R.H. Dadd

Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes

Abstract Digestive enzyme pH optima were determined in vitro for the larvae of three mosquito spp., Culex pipiens, Aedes aegypti, and Anopheles stephensi. All had optimal amylase activity at about pH 8 and optimal protease activity over a broad range between pH 10 and 12. pH within the digestive tract of intact live larvae was determined from the colours of indicator dyes ingested with kaolin and visible in the gut through the transparent or translucent body tissues. In all three spp. the contents of the anterior midgut were held at a pH just exceeding 10, with an abrupt fall to about pH 7·5 in the posterior third midgut.

Dietary amino acid requirements of the aphid, Myzus persicae

Abstract Growth experiments with synthetic diets lacking single amino acids established that three, methionine, histidine, and isoleucine, are essential for Myzus persicae . Growth ceased in one generation on omission of methionine. Two generations on deficient diet were necessary to demonstrate the essentiality of histidine and isoleucine. Omission of cysteine halted growth after one generation, but this could be offset by inorganic sulphate or extra methionine, showing that cysteine was needed only as a source of sulphur. No other amino acids were individually required for growth over a test period of two generations, although omission of lysine caused a slowing in growth in the first generation. All twenty amino acids were individually tolerated over a wide concentration range without adverse effect. Phenolic acids appear unnecessary, as growth was unimpaired by the omission together of tyrosine and phenylalanine. Omission of aspartic acid, glutamic acid, asparagine, and glutamine, as a group, largely prevented growth, even though their omission, individually, had been without effect. This group requirement was partially spared by any of the four substances, alone or in various combinations, or by equivalent amounts of alanine or serine, but not by glycine. Very little growth occurred with a mixture of the ten amino acids generally found essential for animals plus cysteine, even when these were at an overall concentration equivalent to that of the routine mixture of twenty amino acids. At double this concentration, the eleven amino acids mixture was toxic. However, the addition of large amounts of glutamate, alanine, or, best of all, serine, allowed growth approaching that of the complete mixture of twenty amino acids. It was confirmed that 3–4 per cent of the routine amino acid mixture was optimal, and the indications were that if methionine and sulphate were kept at a constant high level, growth would then be optimal with as little as 2 per cent of total amino acids. M. persicae is remarkable for the small number of amino acids that are strictly essential to its diet, and for its ability to grow well with dietary amino acid mixtures of limited or widely variable composition. It is suggested that this extraordinary versatility is a function of its possession of profuse intracellular symbiotes.

Studies on the artificial feeding of the aphid Myzus persicae (Sulzer)—IV. Requirements for water-soluble vitamins and ascorbic acid

Abstract By means of growth experiments using synthetic diets from which individual vitamins were omitted, Myzus persicae was shown to require a dietary source of ascorbic acid and nine water-soluble vitamins: thiamin, riboflavin, nicotinic acid, pyridoxin, folic acid, calcium pantothenate, meso-inositol, choline, and biotin. In all cases the plant-reared mothers of experimental larvae were themselves maintained on the deficient diets for a few days preceding birth of experimental larvae, in order to deplete larvae of maternally derived stores of the particular vitamin being tested. Under these circumstances, deprivation of thiamin, nicotinic acid, and calcium pantothenate greatly reduced first-generation larval growth and prevented the development of mature adults. Individual omission of the other vitamins markedly reduced first-generation growth but did not prevent development to the adult stage. However, in experiments continued through the second generation, little growth occurred in the absence of any of the vitamins. Since anomalies which occurred in the dosage response to ascorbic acid suggested that this easily oxidized nutrient might be deteriorating in diets, a study was made of ascorbic acid loss from diet kept under various conditions approximating to those involved in experimental use and storage. Loss was rapid at experimental temperatures, less so under refrigeration at +5°C, and largely prevented by continuous frozen storage at −20°C. Considerable losses occurred if frozen storage was interrupted by thawing to room temperature for as little as an hour per day. These findings are discussed in relation to the importance of stringent care in the pre-experimental storage of diets that may contain labile nutrients.

Studies on the artificial feeding of the aphid Myzus persicae (Sulzer)—I. Relative uptake of water and sucrose solutions

Abstract A method is described for feeding the green peach aphid Myzus persicae (Sulzer) on liquids accessible via artificial membranes of stretched Parafilm ‘M’ (R) . The aphids ingest considerable amounts of water and sugary fluids entirely by their own sucking efforts. A simple colour scoring method is described which is convenient for routinely assessing the relative uptake by the aphids of various diets. The numbers and kinds of salivations produced by the aphids when penetrating and feeding through membranes are related to the amount of liquid imbibed. The numbers of larvae born to adult apterous aphids are dependent on both the amount of fluid imbibed and its composition. Probing and associated feeding behaviour under the artificial feeding conditions is discussed in relation to the normal feeding behaviour of aphids on their host plants. On the basis of the different criteria of uptake considered in this study, sucrose is distinctly phagostimulatory to M. persicae , 10 to 20 per cent being the optimal concentration range.

Studies on the artificial feeding of the aphid Myzus persicae (Sulzer)—III. Some major nutritional requirements

The development of synthetic diets on which both apteriform and alatiform larvae of Myzus persicae (Sulzer) could be reared to the adult stage is discussed. By the deletion or modification in concentration of particular dietary components, their effects on larval growth and on adult survival and larviposition were determined. Sugar was essential for survival, and sucrose concentrations between 10 and 20% were optimal for both larvae and adults. Larval growth was good with concentrations between 212 and 40% of sugar, but with adults, survival and larviposition deteriorated markedly with concentrations below 10%. No larval growth occurred without amino acids in the diet; with less than 2 per cent of amino acids growth rates fell sharply, the optimal level being about 3 per cent of the diet. The longevity of adults was unaffected by the absence of amino acids, but high rates of larviposition depended upon their presence; a maximal effect on larviposition was achieved with 0·5 per cent of amino acids in the diet. No larval growth occurred if either of the two salts in the diet were omitted, owing to stringent requirements for potassium, magnesium, and phosphorus; adult survival and larviposition were adversely affected if any of these were omitted. A requirement for water-soluble vitamins could be demonstrated only in larvae from parents which had themselves been maintained on the vitamin-deficient regime before bearing the larvae. Under these circumstances, the deleterious effect of vitamin deprivation on the larvae was shown to depend upon the duration of prior maternal deprivation. Apterous adults reared from birth on synthetic diet were able to deposit a small number of live larvae, but these were unable to grow and develop properly although many lived for considerable periods. Adults reared from birth on diet and then transferred to plants deposited larvae which grew and developed normally. From this it is argued that a comparatively simple deficiency condition is involved in the failure of diet-reared aphids to reproduce. The foregoing nutritional information is discussed in relation to the circumstance that the same major dietarily essential components also play an important role in phagostimulation, and the bearing of this on ideas about the relationship of aphids to their natural host plants is considered.

Essential fatty acid for the mosquito Culex pipiens: arachidonic acid.

A nutrient associated with animal-derived phospholipids has previously been found essential for newly-emerged adults of the mosquito Culex pipiens to fly and survive more than a few days. Pure arachidonic acid was completely effective in supporting the emergence of viable flying adults; in combination with synthetic dipalmitoyl lecithin, which slightly improves larval growth rate without inducing adult flight, it wholly adequately replaces animal phospholipids. Linoleic and linolenic acids, which have satisfied the needs of all insects hitherto shown to require an essential fatty acid, were ineffective for C. pipiens, with or without synthetic lecithin. An optimal effect on adult flight was obtained with 0.05 mg of arachidonic acid per 100 ml of dietary medium, a concentration much lower than the linoleic/linolenic concentrations needed by other insects with an essential fatty acid requirement. The relationship of this unique mosquito fatty acid requirement to the essential fatty acid needs of both vertebrates and insects in general is discussed.

Improvement of synthetic diet for the aphid Myzus persicae using plant juices, nucleic acids, or trace metals

Growth on synthetic diet was markedly, though very variably, improved by the incorporation of various boiled and filtered juices pressed from mustard or lettuce. Optimal effects were obtained when mustard and lettuce juices constituted 2 and 10 per cent, respectively, of the complete diet. At higher concentrations growth was adversely affected, and radish juice at 5 per cent of the diet completely inhibited growth and caused high mortality. Greater and more consistent enhancement of growth was brought about by incorporating crude yeast or sperm nucleic acids at optimal levels of 400 to 600 mg per 100 ml of diet. Since a mixture of nucleotides (adenylic, guanylic, uridylic, and cytidylic acids) failed to substitute for crude nucleic acids, whereas a mixture of iron, zinc, manganese, and copper sequestrenes was a completely adequate replacement, it was concluded that the improvements in growth brought about by both plant juices and nucleic acids resulted from the presence in them of traces of these metals. Requirements for iron, zinc, and manganese were demonstrated individually. Deficiencies of iron or zinc become apparent in the first generation of larval growth, and of manganese, in the second generation. An apparent requirement for copper was demonstrated in one experiment carried through three generations. With the addition of these four metals, synthetic diet is apparently able to support Myzus persicae indefinitely. A culture is now, after 10 months, in its twentieth successive generation, with a stable generation period of about 2 weeks and with adults averaging about 350 μg in weight for the second and subsequent generations.

Arachidonic and other tissue fatty acids of Culex pipiens reared with various concentrations of dietary arachidonic acid

Abstract Chloroform/methanol extracts were prepared from groups of Culex pipiens reared in synthetic dietary media provided with various concentrations of arachidonic acid. Extracts were analyzed by gas-liquid chromatography to determine the fatty acid composition of whole extracts and also of phospholipid and triacylglycerol fractions separated by thin-layer chromatography from the whole extracts. The same extracts were also tested for their ability to support flight of adult C. pipiens reared in basal synthetic diet containing various concentrations of the extracts: this provided a bioassay for the presence of arachidonic acid or related polyunsaturates in the extracted lipid, since adults can fly only if provided, as larvae, with dietary arachidonic or related fatty acids. For comparison, chromatographic and bioassay data obtained from normal stock mosquitoes, reared in crude septic medium, are also presented. All extracts were shown by gas-liquid chromatography to contain some arachidonic acid and other polyunsaturated fatty acids. The proportions of arachidonic acid in extracts from mosquitoes reared in synthetic media were greater the greater the concentration of dietary arachidonic acid provided; and in the bioassay, extracts induced more flight activity in test mosquitoes the higher the dietary arachidonic acid provided for extracted mosquitoes. Extracts from stock-reared mosquitoes were more active in the bioassay than synthetic dietreared extracts, even though gas-liquid chromatography indicated lower proportions of arachidonic acid in stock-reared extract. However, stock-reared extract contained a substantial proportion of gammalinolenic acid, which is flight active for C. pipiens , as well as more linolenic acid and a large amount of linoleic acid, both of which are semi-active for flight; thus, stock-reared extract contained a higher overall proportion of flight-inducing fatty acids. Proportions of polyunsaturates in the phospholipid fractions of extracts from synthetic diet-reared mosquitoes were much greater than in the unfractionated extracts, whereas polyunsaturates were virtually absent from the triacylglycerol fractions, indicating a sequestering of polyunsaturates into phospholipids.

Polyunsaturated fatty acids of mosquitos reared with single dietary polyunsaturates

Abstract Stock adults of Culex pipiens and tarsalis reared in crude media had a third of their phospholipid fatty acids as polyunsaturates, mainly 18C but including prominent proportions of arachidonic (20:4n6) and eicosapentaenoic (20:5n3) acids. Adults reared with synthetic media devoid of polyunsaturated fatty acids and therefore unable to fly at emergence contained no more than trace amounts of any polyunsaturate. With synthetic media containing single polyunsaturates the following findings emerged. Of four polyunsaturates known to be highly effective essential fatty acids individually 20:4n6 or 20:5n3 appeared unchanged in tissue phospholipids in proportions reflecting dietary concentrations; dietary 22:4n6 or 22:6n3 (docosahexaenoic acid) appeared also as 20:4n6 or 20:5n3, respectively, retroconverted from the administered dietary fatty acids, which were detected only in traces. Two moderately effective dietary fatty acids, 18:3n6 (γ-linolenic) and 20:3n6 (homo-γ-linolenic), which support weak flight at emergence, appeared in tissue phospholipids respectively as 18:3n6 only, or as similar proportions of 18:3n6 and 20:3n6, this latter indicating shortening to the 18C analogue as well as accumulation of the dietary 20C acid. Six other polyunsaturates [18:2n6 (linoleic), 18:3n3 (linolenic) and their 20C and 22C analogues], all considered slightly effective as essential fatty acids although unable to support proper flight, appeared in tissue phospholipid in dose-related proportions as the 18C basal n6 or n3 family analogues, with only traces of the higher analogues when these were the dietary fatty acids provided, indicating sequential chain shortening within each series, n6 or n3, no interconversion of n6 and n3 members (also shown by all other data), and efficient accumulation of the resultant 18C polyunsaturates. These findings show no capability for de novo synthesis of polyunsaturated fatty acids, afford an insight into the metabolic interrelations of diet-derived polyunsaturates and indicate a primary importance for endogenous arachidonic and eicosapentaenoic acids in mosquito essential fatty acid physiology.

Studies on the artificial feeding of the aphid Myzus persicae (Sulzer)—II. Relative survival, development, and larviposition on different diets

Abstract A study was made of the survival and larviposition of adult apterous Myzus persicae given access to various fluids via a membrane of stretched Parafilm “M” (R) . Aphids fed only on water survived somewhat longer and gave birth to more larvae than starved individuals. The ingestion of sucrose solutions of various concentrations extended survival further and resulted in the birth of a greater number of larvae. Survival and larviposition was greatest on a complex synthetic diet described in detail. Neutral red in the feeding fluids had an adverse effect on survival and larviposition. The diet enabled 50 per cent of instar I larvae to develop into apteriform and alatiform instar IV larvae and 10 per cent to become apterous adults, whereby they increased their original weight fourfold.