STUDIES IN THE MODE OF ACTION OF INSECTICIDES

Homogenates of the thoracic nervous system of Locusta migratoria migratoriodes are able to hydrolyse acetylcholine (ACh) and o -nitrophenylacetate (NPA), and this hydrolysis can be inhibited by tetraethylpyrophosphate (TEPP) at approximately the same molar concentration for both substrates. It is possible that one acetyl-esterase is responsible for the breakdown of the two substances, and there is no reason to assume the existence of a specific acetylcholinesterase. In normal horse serum, on the other hand, the pseudocholinesterase is quite distinct from the enzyme responsible for the breakdown of NPA.
In an attempt to correlate the inhibition of the locust nerve cord acetylesterase with toxic activity to insects and mice, four chlorinated diethyl-phenylphosphates were tested as contact poisons against a number of insects and by injection against locusts and mice, and also as in vitro inhibitors of locust nerve cord acetylesterase and horse-serum pseudocholinesterase. The chemicals were the 2-chloro-, 4-chloro, 2:4-dichloro- and 2:4:5-trichloro- analogues of diethyl-phenylphosphate.
Good correlation exists between their in vitro activity against the nerve-cord acetylesterase and their contact activity to aphids, but not between the former and injection toxicity to locusts. No correlation could be established between the inhibition of horse-serum cholinesterase and injection toxicity to mice. It is thought likely that the inhibition of nerve-cord acetylesterase is of greater importance in aphids than in other insects, where the toxic action of the phosphoric esters is at least partly concerned with other vital processes, and that a detoxication mechanism in the mammal breaks down some of the phosphoric esters, but not others.     

THE EFFECT OF BODY WEIGHT ON THE RESISTANCE TO INSECTICIDES OF THE LAST-INSTAR LARVA OF DIATARAXIA OLERACEA L., THE TOMATO MOTH

At a constant temperature of 24° C. the final larval instar of Diataraxia oleracea lasts about 10 days, during which its resistance to DDT and γ-BHC as contact insecticides progressively increases up to the 5th or 6th day. It then suddenly decreases, this coinciding with cessation of feeding and the beginning of prepupal formation.
Between the 2nd and the 6th days the gross body weight of the last-instar larva increases from about 0.27 to 0.65g. Under the conditions of the experiments, the LD50 of parathion, as a stomach poison, was linearly related to body weight; on the same basis TEPP was slightly less, and lead arsenate, slightly more, toxic to the larger than to the smaller larvae. However, DDT as a stomach or contact insecticide, and γ-BHC as a stomach poison were notably less toxic to the larger larvae. For example, the increase in LD50 for an increase in larval body weight of × 2 was about × 11 for DDT as a stomach poison and about × 12 as a contact insecticide.
The order of effectiveness of the above insecticides as stomach poisons for the last-instar larva of D. oleracea was parathion > DDT > γ-BHC > TEPP = lead arsenate. Zinc fluoarsenate and rotenone were relatively non-toxic. Larvae of D. oleracea were repelled by food leaf treated with an extract of natural pyrethrins.     

STUDIES ON THE MECHANISM OF INSECTICIDAL ACTION OF ORGANO-PHOSPHORUS COMPOUNDS WITH PARTICULAR REFERENCE TO THEIR ANTIESTERASE ACTIVITY

The preparation of an extract of the mealworm larvae, Tenebrio molitor L. which hydrolyses ethyl butyrate and o -nitrophenyl acetate, but not acetylcholine, is described. The inhibition of this esterase by TEPP-containing materials and parathion was determined.
An enzyme that hydrolysed o -nitrophenyl acetate and was inhibited by a TEPP-containing material was demonstrated in the five other insect species used.
The relative toxicities as contact insecticides to adult Tribolium castaneum Hbst. of ten samples of TEPP-containing materials were compared with their relative activities as esterase inhibitors. There was not an exact quantitative correlation between TEPP content estimated chemically, insecticidal activity and anti-esterase activity; but the correlation was sufficient to suggest interdependence of these factors.
Eggs of Diataraxia oleracea L. and Ephestia kühniella Zell, were shown to contain an enzyme that hydrolysed o -nitrophenyl acetate and was inhibited by the TEPP-containing materials. This enzyme was present in eggs less than 24 hr. old, i.e. before there was any visible signs of development. The TEPP was shown to be toxic to these eggs and in high concentrations kills at an early stage of development before differentiation of the nervous system. This, in conjunction with the other evidence, suggests that esterases other than the choline-esterase of the nervous system are important when considering the toxic action of these compounds.
Comparison of the anti-esterase activity and toxicity of parathion and TEPP-containing materials as insecticides showed that although the TEPP materials were the more potent enzyme inhibitors, parathion was the more potent contact insecticide to five species of insects. This appears to be due to the relative instability of TEPP. The study of the rates of action of the two poisons applied at different concentrations supports this view.     

AN ECOLOGICAL STUDY OF THE ENCHYTRAEID WORM POPULATION OF SEWAGE BACTERIA BEDS LABORATORY EXPERIMENTS

This paper records the results of the experimental phase of an ecological study of the enchytraeid population of the sewage beds of the Huddersfield Corporation. The sewage in this drainage area is heavily charged with chemical wastes which have a marked effect on the fauna. Enchytraeus albidus Henle was the dominant worm while Lumbricillus lineatus Mull., the common bacteria bed-worm, was relatively scarce. The ability of these two worms to complete their life cycles in the sewage before and after treatment in particular beds was examined.
The crude Huddersfield sewage proved toxic to both E. albidus and L. lineatus but the former was much more resistant. After treatment in the beds the degree of toxicity was much reduced and the final sewage was harmless to all stages of E. albidus but the eggs of L. lineatus were still unable to develop in it.
The eggs of both species were less resistant to the toxic substances than the young worms; the adults were most resistant. High temperatures lowered toxicity by reducing the duration of the more susceptible early stages.
There was no evidence of physiological adaptation of L. lineatus to conditions in the Huddersfield beds. Wastes from both the main chemical works in the drainage area were examined and shown to be toxic; the domestic sewage was non-toxic. Both the distillate and residue of one of the chemical wastes proved toxic, especially the latter.
The toxicity could be entirely removed from the treated sewage by standing it over activated carbon for a few hours, or permitting slow oxidation for some weeks. The eggs of L. lineatus were used for these tests.
The Huddersfield sewage was very acid from time to time and the acidity reached was sufficient to kill young worms and adults of both species. But this factor was not considered important in the beds owing to the persistent effects of the toxic sewage.     

THE TOXICITY OF SOME INSECTICIDES TO FIFTH-INSTAR NYMPHS OF THE DESERT LOCUST

The toxicities of the insecticides γ-BHC, diazinon, dieldrin, DNC, heptachlor and parathion to fifth-instar hoppers of the desert locust were determined. Parathion and dieldrin were the most toxic, followed in descending order by heptachlor, γ-BHC, diazinon and DNC. The resistance of the hoppers to γ-BHC and DNC changed appreciably with age within the instar.
The effects of some factors which might influence the resistance of the insects were examined and are discussed.     

EMBRYONIC DEVELOPMENT AND ESTERASE ACTIVITY OF EGGS OF PIERIS BRASSICAE IN RELATION TO TEPP POISONING

The embryological development of eggs of Pieris brassicae was studied in relation to the occurrence of enzymes hydrolysing phenyl acetate and acetylcholine. Phenyl acetate is hydrolysed at a high rate at all stages of development of the embryo. Hydrolysis of acetylcholine only becomes appreciable in the later stages of development. The first significant level of hydrolysis of acetylcholine can be correlated with the development of a nervous system in the embryo to a stage where it may be functional.
Aqueous solutions of TEPP were applied to eggs soon after they were laid. Low doses of TEPP allowed a high percentage of eggs to develop to the point of hatching before death occurred. Most fully developed embryos became active before they died. As the dose was increased less development took place and with very high doses little development occurred.
The significance of these results is discussed. The available evidence does not indicate that the poison penetrates slowly nor that it is 'locked up' and later released. The explanation that seems to fit the evidence best is that the poison penetrates rapidly and reacts irreversibly with, probably phosphorylates, one or more components of the egg, the extent of subsequent development depending upon the proportion of a biochemical system or the number of systems inactivated. Whilst inhibition of cholinesterase may play a part in the poisoning process, at least under some conditions, the evidence indicates that the death of the embryo may result from some other cause.     

STUDIES OF THE TOXICITY TO WORKER HONEY-BEES (APIS MELLIFERA L.) OF CERTAIN CHEMICALS USED IN PLANT PROTECTION

Laboratory techniques are described for the estimation of the stomach poison, direct and residual film contact poison and fumigant poison effects of chemicals to adult worker honey-bees.
The toxicity of eleven chemicals used in plant protection has been investigated by these methods. The order of effectiveness as stomach and contact poisons was: parathion, TEPP, γ-BHC, dieldrin, aldrin, chlordane, o , o -diethyl- o -ethylmercaptoethyl thiophosphate (constituent of Systox), bisdimethylamino fluorophosphine oxide, toxaphene and the sodium salts of 2:4-D and MCPA: as residual films, dieldrin, aldrin, γ-BHC, parathion, chlordane, and o , o -diethyl- o -ethylmercapto-ethyl thiophosphate (constituent of Systox); toxaphene, TEPP and bisdimethylamino fluorophosphine oxide had no measurable effect; as fumigants, dieldrin, γ-BHC, aldrin, parathion, and chlordane; the remainder had no measurable effect.     

藻类连续培养体系的构建与优化及其在产毒和无毒微囊藻竞争中的应用

利用发酵罐加装外置环形光源构建藻类连续培养系统, 以产毒微囊藻PCC 7806及其无毒突变株PCC 7806 mcyB–为培养材料, 通过对补料时间、接种密度和稀释率参数的优化, 获得最优培养条件, 并应用于产毒与无毒微囊藻的竞争实验中。通过优化得到连续培养的最优培养条件: 补料时间为第4天, 起始接种密度为4×106 cells/mL, 稀释率为0.15/d。在连续培养下, 光照为35 μmol/(m2·s)时, 以1﹕1的起始比例接种产毒与无毒微囊藻, 二者间的竞争会达到平衡, 并以无毒微囊藻占据优势, 且两者以不同的优势度长时间维持不变。在此基础上, 开展了不同光强对产毒与无毒微囊藻竞争影响的实验, 结果表明, 光强为35和80 μmol/(m2·s)时, 无毒株在连续培养中占据优势; 而光强为5和15 μmol/(m2·s)时, 无毒和产毒微囊藻维持起始接种比例不变。研究通过优化连续培养条件为室内藻类竞争实验提供了更为适宜的培养模式。     

Nutritional needs of adult Drosophila: influence of lecithins and of choline chloride on the fecundity, the fertility and the size of ova]

The lecithin or choline chloride deficiency, in Drosophila, results in an important decrease of the number of eggs produced, hatching and egg size. However, the ovogenesis is not completely stopped, even after 15 days. This effects seem caused by the choline deficiency. When used under the chloride form, choline has no toxic effect even at a rate as high as 3 g/litre. However, the lecithin has toxic effects for amounts up to 4 g/litre. In the adults, the optimal concentration could be about 2 g/litre for the lecithin and 300 mg/litre for the cholin chloride.     

Epidural Analgesia in Dogs with Special Reference to the Intraarterial Blood Pressure

In order to investigate whether epidural analgesia in dogs employing mepivacaine chloride gives rise to complications, mainly changes in blood pressure, such analgesia was carried out in 35 dogs brought to the clinic for orthopedic, abdominal or perineal operations. Intra-arterial blood pressure measurements were made in 16 dogs. Three different solutions of mepivacaine chloride were used: 1 % solution with adrenaline 1:200,000; 2% solution with adrenaline 1:100,000 and 2% solution with adrenaline 1:200,000. The various solutions showed no significant difference in analgesic effect. Nor was any blood pressure change found during the epidural blockade. As a solution with a higher concentration causes a higher blood level of the analgesic and also greater toxic effects it is recommended that first preference be given to use of a 1 % solution with adrenaline 1:200,000; second choice is a 2 % solution with adrenaline 1:200,000 and third choice the 2 % solution with adrenaline 1:100,000.     

INHIBITION OF CHOLINE ACETYLTRANSFERASE AND ITS HISTOCHEMICAL LOCALIZATION

Abstract— A method for the histochemical identification of choline acetyltransferase has been investigated further by studying the effects of certain inhibitors of the enzyme both on rat brain homogenates and on the localization of the enzyme in tissue sections.
It was confirmed that acetyl-CoA hydrolase activity both in homogenates and in tissue sections is inhibited by preincubation in 1 mM-DFP. The effects of the choline acetyltransferase inhibitors chloro- and bromoacetylcholine on the appearance of histochemical staining were related to their activity in homogenates and tissue slices. Bromoketone was found to inhibit choline acetyltransferase in homogenates and, less efficiently, in tissue sections but it also inhibited the hydrolysis of acetyl-CoA by some other unknown enzyme which is inactivated by 1 mM-DFP.
The results obtained with the choline acetyltransferase inhibitors provide support for the specificity of the histochemical method.     

Effect of lithium preparations on the toxic effects of adrenaline

Experiments on rats have shown that intravenous injection of adrenaline in a dose of 0.3-0.4 mg/kg causes cardiac arrhythmia. In this case the primary arrhythmia developing immediately after adrenaline injection is followed by the recovery of sinusal rhythm which was replaced by the secondary arrhythmia. Apart from arrhythmias, there developed pulmonary edema. The animals died 2--3 minutes after adrenaline injections. Lithium chloride and lithium hydroxybutyrate removed the secondary arrhythmia and pulmonary edema. Lithium hydroxybutyrate has proved to be more effective.     

Toxicity and extrachromosomal DNA in strains of the cyanobacterium Microcystis aeruginosa

Abstract Correlations were sought between toxicity and the presence of plasmids in toxic and non-toxic strains of Microcystis aeruginosa . Plasmids were present in toxic and non-toxic cultures. Cultivation of the toxic Microcystis PCC7820 in the presence of novobiocin did not influence toxicity, although extrachromosomal DNA was no longer detected after novobiocin treatment. The data indicate that it is unlikely that plasmids are involved in the toxicity of Microcystis PCC7820.     

Choline-based deep eutectic solvents for enzymatic preparation of biodiesel from soybean oil

In this study, we introduced choline-based deep eutectic solvents (such as choline chloride/glycerol at 1:2 molar ratio) as inexpensive, non-toxic, biodegradable and lipase-compatible solvents for the enzymatic preparation of biodiesel from soybean oil. Through the evaluation of different eutectic solvents and different lipases, as well as the study of reaction parameters (i.e. methanol concentration, Novozym 435 loading and reaction time), we were able to achieve up to 88% triglyceride conversions in 24 h. The enzyme could be reused for at least four times without losing much activity. Our results indicate that new benign eutectic solvents can be used as substitutes of toxic and volatile organic solvents in the enzymatic production of biodiesel from real triglycerides (such as soybean oil).     

Salvia lavandulaefolia essential oil inhibits cholinesterase in vivo.

The essential oil of Salvia lavandulaefolia at two dosage levels was administered orally to rats for five days. Choline esterase activity was measured post mortem in three areas of the brain, both in the absence and presence of TEPP, a specific butylcholine esterase inhibitor, and was found to be significantly reduced in the striatum with both doses and also in the hippocampus at the higher dose. The activity of the enzyme in the cortex was not significantly reduced even at the higher dose. Thus it appears that S. lavandulaefolia oil, shown to inhibit choline esterase in vitro, also has an in vivo effect and this may help explain its traditional use for ailing memory.     

EFFECTS OF EXCITATION AND ANAESTHESIA ON THE GLUTAMINE CONTENT OF THE RAT BRAIN WITH A REFERENCE TO THE ADMINISTRATION OF GLUTAMINE

Abstract— A study of the factors affecting the ghtamine content in brain of rats showed that it was significantly decreased by intraperitoneal injection of physiological saline and by the stimulus of decapitation. It fell markedly at the stage of delirium (excitement) of pentobarbitone sodium anaesthesia and returned to the original value in light and deep surgical anaesthesia; after pentylenetetrazol injection the glutamine content showed a tendency to decrease but this change was only significant in relation to its post-saline level when the convulsant and 0.9 % NaCl were given to lightly-anaesthetized animals.
The i.p. administration of glutamine temporarily abolished the decrease in its concentration in the brain caused by injection and decapitation but never raised it above the original level. Many rats previously treated with glutamine showed no signs of excitement during the induction period of pentobarbitone sodium anaesthesia, and those which were excited showed a comparatively smaller decrease of brain glutamine content. whereas in the anaesthetic state there was no change in the content of brain glutamine after glutamine had been administered. Pentylenetetrazol given at the dose level of 200 mg/kg, but not at that of 100 mg/kg, resulted in the uptake of the added glutamine by the brain up to its normal concentration. Hence, the added glutamine did not readily enter normal or anaesthetized brain, but did penetrate the hyperactive brain where its level had fallen.
The brain levels of free glutamic and aspartic acids were not affected, in general, by procedures which increased cerebral activity. Glutamate was decreased by deep anaesthesia, whereas aspartate was not significantly altered in any of the conditions which were tested.
The significance of these results in relation to cerebral ammonia metabolism and possible changes of the permeability of the tissue to glutamine in different functional states is discussed.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.     

THE REACTION OF CHOLINE AND 3,3-DIMETHYL-1-BUTANOL WITH THE ACETYLENZYME FROM ACETYLCHOLINESTERASE

—A comparison was made of the manner in which choline chloride and 3,3-dimethyl-1-butanol react with the acetylenzyme formed during the hydrolysis of esters of acetic acid by acetylcholinesterase. Acetylcholine and acetylthiocholine were the substrates. The ratio of the formation of alkyl acetate to that of acetic acid increased linearly with the concentration of dimethylbutanol, but approached a limiting value as the concentration of choline was increased. Total enzymic activity was inhibited by choline and activated slightly by dimethylbutanol. The effects of varying ionic strength, pH and substrate concentration were examined. The effects of tetraethyl- and tetramethylammonium ions on the reaction of dimethylbutanol with the acetylenzyme were also studied. The results suggest that dimethylbutanol and choline bind to different regions of the active site. A mechanism for the reaction of choline and substrate with acetylcholinesterase is suggested.