Patent Publication Number: US-11638417-B2

Title: Composition comprising arthropods and astigmatid mite eggs

Description:
RELATED APPLICATION DATA 
     This application is a continuation application which claims priority to U.S. patent application Ser. No. 14/410,192, filed on Dec. 22, 2014, which is a National Stage Application under 35 U.S.C. 371 of co-pending PCT application PCT/EP2013/063130 designating the United States and filed Jun. 16, 2013; which claims the benefit of FR application number 1255950 and filed Jun. 22, 2012 each of which are hereby incorporated by reference in their entireties. 
    
    
     The present invention relates to a biological control composition comprising at least arthropod biological control agents and astigmatid mite eggs. 
     The development of pesticides has greatly expanded agriculture by enabling treatment against a wide variety of pests as well as diseases, with a limited number of molecules. The effects of pesticide products depend primarily on their category: insecticides, acaricides, fungicides, herbicides, nematicides, molluscicides, rodenticides, mole killers. 
     The use of these products, however, contributes to disequilibrium of the biotope, notably by eliminating certain beneficials and/or by creating resistance phenomena, two factors that contribute to the development of crop-damaging pests and pathogenic agents. 
     The use of biological control agents constitutes one method for fighting against pests, pathogenic agents or adventitious plants by means of their natural antagonistic organisms, such as phytophagous species (for adventitious plants), parasitoids (for arthropods, etc.), predators (for nematodes, arthropods, vertebrates, mollusks, bats, etc.) or pathogenic agents (for viruses, bacteria, fungi, etc.). The use of these biological control agents also allows a reduction in pesticide use. 
     The use of arthropods, especially Phytoseiidae, as biological control agents has proven the effectiveness of such an approach. Thus, compositions are found on the market comprising populations of  Amblyseius cucumeris , a predator of thrips larvae, which is a phytophagous species (Thysanoptera), and bran, thus making it possible to more easily distribute the product in crops. These ingredients can, optionally, be provided in pre-pierced sachets, in order to release the predators in a controlled manner over time. Similarly, the use of  Neoseiulus californicus  as a predator of species of Tetranychidae and of Tarsonemidae, and the use of  Amblyseius swirskii  as a predator of thrips or of Aleyrodidae, is currently common. 
     With this approach, it is necessary to reintroduce the living biological control agent, in this case the arthropod, in sufficient number to achieve the required effect. As a result, the diet of the biological control agents, in particular the arthropods, is determining critical factor for rearing the biological control agent, or in the preparation of biological control compositions. 
     A large number of documents describe methods for the mass rearing of biological control agents. Traditionally, biological control agents are raised in the presence of a population of prey as food and then are released into crops. The prey populations used can be prey other than natural prey, for example prey that are easier to raise, if they can act as food for the biological control agents. For example, in the field of the mass rearing of arthropods as biological control agents, GB2393890 discloses that  Amblyseius cucumeris  can use  Tyrophagus putrescentiae, T. tropicus  and  Acarus siro  as substitution prey. 
     Thus, compositions comprising a population of arthropods, a population of prey, and, optionally, a substrate providing support and nutrients for the population of prey, have already been described. 
     Nomikou et al. have shown that populations of  Amblyseius swirskii  can grow while having a high predator/prey ratio, if they feed on  Tyrophagus putrescentiae  (Experimental and Applied Acarology 27(1-2), 57-68). 
     The patent application WO2006/057552 discloses that populations of  Amblyseius swirskii  can be raised using astigmatid mites, in particular belonging to the family Carpoglyphidae, Pyroglyphidae, Glyphagidae or Acaridae, as a food source. 
     This method poses a certain number of problems, however. 
     On the one hand, it is first necessary to obtain a population of prey, with all the difficulties related to the rearing thereof. In particular, this prey regularly requires food containing mainly grain and yeast, ingredients likely to allow mold to develop. 
     On the other hand, it is known that the equilibrium between prey and predator populations, which is critical for obtaining the desired number of predators depends on the initial predator/prey ratio. Although this equilibrium is dynamic, it necessarily involves phases of decrease in the predator population. Too high of a prey rate can have a deleterious effect on the evolution of the predators, then leading to a drastic decrease in the population of predators in the composition. 
     Moreover, in a number of cases, particularly when the preys are household dust mites, it has been noticed that some species can be phytotoxic. In addition, some of the mites often used to rear phytoseiids, in particular among astigmatid mites, are particularly allergenic for man. 
     Finally, the existing compositions comprising predators and prey, in particular comprising mites, have a shelf life that is often very short, and last at longest about 3 weeks in the field without deteriorating 
     There is thus still a need for improved nutritional sources that could be used for mass rearing of biological control agents, in particular arthropods. More specifically, there is a need for nutritional sources that have an improved shelf life and can therefore improve the overall shelf life of biological control agents compositions. 
     The inventors have discovered that the use of astigmatid mite eggs to feed populations of arthropods makes it possible to solve the problems of the prior art. 
     The inventors have more particularly discovered that the specific use of astigmatid mite eggs to feed populations of arthropods is more effective than the use of whole populations of astigmatid mites which further comprise larvae, nymphs and adult astigmatid mites. 
     In addition, the inventors have discovered that astigmatid mite eggs have a shelf life at room temperature that is longer than other types of arthropod food, about 4 to 6 weeks. Thus, compositions comprising astigmatid mite eggs have a longer shelf life in the field than compositions of the prior art, which comprise other types of foods. 
     Finally, the inventors have surprisingly discovered that astigmatid mite eggs can be treated in order to prevent them from hatching. The inventors have indeed found that eggs that are treated in a specific way enter a stage wherein they cannot further hatch yet retain a metabolic activity. This stage is herein called sub-lethal stage. Those eggs are particularly advantageous as they have an improved shelf life, and do not deteriorate as fast as eggs treated in different conditions. Moreover, those sub-lethal eggs will not develop in larva and other stages that are not as effective in feeding populations of arthropods. 
     A first object of the invention is the use of astigmatid mite eggs as a nutrient source for arthropod biological control agents. 
     A second object of the invention is a biological control composition comprising:
         at least one population of arthropod biological control agents,   a nutrient source comprising astigmatid mite eggs, and   optionally, a support and/or dissemination substrate.       

     Another object of the invention is a biological control composition comprising:
         at least one population of arthropod biological control agents, a nutrient source comprising at least one astigmatid mite population, and optionally, a support and/or dissemination substrate.       

     Further, the present invention has for object a method for rearing arthropod biological control agents from astigmatid mite eggs as a nutrient source. 
     Finally, the present invention has for object a method for rearing arthropod biological control agents from a nutrient source comprising at least one astigmatid mite population. 
     In the context of the invention, the expression “population of arthropod biological control agents” refers to any population comprising at least two living individuals belonging to at least one of the species of arthropods among the biological control agents. Said population can be comprised of individuals belonging to only one or to several developmental stages of the biological control agent. For example, the population can comprise eggs, larvae, nymphs, adult individuals or mixtures of these various stages. The arthropods used according to the invention are selected, for example, from the class Arachnida, preferentially from mites (order Acari), or from the class Insecta. 
     In an embodiment, the mites are selected from the orders Mesostigmata and Trombidiforms. 
     According to an embodiment, the mites can be selected, for example, from the families of phytoseiids (Phytoseiidae), Macrochelidae or Laelapidae. 
     Advantageously, the phytoseiids can be selected from the subfamily Amblyseiinae, for example  Amblyseius swirskii, Amblyseius largoensis, Amblyseius andersoni ; or from the genus  Neoseiulus , for example  Neoseiulus womersleyi, Neoseiulus californicus, Neoseiulus cucumeris, Neoseiulus fallacis, Neoseiulus longispinosus ; or from the genus  Iphiseius , for example  Iphiseius degenerans ; or from the genus  Amblydromalus , for example  Amblydromalus lailae, Amblydromalus limonicus  or  Amblydromalus manihoti ; or from the subfamily Typhlodrominae, in particular the genus  Typhlodromips , for example  Typhlodromips montdorensis ; or from the genus  Euseius , for example  Euseius ovalis, Euseius scutalis, Euseius finlandicus, Euseius gallicus, Euseius stipulatus, Euseius tularensis, Euseius hibisci.    
     Advantageously, the mites of the family Macrochelidae can be selected from  Macrocheles , for example  Macrocheles robustulus.    
     Advantageously, the mites of the family Laelapidae can be selected from  Hypoaspis , for example  Hypoaspis aculeifer, Hypoaspis miles  or  Hypoaspis scimitus.    
     Preferentially, the mites are selected from  Amblyseius swirskii, Neoseiulus cucumeris, Amblyseius largoensis, Neoseiulus californicus, Neoseiulus fallacis, Neoseiulus longispinosus, Amblydromalus lailae, Typhlodromips montdorensis, Euseius ovalis, Euseius scutalis, Euseius hibisci.    
     According to a particularly advantageous embodiment of the invention, the mites are the species  Amblyseius swirskii.    
     According to an embodiment, the arthropods can be selected from the class Insecta. The insects can be selected from the species of the order Hemiptera, for example family Miridae, genus  Nesidiocoris , for example  Nesidiocoris tenuis  or  Nesidiocoris  spp., or for example the family Anthocoridae, genus  Orius , for example  Orius laevigatus, Orius insidiosus, Orius  spp., or finally, for example, the family Lygaeidae, genus  Geocoris , for example  Geocoris  spp. 
     The insects can be selected from the species of the order Coleoptera, for example the family Coccinellidae, genus  Cryptolaemus , for example  Cryptolaemus montrouzieri  or  Cryptolaemus  spp. 
     The insects can be selected from the species of the order Neuroptera, for example family Chrysopidae, for example genus  Chrysoperla , for example  Chrysoperla  spp. 
     In the context of the present invention, the term “egg” refers to eggs that have been collected and not yet hatched. For example, the eggs can be used fresh or after having been preserved by any mode of preservation that allows maintaining all of the eggs&#39; nutritional properties. Preferentially, the eggs are preserved by any mode of preservation that allows maintaining the eggs in their development stage without altering the eggs&#39; ability to then develop. 
     Further, the eggs can be specifically treated in order to prevent them from hatching by inducing a sub-lethal stage. Thus, by “sub-lethal eggs” it is herein referred to eggs that do not hatch and retain metabolic activity. In order to verify that an egg does not hatch, the person skilled in the art may for example put the egg in normal culture conditions that are appropriate for the development of the egg, and verify that the egg does not develop into larvae, nymph and adult, over time. For example, the skilled person can put the egg at 25° C. and at 75% relative humidity, and verify that the egg does not develop into larvae, nymph and adult over time. 
     Metabolic activity can be evaluated by any technique known in the field. For example, it has been long known that metabolic activity of acari can be evaluated by measuring their respiratory rates, more precisely by measuring the dioxygen (O 2 ) uptake per individual (Szlendak et al.,  Exp Appl Acarol.  10(3-4):221-30, 1991; Block et al.,  J exp Biol  68:69-87, 1977; Block W,  Oikos  26:15-25, 1975). This approach has also been used for evaluating the metabolic activity of mite eggs (D. J. Thurling,  Oecologia,  46:3, 391-396, 1980). 
     Measuring the dioxygen (O 2 ) per egg can easily be performed using a gas analyzer or using a Cartesian Diver micro-respirometer. Such analysis is a routine practice for the person skilled in the art, and does not require to be further explained herein. The inventors have found that sub-lethal eggs can be obtained by exposing them to UV radiations, or to specific temperature conditions. Thus, in order to obtain sub-lethal eggs, the eggs can be exposed to a temperature between −100° C. and 9° C., advantageously between −80° C. and 5° C., even more advantageously between −30° C. and 5° C. 
     For example, the eggs after collection undergo a brief heat shock and are then stored at a temperature between about −90° C. and 5° C., advantageously between about −30° C. and 5° C., even more advantageously between about −5° C. and 5° C. Advantageously, the heat shock is carried out at a temperature between about −100° C. and 0° C., preferentially at a temperature between about −100° C. and −10° C. 
     In addition, the inventors have found that induction of the sub-lethal stage depends both on temperature and exposure time to said temperature. As a general rule, they observed that the lower the temperature is, the shorter the exposure time should be. Indeed, as the inventors have found, if the exposure time is too important, the treatment does not result in sub-lethal eggs, but in dead eggs that tend to collapse with time. 
     For example, one the one hand, when the astigmatid mite eggs are exposed to a temperature between −10° C. and 9° C., the time of exposure could vary between a few days and a few weeks. On the other hand, when the astigmatid mite eggs are exposed to lower temperatures, such as temperatures between −100° C. and −80° C., the time of exposure should be shortened from a few seconds to a few hours. 
     The inventors have discovered that, for a given temperature, the necessary and sufficient exposure time to obtain a maximum of sub-lethal eggs can be determined by a specific equation. They have determined specific combinations of temperature and exposure time that can be safely used in order to obtain at least 60% of sub-lethal eggs. 
     Preferably, the eggs of the invention are exposed to conditions comprised in the list consisting of a temperature comprised between 8° C. and 9° C. for between 7 to 10 weeks, a temperature comprised between 7° C. to −4° C. for between 4 to 6 weeks, a temperature comprised between −5° C. to −14° C. for between 1 to 3 weeks, a temperature comprised between −15° C. to −24° C. for between 2 to 5 days, a temperature comprised between −25° C. to −34° C. for between 12 to 36 hours, a temperature comprised between −35° C. to −54° C. for between 2 to 8 hours, a temperature comprised between −55° C. to −79° C. for between 5 to 60 minutes, a temperature comprised between −80° C. to −100° C. for between 10 seconds to 3 minutes. 
     Yet preferably, the eggs of the invention are exposed to a temperature comprised between 7° C. to −4° C. for between 4 to 6 weeks. Even more preferably, the eggs of the invention are exposed to a temperature comprised between 4° C. to −2° C. for between 4 to 6 weeks. Advantageously, the eggs of the invention are exposed to a temperature of 0° C. for between 4 to 6 weeks. 
     The inventors have further determined specific combinations of temperature and exposure time that can be safely used in order to obtain at least 80% of sub-lethal eggs. 
     Preferably, the eggs of the invention are exposed to conditions comprised in the list consisting of a temperature comprised between 8° C. and 9° C. for 9 weeks, a temperature comprised between 7° C. to −4° C. for 5 weeks, a temperature comprised between −5° C. to −14° C. for 2 weeks, a temperature comprised between −15° C. to −24° C. for between 4 days, a temperature comprised between −25° C. to −34° C. for between 24 hours, a temperature comprised between −35° C. to −54° C. for between 6 hours, a temperature comprised between −55° C. to −79° C. for between 30 minutes, a temperature comprised between −80° C. to −100° C. for between 30 seconds. 
     Yet preferably, the eggs of the invention are exposed to a temperature comprised between 7° C. to −4° C. for between 5 weeks. Even more preferably, the eggs of the invention are exposed to a temperature comprised between 4° C. to −2° C. for between 5 weeks. Advantageously, the eggs of the invention are exposed to a temperature of 0° C. for between 5 weeks. 
     According to the invention, the eggs can be whole or fractionated. The eggs can be treated so as to extract particular nutrients. 
     The eggs can be administered alone or mixed with at least one other substrate. The substrate is selected from substrates containing nutrients. For example, the substrate is selected from cereals, cereal flours, in particular wheat flour or corn flour, yeasts, bran, pollen, fruits, dried fruits, sugar, for example dextrose. 
     In the context of the invention, the term “nutrient source” refers to any embodiment wherein astigmatid mite eggs are used to feed the biological control agents. The nutrient source of the invention is the sole food or is one of the foods used to feed the biological control agents. Preferably, the nutrient source of the invention is the sole nutrient source of animal origin. According to a particularly preferred embodiment, the nutrient source of the invention is the sole food used to feed the biological control agents. 
     The nutrient source of the invention comprises at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% astigmatid mite eggs by weight in relation to the total weight of the nutrient source. Advantageously, the nutrient source of the invention comprises at least a population of astigmatid mites. According to the invention, the population of astigmatid mites consists in astigmatid mites eggs. Thus, the population of astigmatid mites does not contain larvae, nymphs or adult astigmatid mites 
     Preferentially, the nutrient source of the invention consists in astigmatid mite eggs. Thus, in a preferred embodiment, the nutrient source of the invention does not contain larvae, nymphs or adult astigmatid mites. 
     According to an embodiment, the term “egg” excludes any other stage: for example, astigmatid mite eggs do not contain larvae, nymphs or adult astigmatid mites. 
     The astigmatid mite eggs of the invention can be selected from eggs of Pyroglyphidae, Acaridae, Carpoglyphidae or Glyciphagidae. 
     According to an advantageous embodiment, the astigmatid mite eggs are selected from eggs of at least one species of the family Acaridae, for example at least one species of the genus  Acarus , for example  Acarus siro, Acarus farris, Acarus immobilis  or  Acarus chaetoxysilos ; or, according to another example, at least one species of the genus  Tyrophagus , such as  Tyrophagus longior, Tyrophagus similis  or  Tyrophagus putrescentiae ; or even at least one species of the genus  Aleuroglyphus , such  Aleuroglyphus ovatus ; or at least one species of the genus  Lardoglyphus , such as  Lardoglyphus konoi , for example; at least one species of the genus  Caloglyphus , such  Caloglyphus mycophagus  or  Caloglyphus berlesei ; at least one species of the genus  Suidasia , such as  Suidasia nesbitti , for example; at least one species of the genus  Thyreophagus , such  Thyreophagus entomophagus ; at least one species of the family Carpoglyphidae, such as, for example, at least one species of the genus  Carpoglyphus , for example  Carpoglyphus lactis, Carpoglyphus munroi ; at least one species of the family Glycyphagidae, such as, for example, at least one species of the genus  Glycyphagus , such as  Glycyphagus domesticus ; at least one species of the genus  Lepidoglyphus , such as  Lepidoglyphus destructor ; at least one species of the genus  Blomia , such as, for example,  Blomia freemani ; at least one species of the family Chortoglyphydae, at least one species of the genus  Chortoglyphus , for example  Chortoglyphus arcuatus.    
     Advantageously, the astigmatid mite eggs are selected from eggs of at least one species of the genus  Aleuroglyphus . According to a particularly advantageous embodiment, the astigmatid mite eggs are selected from eggs of at least  Aleuroglyphus ovatus.    
     According to an embodiment, the nutrient source of the invention comprises Acaridae eggs. According to a preferred embodiment, the nutrient source of the invention comprises  Aleuroglyphus ovatus  eggs. 
     The inventors further discovered that, surprisingly, mixtures of astigmatid mite eggs and lepidopteran eggs act synergistically, leading to particularly significant arthropod growth. 
     Thus, another object of the invention is the use of astigmatid mite eggs and lepidopteran eggs as a nutrient source for arthropod biological control agents. 
     Preferably, the nutrient source of the invention further comprises eggs of at least one lepidopteran species. Preferably, the nutrient source of the invention further comprises at least one lepidopteran population. More preferably said lepidopteran population consists in eggs. 
     According to an embodiment of the invention, the lepidopteran eggs are selected, for example, from the family Pyralidae, for example  Ephestia kuhniella, Ephestia elutella, Ephestia cautella, Corcyra cephalonica  or  Plodia interpunctella , or for example from the subfamily Gelechiidae, for example  Sitotroga cerealella . According to a particularly advantageous embodiment, the lepidopteran eggs are  Ephestia kuhniella  eggs. 
     Thus, according to a particularly advantageous embodiment of the invention, the composition of the invention comprises at least one population of the arthropod species  Amblyseius swirskii, Aleuroglyphus ovatus  eggs and  Ephestia kuhniella  eggs. 
     Preferably, the nutrient source of the invention further comprises 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% of lepidopteran eggs by weight in relation to the total weight of the nutrient source. 
     According to a preferred embodiment of the invention, the nutrient source of the invention is comprised of astigmatid mite eggs and lepidopteran eggs, for example in a ratio of astigmatid mite eggs to lepidopteran eggs of between 1:2 and 2:1 by weight. 
     According to a preferred embodiment, a nutrient source comprising  Aleuroglyphus ovatus  eggs is used for at least one population of arthropods whose species belongs to the family Phytoseiidae. 
     According to a preferred embodiment, a nutrient source comprising  Aleuroglyphus ovatus  eggs is used for at least one population of arthropods of the species  Amblyseius swirskii.    
     The astigmatid mite eggs can be collected by any method known to persons skilled in the art. 
     According to an embodiment of the invention, the ratio of the weight of the arthropods to the weight of the eggs is preferentially between 1:1 and 1:50. Advantageously, this ratio can be between 1:2 and 1:10. In a particularly advantageous embodiment, this ratio is between 1:3 and 1:7. 
     According to an embodiment of the invention, the ratio of the weight of the arthropods to the weight of the astigmatid mite eggs is preferentially between 1:1 and 1:50. Advantageously, this ratio can be between 1:2 and 1:10. In a particularly advantageous embodiment, this ratio is between 1:3 and 1:7. 
     Thus, according to an embodiment, the invention has as an object a method for rearing arthropods from astigmatid mite eggs, comprising the steps of:
         a. collecting astigmatid mite eggs,   b. determining the weight of the astigmatid mite eggs obtained,   c. bringing together a weight of astigmatid mite eggs and a weight of arthropods,
 
characterized in that the ratio of the weight of the arthropods to the weight of the astigmatid mite eggs is between 1:3 and 1:7.
       

     According to a preferred embodiment, the invention has as an object a method for rearing arthropods from astigmatid mite eggs and lepidopteran eggs, comprising the steps of:
         a. collecting astigmatid mite eggs and lepidopteran eggs,   b. determining the weight of the astigmatid mite eggs obtained and the weight of the lepidopteran eggs obtained,   c. bringing together a weight of astigmatid mite eggs, a weight of lepidopteran eggs and a weight of arthropods,
 
characterized in that the ratio of the weight of the arthropods to the sum of the weight of the astigmatid mite eggs and the weight of the lepidopteran eggs is between 1:3 and 1:7.
       

     According to an embodiment of the invention, a support or dissemination substrate is added to the mixture comprising the population of arthropod biological control agents and the nutrient source. The support or dissemination substrate is selected from inert substrates, such as, for example, particles of vermiculite, particles of pearlite, or mixtures thereof. 
     The composition of the invention can further comprise traces of the arthropod rearing medium. 
     Advantageously, the arthropods are reared at a temperature between 5° C. and 40° C., preferentially between 15° C. and 35° C. In a particularly advantageous manner, the arthropods are reared at a temperature between 20° C. and 30° C. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG.  1   : Combined rates of dead or escaped  Amblyseius swirskii  at different stages of development (in percent of the original population), depending on the food proposed, at 25° C., 75% HR and 16/8 (L/D). 
         FIG.  2   :  Amblyseius swirskii  fecundity depending on the food proposed expressed in eggs per female for the whole adult period at 25° C., 75% HR and 16/8 (L/D). 
         FIG.  3   :  Amblyseius swirskii  longevity in days depending on the food given at 25° C., 75% HR and 16/8 (L/D). 
         FIG.  4   :  Amblyseius swirskii  escape rate (in % of the original population), while submitted to starving, at 25° C., 75% HR and 16/8 (L/D). The populations represented were fed either  A. ovatus  sub-lethal eggs (balck plot), or a mixture of all stages of  A. ovatus  (light grey plot). 
         FIG.  5   :  A. ovatus  eggs (A) and all stages of  A. ovatus  (B) after two months of cold storage and then one week at ambient temperature (eggs are in mean 132.5 μm long and 86.2 μm large). (A) 
     
    
    
     EXAMPLES 
     Example 1: Effect of a Diet Consisting in Astigmatid Mite Eggs on Growth and Health of Arthropod Biological Control Agents 
     Eggs of  A. swirskii  were deposited in distinct cells and raised at 25° C., 75% HR (for “relative Humidity”) and 16/8 (L/D, for “Light/Dark”, indicating the time in hours spent in the referred condition). 
     The resulting  A. swirskii  populations were fed with one of the two following conditions:
         a mixture of all stages of  A. ovatus,      only  A. ovatus  sub-lethal eggs.       

     Every 12 hours, the  A. swirskii  individuals that escaped or died were noted, along with their developmental stage. 
     Results are represented  FIG.  1   . 
     The combined  A. swirskii  rate of juveniles that died or escaped when only eggs are proposed is lower (37%) than when a mixture of all stages of  A. ovatus  is given to  A. swirskii  (63%). 
     Conclusion: populations of  Amblyseius swirskii  fed with  Aleuroglyphus ovatus  eggs suffer less death and escapes from individuals than populations of  Amblyseius swirskii  fed with all stages of  Aleuroglyphus ovatus . Populations of arthropod biological control agents reared on astigmatid mite eggs are likely to be more healthy and numerous than populations of arthropod biological control agents reared on all stages of astigmatid mite. 
     Example 2: Effect of a Diet Consisting in Astigmatid Mite Eggs on Fecundity of Arthropod Biological Control Agents 
     Young  A. swirskii  females of the same cohort were individually deposited in experimental cells at 25° C., 75% HR and 16/8 (L/D), and a male was given to each female. 
     The  A. swirskii  females were fed with one of the two following conditions:
         a mixture of all stages of  A. ovatus,      only  A. ovatus  sub-lethal eggs.       

     The fecundity (mean number of eggs/female) of 30 to 60  A. swirskii  was then noted daily between the first mating and their death. 
     Results are represented  FIG.  2   . 
     The longevity of those females was also noted for the two modalities given above, and is represented  FIG.  3   . 
     It appears that when the food given to  A. swirskii  females only consist of sub-lethal eggs, longevity is much higher than when the food is composed of a mixture of all stages of  A. ovatus . The inverse trend is observed about fecundity for the two modalities tested. No global performance is lost while giving only  A. ovatus  sub-lethal eggs to  A. swirskii , but an advantage is obtained in term of longevity. 
     Conclusion: The fecundity of  Amblyseius swirskii  females fed with  Aleuroglyphus ovatus  eggs is more important than the fecundity of the same females fed with all stages of  Aleuroglyphus ovatus.    
     The fecundity of arthropod biological control agents reared on astigmatid mite eggs are likely to be more important than the fecundity of arthropod biological control agents reared on all stages of astigmatid mite. 
     Example 3: Effect of a Diet Consisting in Astigmatid Mite Eggs on Survival of Arthropod Biological Control Agents 
     Young  A. swirskii  females, were maintained at 25° C., 75% HR and 16/8 (L/D). 
     The  A. swirskii  females were fed with one of the two following conditions for at least 3 days:
         a mixture of all stages of  A. ovatus,      only  A. ovatus  sub-lethal eggs.       

       Amblyseius swirskii  females were then transferred to individual cells. They were then starved. 
     Escape was noted daily from the first day of starvation until the end of the experiment. 
     Results are represented  FIG.  4   . 
     The escape rate was higher when  A. swirskii  were given a mixture of all the stages of  A. ovatus  as food, compared to eggs. 
     Conclusion: populations of  Amblyseius swirskii  fed with  Aleuroglyphus ovatus  eggs suffer less death and escapes from individuals than populations of  Amblyseius swirskii  fed with all stages of  Aleuroglyphus ovatus , even after they have been deprived of food. Populations of arthropod biological control agents reared on astigmatid mite eggs are likely to be more healthy and numerous than populations of arthropod biological control agents reared on all stages of astigmatid mite. 
     Example 4 Sub-Lethal Astigmatid Mite Eggs do not Develop Fungus 
     A population of  A. ovatus  fed with yeast and wheat bran was prepared. At the end of the preparation, the population of  A. ovatus  contained a mixture of all stages of  A. ovatus.    
     A sample of the prepared population was taken, from which eggs were isolated. The isolated eggs where put in a cold storage, in the appropriate conditions to induce a sub-lethal state. 
     Another sample of the prepared population was taken, and put in a cold storage. This second sample thus contained a mixture of all stages of  A. ovatus.    
     After two months, the isolated eggs (A) and the mixture of all stages of  A. ovatus  (B) were taken out of the storage and put back to ambient temperature for a week. 
     Fungus development was then observed on the sample containing all the stages of  A. ovatus  (B), while no microorganisms&#39; development was observed on the sample containing only sub-lethal eggs (A). 
     After a week, a picture was taken to illustrate the evolution of both samples. 
     Results are given  FIG.  5   . 
     Conclusion: These results show that sub-lethal astigmatid mite eggs do not develop fungus, and therefore do not deteriorate as fast as populations comprising all stages of astigmatid mites that had been stored in the same conditions. 
     Example 5: Specific Temperature Exposure and Duration of Exposure to Such Temperature Lead to Sub-Lethal Eggs 
     Eggs of  A. ovatus  were isolated from a lab population and stored at different temperatures in an experimental cell (30 eggs/cell/treatment) for 4, 5 or 6 weeks
         −5° C.   0° C.   8° C.       

     After storage, all eggs were put back to 25° C. and 75% HR conditions. 
     Every day during a week, every hatched or collapsed egg was noted, resulting in egg hatching, collapsing and edible rates. 
     Results are represented Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                   A. ovatus  egg collapsing and hatching rates depending on 
               
               
                 the storage temperature and on storage time. 
               
            
           
           
               
               
               
               
               
            
               
                 Exposure 
                   
                 Collapsing 
                 Hatching 
                 Edible 
               
               
                 time 
                 Treatment 
                 (A) 
                 (B) 
                 (1 − A − B) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 4 weeks 
                 −5° 
                 C. 
                 43.3% 
                 0% 
                 56.7% 
               
               
                   
                 0° 
                 C. 
                 32.3% 
                 3.2%     
                 64.5% 
               
               
                   
                 8° 
                 C. 
                     60% 
                 23.3%   
                 16.7% 
               
               
                 5 weeks 
                 −5° 
                 C. 
                 56.7% 
                 0% 
                 43.3% 
               
               
                   
                 0° 
                 C. 
                 13.3% 
                 0% 
                 86.7% 
               
               
                   
                 8° 
                 C. 
                 61.3% 
                 29%  
                  9.7% 
               
               
                 6 weeks 
                 −5° 
                 C. 
                  100% 
                 0% 
                   0% 
               
               
                   
                 0° 
                 C. 
                     40% 
                 0% 
                     60% 
               
               
                   
                 8° 
                 C. 
                 36.6% 
                 56.7%   
                  6.7% 
               
               
                   
               
               
                 Storage at 0° C. provokes a massive egg’s collapse and no hatching. 
               
               
                 Storage at 8° C. still induces hatching and a high collapsing rate. 
               
               
                 Storage at 0° C. conserves the egg turgescence, and reduces hatchability to zero after 5 week of exposure. 
               
            
           
         
       
     
     Conclusion: Astigmatid mites eggs exposed to a temperature of 0° C. for 4 to 6 weeks do not hatch, proving that the obtained eggs are sub-lethal eggs. Moreover, in those conditions, collapsing is minimized and it is possible to obtain more than 60% edible eggs.