Food composition for hemophagous insects   

Abstract: A composition suitable for feeding hemophagous insects includes peptides, salt, and a CO2 generator.

SUMMARY

In one aspect, a food composition includes a protein mixture including albumin and whey protein in a ratio between about 3:1 and about 12:1 dry weight, respectively, sodium chloride, and a CO2 generator. The CO2 generator may include sodium bicarbonate or an edible acid (e.g., citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid). The food composition may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant, or a colorant. The food composition may be water-soluble, and may be dissolved in a liquid (e.g., water or blood plasma) or a gel, which may include a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant. Protein may be present in the liquid in a concentration between about 40 g/L and 100 g/L, and the liquid may be equivalently isotonic, substantially isotonic, or isotonic.

In another aspect, a food composition includes a peptide component, sodium chloride, and a CO2 generator. The peptide component includes amino acids in dry-weight proportions of about 6.4%-about 6. 5% isoleucine, about 8.6%-about 9.0% leucine, about 6.2%-about 6.8% lysine, about 5.8%-about 6.4% phenylalanine, about 3.9%-about 4.5% threonine, about 1.1%-about 1.3% tryptophan, and about 6.9%-about 7.2% valine. The food composition is characterized in that upon dilution to an isotonic solution, the peptide component is present in a concentration of about 40 g/L to about 100 g/L. The peptide component may further include about 4.5%-about 5.0% arginine, about 1.5%-about 1.7% cysteine, about 2.2% histidine, or about 4.3%-about 4.8% methionine. The peptime component may consist essentially of individual amino acids, or may include oligopeptides or polypeptides. The peptide component may include albumin or whey protein. The CO2 generator may include sodium bicarbonate or an edible acid (e.g., citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid). The food composition may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant, or a colorant. The food composition may be water-soluble, and may be dissolved in a liquid (e.g., water or blood plasma) or a gel, which may include a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant. Protein may be present in the liquid in a concentration between about 40 g/L and 100 g/L, and the liquid may be equivalently isotonic, substantially isotonic, or isotonic.

In another aspect, a food composition includes a peptide component, sodium chloride, and a CO2 generator. The peptide component includes amino acids isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, and valine in a relative proportion substantially equal to the proportions the same amino acids would have in a mixture of albumin and whey protein at a ratio between about 3:1 and about 12:1 dry weight. The food composition is characterized in that, upon mixing with water to form an isotonic solution, the resulting solution includes about 40 to about 100 grams of peptide component per ml. The peptide component may be composed principally of amino acids, or may include oligopeptides or polypeptides. The peptide component may include albumin or whey protein. The CO2 generator may include sodium bicarbonate or an edible acid (e.g., citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid). The food composition may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant, or a colorant. The food composition may be water-soluble, and may be dissolved in a liquid (e.g., water or blood plasma) or a gel, which may include a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant. Protein may be present in the liquid in a concentration between about 40 g/L and 100 g/L, and the liquid may be equivalently isotonic, substantially isotonic, or isotonic.

In another aspect, a food composition includes a first portion including an edible acid (e.g., citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid), a second portion separate from the first portion containing sodium bicarbonate. At least one of the first and second portions includes a peptide component including amino acids isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, and valine, these amino acids having relative proportions substantially equal to the proportions of a mixture of albumin and whey protein at a ratio between about 3:1 and about 12:1 dry weight. The peptide component may be present in an amount sufficient that upon mixing the first portion, the second portion, and optionally water to form a mixture equivalently isotonic with blood (or substantially isotonic with blood, or isotonic with blood), the peptide component is present in a concentration of about 40-100 g/L. The food composition may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, an insect phagostimulant, or a colorant.

In another aspect, a method of feeding an insect includes providing a solution including a mixture of albumin and whey protein in a ratio between about 3:1 and about 12:1, respectively, sodium chloride, water, and a source of carbon dioxide, and placing the solution in a container including a barrier surface, wherein the barrier surface is configured to be penetrated by the insect to feed upon the solution. The method may further include substantially maintaining the solution at a selected temperature, or inducing convection (e.g., free or forced convection) in the solution; The method may further include exposing the solution to an insect such as a mosquito (e.g., of genus Anopheles, Aedes, or Culex). At least one of the barrier surface, the aqueous solution, and the container may include an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, or an insect phagostimulant. The source of carbon dioxide may be a chemical reaction (e.g., a reaction between sodium bicarbonate and an edible acid such as citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid). The solution may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, or a colorant. The concentration of protein in the solution may be from about 40 g/L to about 100 g/L. The solution may be equivalently isotonic, substantially isotonic, or isotonic.

In another aspect, a method of feeding an insect includes providing a solution including a peptide component, sodium chloride, water, and a source of carbon dioxide, and placing the solution in a container including a barrier surface, wherein the barrier surface is configured to be penetrated by the insect to feed upon the solution. The peptide component includes amino acids isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, and valine, these amino acids having relative proportions substantially equal to the proportions the same amino acids would have in a mixture of albumin and whey protein at a ratio between about 3:1 and about 12:1 dry weight. The peptide component may be composed principally of amino acids, or may include oligopeptides or polypeptides. The peptide component may include albumin or whey protein. The method may further include substantially maintaining the solution at a selected temperature, or inducing convection (e.g., free or forced convection) in the solution. The method may further include exposing the solution to an insect such as a mosquito (e.g., of genus Anopheles, Aedes, or Culex). At least one of the barrier surface, the aqueous solution, and the container may include an insect attractant, an insect repellent, a pheromone, a kairomone, an allomone, or an insect phagostimulant. The source of carbon dioxide may be a chemical reaction (e.g., a reaction between sodium bicarbonate and an edible acid such as citric acid, acetic acid, ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid). The solution may further include a sugar (e.g., glucose), a blood component (e.g., plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate, glucose, or cholesterol), which may be at a concentration approximately equal to a physiological level for human blood, a preservative, a stabilizer, or a colorant. The concentration of the peptide component in the solution may be from about 40 g/L to about 100 g/L. The solution may be equivalently isotonic, substantially isotonic, or isotonic.

The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

DETAILED DESCRIPTION

As used herein unless context dictates otherwise, the terms “feeding medium” and “food composition” include compositions suitable for feeding hemophagous insects. As used herein unless context dictates otherwise, the term “artificial diet” includes food compositions other than whole blood of a person or animal. Artificial diets may include blood components, such as but without limitation plasma, hemoglobin, or red blood cells. Food compositions, feeding media, or artificial diets may, but need not, support egg-laying in hemophagous insects.

When raising hemophagous insects (e.g., for research purposes), it may be inconvenient or expensive to provide blood meals. In some cases, insects may be kept on artificial diets which may be more readily stored and handled in a laboratory. In some cases, an artificial diet (e.g., glucose water or water-soaked raisins) has been found to be adequate for maintaining health of hemophagous insects (e.g., mosquitoes), but it has not been adequate in order to stimulate egg-laying.

While the following discussion is in the context of feeding of mosquitoes, the formulations described herein, as well as the methods of determining appropriate formulations, are expected to be applicable to other hemophagous insects as well (e.g., tsetse flies, lice, bed bugs, no-see-urns, fleas, sand flies, midges, snipe flies, horse flies, stableflies, or sheep flies).

Uchida describes a set of seven “absolutely essential” amino acids for stimulating oögenesis in Culex pipiens pallens mosquitoes (isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, and valine), and identifies four more that “may be essential” or “are required for a full level of initiation and promotion of egg development” (arginine, cysteine, histidine, and methionine). See, Uchida, “Balanced. Amino Acid Composition Essential for Infusion-induced Egg Development in the Mosquito (Culex pipiens pallens),” J. Insect Physiol., 39(7):615-621 (1993). See also, Uchida, et al., “Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids,” J. Insect Physiol., 44:525-528 (1998) (stimulating oögenesis in decapitated females with a combination of 20-hydroxyecdysone hormone and a mixture of 17 amino acids). Chang, et al. found that supplementation with additional isoleucine enhanced egg production rate in Aedes aegypti mosquitoes fed with human red blood cells. See, Chang et al., “Amino Acid Composition of Human and Guinea Pig Blood Proteins, and Ovarian Proteins of the Yellow Fever Mosquito Aedes aegypti; and Their Effects on the Mosquito Egg Production,” Comp. Biochem. Physiol., 62A:753-755 (1979). See also, Greenberg, “Some Nutritional Requirements of Adult Mosquitoes (Aedes aegypti) for Oviposition,” J. Nutr. 43(1):27-35 (1951) (isoleucine supplementation enhanced egg-laying after blood feeding). Arsic, et al., found that albumin-containing substances would support follicular development in Anopheles gambiae mosquitoes, but did not support complete egg development without addition of red blood cells in this species. Griffith, et at, were able to support a population of Culex quinquefasciatus mosquitoes for more than fifty generations on a blood substitute artificial diet including ovalbumin, soya infant formula, globulins, and adenosine triphosphate. See, “Culturing Culex quinquefasciatus mosquitoes with a blood substitute diet for the females,” Med. and Vet. Entomology 10:265-268 (1996). Each of the above-mentioned publications is incorporated by reference herein to the extent not inconsistent herewith.

A population of Anopheles mosquitoes may be maintained through an indefinite number of generations on our artificial feeding medium without any blood feeds. Each of the experiments described below used a combination of ovalbumin (Sigma, Grade II, A5253, CAS 9006-59-1, containing 75%±7% protein plus carbohydrate and phosphate portions) and whey protein (SIMPLESSE™ from CP Kelco, a microparticulated whey protein concentrate containing 53% bovine whey protein plus food-grade xantham gum). In some embodiments, the dry weight ratios of albumin to whey protein may be about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1 or about 12:1, respectively. In addition, dry weight ratios in various ranges between any of these values is contemplated. For example, and without limitation, ratios of about 2.25:1, about 3.25:1, about 4.25:1, about 5.5:1, about 6.5:1, about 7.5:1, about 8.5:1, about 9:5:1, about 10.5:1, and about 11.5:1. For example, and without limitation, dry weight ratios of albumin to whey protein may be about 3:1 to about 6:1, respectively; or about 3:1 to about 12:1, respectively. In addition to protein, in some embodiments a food composition may contain other macronutrients such as carbohydrates or lipids, or micronutrients such as vitamins or minerals.

Food compositions that are substantially isotonic with blood (reference osmolality of 275-300 mOsm/kg) can help avoid insect death by osmotic imbalance after feeding. Isotonicity can be achieved, for example, by either adding Ringer\'s salts or the formula of Griffith, et al. (cited above). As used herein, an “isotonic” solution is one having a tonicity within ±5% of the tonicity of blood (e.g., 5% less than bottom of reference range, 4% less, 3% less, 2% less, 1% less, within reference range, 1% more than top of reference range, 2% more, 3% more, 4% more, or 5% more), while a “substantially isotonic” solution is one having an tonicity within ±10% of the tonicity of blood (e.g., 10% less than bottom of reference range, 9% less, 8% less, 7% less, 6% less, 6% more than top of reference range, 7% more, 8% more, 9% more, or 10% more). An “equivalently isotonic” solution has an tonicity within ±20% of the tonicity of blood (e.g., 20% less than bottom of reference range, 19% less, 18% less, 17% less, 16% less, 15% less, 14% less, 13% less, 12% less, 11% less, 11% more than top of reference range, 12% more, 13% more, 14% more, 15% more, 16% more, 17% more, 18% more, 19% more, or 20% more).

In addition to the protein mixtures discussed above, attractants can be added to the food composition to increase mosquito feeding rates. In some embodiments, an attractant is CO2. CO2 can be produced within the food composition either by mixing proteins and salts with carbonated “sparkling” water, or by using a chemical CO2 generator, for example, citric acid (or another acid edible by hemophagous insects, such as ascorbic acid, malic acid, lactic acid, tartaric acid, phosphoric acid, oxalic acid, benzoic acid, or butyric acid) mixed with sodium bicarbonate. Some embodiments may use other CO2 generators; for example, other chemical reactions that produce CO2, bubbling CO2 through or blowing it onto a feeding medium, or biological CO2 generators (e.g., yeast). In some embodiments, the feeding medium may be in two separate parts, which can be mixed together (e.g., at feeding time) to begin the CO2-generating reaction (e.g., a reaction between an edible acid and sodium bicarbonate). In some embodiments, other attractants may be included (e.g., lactic acid, pentyl vinyl carbinol, or isolaveric acid), or other insect-behavior-modifying ingredients may be used (e.g., repellents that discourage undesired insects, pheromones, allomones, kairomones, or phagostimulants). Insect-behavior-modifying ingredients may, for example, encourage or discourage feeding of a particular species, or of insects with certain conditions (e.g., gravid females), or might encourage or discourage crop feeding or midgut engorgement.

In addition to, or instead of, attractants and other insect-behavior-modifying components, a food composition may include other active ingredients that affect mosquito metabolism or physiology. In one embodiment, a food composition may be used to control mosquito population by including a toxin, parasite, microorganism, or biocontrol agent (e.g., Ascogregarine trophozoites, Bacillus thuringiensis strains, Bacillus sphaericus, Wolbachia, Microsporidia such as Nosema sp., Coelomomyces sp. fungi, Lagenidium, Saccharaopolyspora spinosa, Metarrhizium anisopliae, Edhazardia aedes, Beauveria bassiana spores, and mermithid nematodes). See, e.g., Reyes-Villanueva, et al., “Susceptibility of Aedes aegypti and Aedes albopictus larvae to Ascogregarina culicis and Ascogregarina taiwanensis” J. Inverteb. Path. 84:47-53 (2003); Saridaki, et al., “Wolbachia: more than just a bug in insects genitals,” Curr. Opinion in Microbiol. 13:67-72 (2010); Axtell, et al., “Encapsulation of the Mosquito Fungal Pathogen Lagendium giganteum (Oomyetes:Lagenidiales) in Calcium Alginate,” J. Am. Mosq. Control Assoc. 3(3):450-459 (1978); Lichtwardt, et al., “A new Coelomomyces pathogenic to mosquitoes in Costa Rica,” Rev. Biol. Trop. 41(3):407-410 (1993); Schenker, et al., “The effects of Nosema algerae on the development of Plasmodium yoelii nigeriensis in Anopheles stephensi,” Parasitol. Res. 78:56-59 (1992); Bukhari, et al., “Factors affecting fungus-induced larval mortality in Anopheles gambiae and Anopheles stephensi,” Malaria J. 9(22):1-15 (2010); Perez, et al., “Spinosad, a Naturally Derived Insecticide, for Control of Aedes aegypti (Diptera: Culicidae): Efficacy, Persistence, and Elicited Oviposition Response,” J. Med. Entomol. 44(4):631-638 (2007); Cetin, et al., “Evaluation of the naturally-derived insecticide spinosad against Culex pipiens L. (Diptera: Culicidae) larvae in septic tank water in Antalya, Turkey,” J. Vect. Ecol. 30(1):151-154 (2005); Becnel, et al., “Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae),” J. Invert. Pathol. 60(3):299-303 (1992); Petersen, “Role of mermithid nematodes in biological control of mosquitoes,” Exp. Parasitol. 33(2):239-247 (1973), each of which is incorporated by reference herein to the extent not inconsistent herewith.

In some embodiments, it may also be useful to deliver an agent to the insect. For example, for the study of malaria, Plasmodium parasites (e.g., P. falciparum gametocytes) can be added to the food composition. In some embodiments, the agent added can be a toxin, parasite, a beneficial agent, etc. In some embodiments, if toxins are added, the toxins may be sequestered (e.g., separated from the food composition or from immediate contact with the insect). In some such embodiments, these sequestered toxins may become unsequestered over a period of time, for example upon return to a breeding site, potentially killing immature insects (for example, to control a population). For example, toxins (or other agents) may include a dissolvable coating, or a cleavable blocking agent, so that they can be ingested and then excreted in a location where the coating will dissolve and release the toxin or other agent. In some embodiments, the medium may include enzymes, antibiotics, or antiseptics.

The feeding medium may also contain “inactive” ingredients that do not act directly upon the mosquito metabolism, (e.g., by providing nourishment or affecting a biological function of the mosquito). For example, the formulations described below made with SIMPLESSE™ include xantham gum. In some embodiments, this ingredient may modify the texture or rheological properties of the feeding medium. In some embodiments, other rheological agents may be added or substituted, while some embodiments may not include rheological agents. In some embodiments, the medium may include stabilizers, preservatives, colorants, pH modifiers, or other components that do not act directly upon the mosquito metabolism. In some embodiments, the medium may include blood components, such as plasma, hemoglobin, gamma globulin, red blood cells, adenosine triphosphate (ATP), glucose, or cholesterol. These ingredients may be added for their physical effects on the medium, or for their influence on insect behavior.

In some embodiments the feeding medium is a liquid having active or inactive ingredients dissolved therein. In some embodiments, one or more of the ingredients may be in suspension. In some embodiments, the feeding medium may be a gel, or may include a mixture of phases (e.g., a liquid-soaked sponge).

In some embodiments, rather than using whole albumin or whey proteins, these proteins may be fully or partially hydrolyzed. In some embodiments, peptide mixtures may further include proportions of any of the four additional amino acids that Uchida suggests “may be essential” or “are required for a full level of initiation and promotion of egg development.” These proportions have been calculated using the protein sequence of albumin (see, Nisbet, et al., “The Complete Amino-Acid Sequence of Hen Ovalbumin,” Eur. J. Biochem., 115:335-345 (1981), which is incorporated by reference herein) and an amino acid breakdown for whey protein isolate published by Davisco Foods (see, “Whey Protein Concentrate 80%,” Version 07F-0604, included herewith and incorporated by reference herein). (The amino acid breakdown of SIMPLESSE™ was not readily available, but is expected to be substantially similar to the Davisco product). Table 1 shows a breakdown by weight percent of amino acids of each of these proteins, as well as a calculated value for ratios of 4.25:1 and 8.5:1 of albumin to whey protein.

TABLE 1 Whey Albumin protein 3:1 4.25:1 8:5:1 12:1 wt % wt % albumin:whey albumin:whey albumin:whey albumin:whey Leucine 8.43% 10.8%  9.0% 8.9% 8.7% 8.6% Valine 7.29% 5.8% 6.9% 7.0% 7.1% 7.2% Isoleucine 6.59% 5.8% 6.4% 6.4% 6.5% 6.5% Phenylalanine 6.64% 3.3% 5.8% 6.0% 6.3% 6.4% Lysine 5.87% 9.6% 6.8% 6.6% 6.3% 6.2% Threonine 3.59% 7.2% 4.5% 4.3% 4.0% 3.9% Tryptophan 1.09% 1.8% 1.3% 1.2% 1.2% 1.1% Methionine 5.09% 1.9% 4.3% 4.5% 4.8% 4.8% Arginine 5.25% 2.1% 4.5% 4.6% 4.9% 5.0% Histidine 2.18% 2.2% 2.2% 2.2% 2.2% 2.2% Cysteine 1.46% 2.3% 1.7% 1.6% 1.5% 1.5% Serine 8.02% 4.7% 7.2% 7.4% 7.7% 7.8% Alanine 6.26% 4.9% 5.9% 6.0% 6.1% 6.2% Glutamic acid 9.76% 16.7%  11.5%  11.1%  10.5%  10.3%  Glycine 2.87% 1.8% 2.6% 2.7% 2.8% 2.8% Asparagine 4.55% unk unk unk unk unk Glutamine 4.43% unk unk unk unk unk Aspartic acid 3.74% 10.8%  5.5%

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