodide caused a small diminution in the R value when the time between injecting [¹³¹I]iodide and killing the animal was 16hr. or more. 4. Iodothyronines represented a greater percentage of the total thyroid-gland radioactivity in the iodine-deficient animals than in animals fed on the pellet diet. Thyroid-stimulating hormone had little effect, if any, on the iodothyronine contents.