EP4195933A1 - Verfahren und zusammensetzungen zur aufzucht von prädatorinsekten - Google Patents

Verfahren und zusammensetzungen zur aufzucht von prädatorinsekten

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Publication number
EP4195933A1
EP4195933A1 EP21782739.3A EP21782739A EP4195933A1 EP 4195933 A1 EP4195933 A1 EP 4195933A1 EP 21782739 A EP21782739 A EP 21782739A EP 4195933 A1 EP4195933 A1 EP 4195933A1
Authority
EP
European Patent Office
Prior art keywords
predator
population
insect
food
strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21782739.3A
Other languages
English (en)
French (fr)
Inventor
Enrique Vila Rifá
Pablo BIELZA LINO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AGROBIO SL
Original Assignee
AGROBIO SL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AGROBIO SL filed Critical AGROBIO SL
Priority to EP23166377.4A priority Critical patent/EP4223125A3/de
Publication of EP4195933A1 publication Critical patent/EP4195933A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A01N63/14—Insects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/02—Acaricides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides

Definitions

  • the present invention belongs to the field of methods for rearing predator insects, in particular, insects from the family Miridae or from the family Anthocoridae, as well as methods for controlling crop pests using said insects.
  • a successful ratio is best, often only, achieved if a reasonable breeding population of the biocontrol agent can be established on the crop before the first pests invade.
  • predators are introduced before the pest arrives, they will quickly die of starvation, especially if the crop itself is unable to provide nutrients, such as pollen or nectar.
  • Many valuable crops, e.g. cucumbers do neither have flowers with pollen, nor possess nectar sites, and so do not have an alternative nutrient source.
  • Other crops, such as young sweet pepper plants or ornamentals will have yet to flower.
  • pollen or flowers are present, not all predators can efficiently use pollen or nectar as food sources.
  • Polyphagous natural enemies include some species of Heteroptera and predatory mites of the family Phytoseiidae.
  • pollen represents a viable proposition as an alternative pre-pest food, whereas others defer. This is probably not due to different approaches in methodologies but by the fact that there are very different qualities of “pollen”.
  • the pollen profile for each flowering plant is unique, thus to one predator species a specific pollen may represent a natural food, whilst to another it will be factitious, unknown to it in its natural environment.
  • Other drawbacks associated with pollen as an alternative food are in themselves major problems, for example:
  • pollen also provides a source of nutriment for many pest species
  • the zoophytophagous predator bugs is still lacking.
  • the bugs of the Miridae family are the most used for pest control in various crops, especially tomato and eggplant.
  • the species that are massively released in greenhouses are Macrolophus pygmaeus in northern Europe, Nesidiocoris tenuis in Mediterranean countries and some Asians (South Korea) and Australia, and Dicyphus hesperus in Canada.
  • These mirids have a longer period of development compared to most of the phytoseiid mites, and they need several weeks until establishment in the crop.
  • Another problem is the drop of the populations when pests are at low levels, when temperatures are low (most of the plastic houses and tunnels have no heating system) and/or chemicals are applied to control diseases or some specific pests without biocontrol solutions. In these situations, biocontrol is disrupted and a cost/effective feeding program would help to overcome these problems.
  • the eggs of the moth Ephestia kuehniella is by far the best factitious food used, because they are considered a high-quality food, used in most of the commercial mass rearings.
  • the efficacy of E. kuehniella eggs as “factitious prey” for many polyphagous predatory insects has been demonstrated in numerous studies and in some of them it has been even shown that these predators perform better than when fed on natural prey (Riddick, BioControl 2009, 54: 325-339).
  • the use of E. kuehniella eggs as supplemental food is expensive, with a cost higher than 800 euros per kg, and this limits greatly its use. For this reason, several studies have been carried out to find an alternative food to E. kuehniella eggs.
  • Artemia cysts are the encapsulated eggs of the brine shrimp Artemia spp., and when de-capsulated form a basic food for exotic ornamental fish species, hence large quantities are sold across the world each year. Artemia sp. cysts have been shown to be a good alternative food for rearing M. pygmaeus and N. tenuis, and its consumption allows preimaginal development and reproduction of the predator (Castane et al. Biological Control 2006, 38: 405-412; Vandekerkhove et al. Journal of Applied Entomology, 2009, 133: 133-142).
  • polyphagous predators that has been the key to the success of augmentative biological control programs are the bugs of the Anthocoridae family.
  • various species of the Orius genus known as flower bugs, are inoculatively introduced into horticultural crops, such as bell peppers, to control thrips.
  • Polyphagous predators O. majusculus and O. laevigatus are two Paleartic species and two of the natural enemies widely used in biological control programs, both in inoculation and conservation strategies, for the control of thrips, especially the pest Frankliniella occidentalis.
  • these predators are polyphagous, also feeding on food of vegetable origin, especially pollen, and obtaining nutritional benefits from them (Pumariho and Alomar, Biological Control, 2012, 62(1): 24-28).
  • Orius laevigatus is by far the most used species in Europe, it is released in all the sweet pepper crops as soon as flowering is above 50%, so individuals can feed on pollen as an alternative food. Nevertheless, it takes some time for predators to build up a significant population to cope with pests, especially feeding on a non-prey food like pollen. Therefore, an even earlier release would be very advantageous. In this sense, providing artificially with alternative food would allow predator release without the presence of prey or flowers. Moreover, innovations in varieties adapted to modern trends of healthy convenience food, like seedless baby peppers, bring about some side-effects for biocontrol, since these pepper plants do not produce pollen, hindering establishment of biological control agents, and so demanding application of supplemental food.
  • predator-prey populations interact and eventually the prey may disappear, and consequently the predator may also vanish due to a lack of prey. This situation would leave the crop without protection against immigrant pest populations.
  • Presence of alternative food provided by the plant may reduce the impact on predator populations.
  • the number of flowers and the amount and quality of pollen vary throughout the season.
  • the same constrains that affect mirids are also limiting the success of anthocorids.
  • Predator populations may drop due to a number of environmental and chemical stresses, such as extreme temperature and humidity. Also, in these cases, artificial supplementing of alternative food may help the recovery and persistence of the populations.
  • insects from the families Miridae or Anthocoridae which are natural predators for common crop pests, are capable to feed on crops on astigmatid mites, a factitious prey for these predators (see Examples 1 , 2, 3).
  • the predator insects performed better when fed on astigmatid mites than when fed on other expensive factitious prey like cysts of Artemia or eggs of Ephestia kuehniella (see Examples 2 and 3). Therefore, astigmatid mites can be used to feed these predator insects directly on the crop as a part of a biological control strategy, resulting in a successful control of the pests (see Example 2).
  • the inventors have selected a strain of the insect Orius laevigatus with an increased ability to develop and reproduce when fed on pollen (Example 4) and have shown that, surprisingly, this new strain has also an increase tolerance to factitious prey, in particular, astigmatid mites (see Examples 5, 6 and 7).
  • the invention relates to a method for rearing a predator insect from the family Miridae and/or a predator insect from the family Anthocoridae on a crop comprising:
  • the invention relates to a method for controlling a pest in a crop comprising:
  • the invention relates to a use of an astigmatid mite for rearing a predator insect from the family Miridae or from the family Anthocoridae on a crop, wherein said astigmatid mite is not Tyrophagus putrescentiae.
  • the invention in another aspect, relates to a composition
  • a composition comprising at least a breeding population of a predator insect from the family Miridae or from the family Anthocoridae and a first source of food for said predator insect, wherein said first source of food comprises a population of an astigmatid mite, wherein said population of an astigmatid mite is not a population of Tyrophagus putrescentiae or Carphoglyphus lactis, wherein the breeding population of the predator insect and the first source of food are physically separated so that there is no contact between the population of the predator insect and the population of the astigmatid mite.
  • the invention relates to the use of the composition according to the previous aspect for controlling a pest in a plant, where the pest is a prey for the predator insect.
  • the invention relates to a method for controlling a pest in a crop comprising providing the crop with at least a breeding population of a predator insect from the family Miridae and/or a predator insect from the family Anthocoridae, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by:
  • the invention in another aspect, relates to a method for controlling a pest in a crop comprising providing the crop with at least a breeding population of a predator insect from the species Orius laevigatus, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained in at least 70% of the individuals of the strain: (a) a fragment of 780-840 base pairs or
  • the invention relates to the use of a predator insect from the family Miridae and/or a predator insect from the family Anthocoridae for controlling a pest in a crop, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by:
  • the invention relates to the use of a predator insect from the species Orius laevigatus for controlling a pest in a crop, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained in at least 70% of the individuals of the strain:
  • Figure 1 Mean ( ⁇ SE) of nymphs and adults of O. laevigatus counted between June and August 2019 in a commercial crop.
  • a pepper crop of a variety type mini Capia was planted in May the 10 th and 4 treatments were established according to the factitious prey introduced: (1) prey mite C. lactis, (2) C. lactis + cysts of Artemia, (3) C. lactis + eggs of S. cerealella and (4) control without factitious prey.
  • Figure 2 Mean ( ⁇ SE) of nymphs and adults of N. tenuis (a), larvae of Tuta absoluta (b) and nymphs, pupae and adults of whiteflies (c) counted between September 2017 and May 2018 in a commercial greenhouse.
  • a tomato crop of a variety Genio was planted in August the 23rd and 3 treatments were established according to the factitious prey introduced: (1) prey mite S. medanensis, (2) cysts of Artemia and (3) control without factitious prey.
  • Figure 3 Mean ( ⁇ SE) of nymphs and adults of D. bolivari counted between November 2018 and March 2019 in an experimental greenhouse. A tomato crop of a variety Caniles was planted in October the 31 st , and 4 treatments were established according to the factitious prey introduced: (1) prey mite T. casei, (2) T. casei complemented with cysts of Artemia, (3) eggs of E. kuehniella and (4) control without factitious prey.
  • Figure 4 Longevity and lifetime fecundity (mean ⁇ SE) of females of a commercial population (Agrobio) of O. laevigatus and a new strain (POL11) feeding Astigmatid mites.
  • Figure 5. Mean ( ⁇ SE) of nymphs and adults of O. laevigatus (a) and larvae and adults of thrips (b) counted between March and April 2020 in a commercial crop.
  • a pepper crop of the variety mini Capia ‘Pepperito’ was planted in February the 2 on and 2 treatments were established: (1) releases of a new strain of O. laevigatus and (2) releases of standard commercial populations of O. laevigatus.
  • Figure 6 Mean ( ⁇ SE) of nymphs and adults of O. laevigatus (a) and larvae and adults of thrips (b) counted between April and May 2020 in a commercial crop.
  • a Dutch type cucumber crop was planted in February the 27th and 2 treatments were established: (1) releases of a new strain of O. laevigatus and (2) releases of standard commercial populations of O. laevigatus.
  • Figure 7 Mean ( ⁇ SE) of nymphs and adults of O. laevigatus counted between February and March 2020 in a commercial crop. A chrysanthemums crop was planted in January the 28th and 2 treatments were established: (1) releases of a new strain of O. laevigatus and (2) releases of standard commercial populations of O. laevigatus.
  • Figure 8 Survival from egg to adult of females of a commercial population (Agrobio) and a new strain (POL11) feeding two Astigmatid mite species. Bars with the same letter within each mite species are not significantly different (p>0.05).
  • Figure 9 Lifetime fecundity of females of a commercial population (Agrobio) feeding different Astigmatid mite species.
  • FIG. 10 PCR program used for amplification of the SSR marker RP02.
  • FIG. 11 Electrophoresis images from PCR amplification with RP02 SSR marker in 13 adults of each improved strain, Orius PLUS (A), Orius COLD-PLUS (B) and in 48 individuals from a wild population, Orius Wild-type (C).
  • line 1 is placed 100bp DNA Ladder. The ladder consists of fragments ranging in size from 100-1.000 bp in 100 bp increments.
  • the invention relates to a method for rearing a predator insect from the family Miridae and/or a predator insect from the family Anthocoridae on a crop comprising:
  • breeding broadly refers to breeding, reproducing, surviving and growing of individuals, and includes the propagation and increase of a population by sexual reproduction.
  • the first method of the invention is directed to rear a predatory insect from the families Miridae or Anthocoridae on a crop.
  • the expression “rearing on a crop”, as used herein, means that these predatory insects are located on the surface of any plant, including pot plants and crops, including an open field crop or on a greenhouse crop.
  • the term “crop”, as used herein, refers to a plant of economic importance and/or a men- grown plant.
  • the plant or crop is selected from the group consisting of vegetables (including cucumber, pepper, eggplant, zucchini, melon, watermelon, tomato, leaf vegetables like lettuce or rocket, potato, beans and onion), orchards, vineyard, olive trees, citrus trees, stone fruit trees, berries, ornamental plants, including pot plants and cut flowers, aromatic plants, and plants for pharmaceutic use like cannabis.
  • the plant or crop is selected from the group consisting of pepper, tomato, cucumber and chrysanthemums.
  • pepper refers to a plant of the genus Capsicum, identified in the NCBI database by the Taxonomy ID: 4071.
  • the term “pepper” or “Capsicum” includes any species, for example.
  • the pepper crop is from the type “Capia” and from the variety “Pepperito”.
  • tomato refers to a plant of the species Solanum lycopersicum, identified in the NCBI database by Taxonomy ID: 4081.
  • the term “tomato” or “Solanum lycopersicum” includes any tomato variety.
  • the tomato crop is from the variety “Genio” (from Clause) or from the variety “Caniles”.
  • cucumber refers to a plant of the species Cucumis sativus, identified in the NCBI database by Taxonomy ID: 3659.
  • the term “cucumber” or “Cucumis sativus” includes any variety.
  • the cucumber is from the Dutch type variety Litoral (Rijk Zwaan).
  • family Miridae refers to a family of insects belonging to the suborder Heteroptera commonly known as capsid bugs or mirid bugs.
  • the family Miridae corresponds to the family identified in the NCBI database by the Taxonomy ID: 30083. Most widely known mirids are species that are notorious agricultural pests. However, some species are predatory.
  • predatory insect refers to an insect that eats other animals, called prey.
  • Predator insects from the family Miridae also known as “predatory mirids”
  • Predatory mirids are considered zoophytophagous or true omnivore predators, as these organisms consume both plant and animal resources during their life cycle.
  • Predatory mirids are important predators for plant pests, like whiteflies, thrips, aphids, leafminers, spidermites and lepidoptera.
  • Illustrative non-limitative examples of predatory insects from the family Miridae includes insects from the subfamilies Bryocorinae, Deraeocorinae, Phylinae and Orthotylinae, preferably, from the subfamily Bryocorinae and the tribe Dicyphini, from the subfamily Deraeocorinae and the tribe Deraeocorini, from the subfamily Phylinae and the tribes Pilophorini and Nasocorini, from the subfamily Orthotylinae and the tribe Orthotylini. More preferably, the insects from the tribe Dicyphini are selected from the genus Nesidiocoris, such as N. tenuis, N. volucerand N.
  • the genus Macrolophus such as M pygmaeus (formerly described as M. caliginosus), M. melanotoma, M. costalis and M. basicornis
  • the genus Engytatus such as E. varians and E. modestus
  • the genus Dicyphus such as D. tamaninii, D. bolivari, D. errans, D. hesperus and D. marrocanus
  • Tupiocoris such as T. cucurbitaceus
  • the genus Campyloneuropsis such as C. infumatus
  • Cyrtopeltis such as C. callosus.
  • insects selected from the tribe Deraecorini are preferably selected from the genus Deraeocoris, such as D. brevis and D. nebulosus
  • the insects selected from the tribe Pilophorini are preferably selected from the genus Pilophorus, such as P. typicus and P. gallicus
  • the insects selected from the tribe Nasocorini are preferably from the genus Campylomma, such as C. verbasci and C. chinensis.
  • insects selected from the tribe Orthotylini are preferably from the genus Cyrtorhinus, Orthotylus and Eurotas, such as Cyrtorhinus lividipennis, Orthotylus marginalis and Eurotas brasilianus.
  • the predator insect from the family Miridae is from a genus selected from the group consisting of Macrolophus, Nesidiocoris, Dicyphus, Deraeocoris, Engytatus, Tupiocoris, Campyloneuropsis, Cyrtopeltis, Pilophorus, Campylomma, Cyrtorhinus, Orthotylus and Eurotas.
  • the predator insect from the family Miridae is from a species selected from the group consisting of Macrolophus pygmaeus, M. costalis, M. basicornis, Nesidiocoris tenuis, N. volucer, N. callani, Dicyphus bolivari., D.
  • the predator insect from the family Miridae is from the genus Macrolophus, Nesidiocoris or Dicyphus, more particularly from the species Nesidiocoris tenuis, M. pygmaeus, D. hesperus or Dicyphus bolivari.
  • family Anthocoridae refers to a family of insects belonging to the order Hemiptera and the superfamiliy Cimicoidea, and are commonly known as minute pirate bugs or flower bugs.
  • the family Anthocoridae corresponds to the family identified in the NCBI database by the Taxonomy ID: 82738. Insects from the family Anthocoridae can feed on plants, but are mostly predatory, feeding on other small- soft bodied arthropods, some of which can be agricultural pest, such as mites and thrips.
  • Illustrative non-limitative examples of predatory insects from the family Anthocoridae include the tribe Anthocorini, Orinii and Xylocorini.
  • the insects from the Anthocorini are selected from the genus Anthocoris, such as A. nemorum, A. nemoralis, A. confusus and A. minki, the genus Wollastoniella, such as W. rotunda, and the genus Blaptostethus, such as B. pallescens
  • the insects from the tribe Orinii are selected from the genus Orius, such as O. laevigatus, O. insidiosus, O. majusculus, O.
  • the insects from the tribe Xylocorini are preferably selected from the genus Xylocoris, such as X. flavipes.
  • the predator insect from the family Anthocoridae is an insect from a genus selected from the group consisting of Orius, Anthocoris, Blaptostethus, Montandoniola and Xylocoris.
  • the predator insect from the family Anthocoridae is an insect from a species selected from the group consisting of Orius laevigatus, O. insidiosus, O. majusculus, O. niger, O. albidipennis, O. minutus, O. thripoborus, O. naivashae, O. strigicollis, O. sauteri, O. tristicolor, O. nagaii, O. antillus, O. limbatus, O. thripoborus, O. naiashae, O. horvathi, O. vicinus, O. pumilio, Orius laticollis, O.
  • the predator insect from the family Anthocoridae is Orius laevigatus.
  • Orius laevigatus corresponds to a species identified in the NCBI database by the Taxonomy ID: 82742.
  • the predator insect from the family Anthocoridae is an insect from a strain from the species Orius laevigatus with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by the method for obtaining a strain with increased tolerance to a diet based on a factitious food of the invention.
  • the predator insect from the family Anthocoridae is an insect from a strain from the species Orius laevigatus with increased tolerance to a diet based on a factitious food, wherein said strain is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained in at least 70% of the individuals of the strain:
  • the fragment of 780-840 base pairs has 800-820 base pares.
  • the fragment of 1280-1360 bp has 1300-1340 bp, more particularly 1300 bp.
  • the fragment of 580-640 bp has 600-620 bp, more particularly 600 bp.
  • the fragment of 420-480 bp has 440-460 bp, more particularly 450 bp.
  • the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by the methods for obtaining a strain with increased tolerance to a diet based on a factitious food of the invention.
  • the first method of the invention comprises a step of providing the plant with a breeding population of the predator insect from the family Miridae or from the family Anthocoridae.
  • breeding population refers to a population that can increase their number by sexual reproduction.
  • a breeding population can comprise alive sexually mature individuals of both sexes and/or other stages of life, for example, eggs and/or nymph from both sexes that can mature into sexually mature adults.
  • a breeding population can comprise one or more fertilized females.
  • nymph refers to an individual sexually immature, similar to the adult and found in such insects which have incomplete, or hemimetabolic, metamorphosis. Wings, if present, develop from external wing buds after the first few molts. The body proportions of the first nymphal stages are quite different from those of the adult. During each successive growing stage (instar) the nymph begins to resemble the adult more closely. The predatory Mirids and Anthocorids have 5 nymphal instars before reaching the adult stage.
  • Eggs of Miridae and Anthocoridae generally are moderate to large in size relative to adults and have a smooth to finely sculptured surface, sometimes with a colour pattern and often with slender projections. Eggs contain sufficient nutrients to permit the embryo to develop into a free-living, sexually immature, wingless nymph. Predatory mirids and anthocorids generally insert the eggs into the tissue of selected host plants.
  • substrates containing eggs can optionally be introduced, either with nymphs, adults or mixtures.
  • substrates containing eggs can optionally be introduced, either with nymphs, adults or mixtures.
  • pieces of vegetables for instance beans, with the eggs embedded into the tissue, but also some artificial substrates containing the eggs can be introduced.
  • the pieces of vegetables have the advantage that are also a source of water and nutrients for the predators.
  • the breeding populations comprises between 30 and 100% of females, preferably between 40 and 80%, more preferably between 45 and 65%.
  • the females need a preoviposition period to start laying eggs, which may vary according to the species.
  • the breeding population comprises adults between 1 hour and 10 days old.
  • the age of the adults refers to the time elapsed since molting.
  • the needed transport time from the mass-rearing facilities to the crop is also conditioning the age of the females when released.
  • the released adults are preferably between 1 and 7 days old, more preferably between 2 and 6 days old, most preferably between 3 and 5 days old.
  • the released adults are between 3-5 days if the adults have been maintained at a temperature comprised between 8°C and 20°C and between 2-4 days if the adults have been maintained at a temperature between 20°C and 30°C.
  • Predators are introduced into the plant or crop preferably with a carrier material.
  • the carrier material or substrate is also commonly necessary to minimize the mortality produced by cannibalistic behavior of the living predators during storage and transport from the rearing facilities to the crop.
  • Substrates can be any material that provides a surface where predators can hide from each other. Preferably, the materials contain cavities where the predators can hide. Examples of substrates are cardboard or wrinkled paper, sawdust, shells of seeds, for example buckwheat, popcorn, and vermiculite.
  • populations can also be supplied without substrate and, optionally, they can be mixed with a carrier material after transport, just before the distribution in the crop.
  • Predators are introduced on the plants before or after transplanting the crop or both.
  • the predators are introduced between 15 days and 1 hour before transplanting the crop when a fast establishment is desired. More preferably the predators are introduced between 12 days and 1 day before transplanting, and even more preferably between 10 days and 7 days before transplanting.
  • When predators are introduced before transplanting between 1 and 4 introductions are performed, preferably between 1 and 3, most preferably between 1 and 2 introductions.
  • predators are introduced after transplanting, preferably between 1 and 30 introductions are performed, more preferably between 1 and 10 introductions, most preferably between 1 and 4 introductions.
  • a ratio of between 0.05 and 50 individuals (nymphs, adults or mixtures) per plant are introduced each releasing time.
  • the population of the predator insect is released in the plant or crop at a plurality of ‘spots’.
  • the density of releasing spots of predators in the crop is preferably between 1000 spots/m 2 and 1 spot/3500 m 2 .
  • the material is sprayed by machinery in the seedlings before transplanting.
  • the populations are introduced in the crop after transplanting, more preferably the material is spread by hand into cardboard cages that are hang on the plants, being considered each cage a spot or releasing point.
  • the preferred density is between 1 releasing spot/ 5 m 2 and 1 spot/ 2000 m 2 , most preferably between 1 spot/ 10m 2 and 1 spot/100 m 2 , and even more preferably between 1 spot / 12m 2 and 1 spot / 25 m 2 .
  • the first method of the invention comprises a step of providing the plant with a first source of food for the predator insect, wherein said first source of food comprises a population of an astigmatid mite.
  • Astigmatid mite refers to a mite belonging to the Astigmata.
  • Astigmata refers to the infraorder Astigmata, belonging to the suborder Sarcoptiformes and the order Acariformes.
  • Astigmata was classified as an Order, but recently a modification to infraorder was proposed by Mironov & Bochkov, Entomological Review, 2009, 89 (8): 975-992.
  • Astigmata is identified in the NCBI database by the Taxonomy ID: 6951.
  • population of an astigmatid mite refers to a group of individuals that can include individuals of both sexes and of any stages of life, for example, adults and/or nymphs and/or eggs.
  • the population of the astigmatid mite can be, a breeding population as previously defined, but it can also comprise dead individuals or can even be formed in its entirety by dead individuals.
  • At least part of the population of the population of the astigmatid mite is not alive, for example, at least a 5 %, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60 %, at least a 70%, at least a 80%, at least a 90% or the 100% of the population.
  • at least part of the population of the astigmatid mite is dead by a human action, for example, radiation exposure, freezing or exposure to high carbon dioxide levels.
  • the astigmatid mite is a storage food mite.
  • storage food mite refers to astigmatid mites that feed on stored food. These mites can be found in environments where there is moisture or increased humidity, but are most frequently found in dry food items such as flour, grains, dried fruits, cereal and dry dog and cat foods.
  • Illustrative non-limitative examples of storage food mites include the species described as storage food mites by Hughes, (1976), in her book entitled - 'The mites of stored foods and houses' (2nd edition, London. Ministry of Agriculture, Fisheries and Food, Technical Bulletin 9, iv, 400 p).
  • the storage food mite is selected from the group consisting of Acarus siro, A. farris, A. immobilis, Lardoglyphus konoi, Aleuroglyphus ovatus, Mycetoglyphus fungivorus, Tyrolichus casei, Tyrophagus tropicus, T. brevicrinatus, T. putrescentiae, T.
  • Austroglycyphagus geniculatus Diamesoglyphus intermedius, Ctenoglyphus palmifer, C. plumiger, C. canestrinii, Aeroglyphus robustus, Gohieria fusca, Chortoglyphus arcuatus, Carpoglyphus munroi, C. lactis, Carpoglyphus wardleorum, Pyroglyphus africanus, Euroglyphus longior, E. maynii, Histiomidae feroniarum.
  • the storage food mite is from a family selected from the group consisting of Acaridae, Carpoglyphidae, Pyroglyphidae, Glycyphagidae, Chortoglyphagidae and Suidasiidae.
  • the storage food mite is from a genus selected from the group consisting of Acarus, Tyrophagus, Aleuroglyphus, Lardoglyphus, Caloglyphus, Suidasia, Thyreophagus, Carpoglyphus, Glycyphagus, Tyrolichus, Tyroglyphus, Lepidoglyphus, Blomia and Chortoglyphus.
  • the storage food mite is from a species selected from the group consisting of Suidasia medanensis, Suidasia nesbitti, Lepidoglyphus (Glycyphagus) destructor, Blomia freeman, Carpoglyphus lactis, Carpoglyphus munroi, Carpoglyphus wardleorum, Lardoglyphus konoi, Aleuroglyphus ovatus, Tyrolichus casei, Thyreophagus entomophagus, Chortoglyphus arcuatus and Acarus siro.
  • the storage food mite is from a family selected from the group consisting of Acaridae, Carpoglyphidae and Suidasiidae, more particularly from a genus selected from the group consisting of Carpoglyphus, Suidasia, Tyreophagus, Aleuroglyphus and Tyrolichus, even more particularly from a species selected from the group consisting of Carpoglyphus lactis, Aleuroglyphus ovatus, Suidasia medanensis, Tyreophagus entomophagus and Tyrolichus casei.
  • Carpoglyphus lactis refers to a species identified in the NCBI database by the Taxonomy ID: 223459.
  • asia medanensis refers to a species identified in the NCBI database by the Taxonomy ID: 2236625.
  • Taxonomy casei refers to a species identified in the Global Biodiversity Information Facility database by the Taxonomy ID: 2181934.
  • Tyreophagus entomophagus refers to a species in the Global Biodiversity Information Facility database by the Taxonomy ID: 2181946.
  • Aleuroglyphus ovatus refers to a species in the Global Biodiversity Information Facility database by the Taxonomy ID: 5860704.
  • the astigmatid mite is not an astigmatid mite that has plants as its natural habitat or that is harmful to agricultural crops.
  • Illustrative non- limitative examples of astigmatid mites that have plants as their natural habitat and/or are harmful to agricultural plants include Tyrophagus putrescentiae, Tyrophagus neiswanderi, Tyrophagus similis, Tyrophagus perniciosus, Tyrophagus robertsoni, Tyrophagus longior, Acarus farris, Rhizoglyphus echinopus, R. robini, Caloglyphus sp., Mycetoglyphus fungivorus or Schwiebea sp.
  • the population of the astigmatid mite is not a population of Tyrophagus neiswanderi, Tyrophagus similis, Tyrophagus perniciosus, Tyrophagus robertsoni, Tyrophagus longior, Acarus farris, Rhizoglyphus echinopus, R. robini, Caloglyphus mycophagus, Mycetoglyphus fungivorus or Schwiebea sp.
  • the population of the astigmatid mite is not a living population of Tyrophagus neiswanderi, Tyrophagus similis, Tyrophagus perniciosus, Tyrophagus robertsoni, Tyrophagus longior, Acarus farris, Rhizoglyphus echinopus, R. robini, Caloglyphus mycophagus, Mycetoglyphus fungivorus or Schwiebea sp.
  • the population of the astigmatid mite is not a population of Tyrophagus.
  • the population of the astigmatid mite is not a population of Carpoglyphus lactis.
  • living population refers to a population where substantially all individuals are alive, or at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the individuals are alive.
  • Taxophagus putrescentiae refers to a species identified in the NCBI database by the Taxonomy ID: 59818.
  • the fist source of food for the predator insect can comprise more than one population of an astigmatid mite, for example 2, 3, 4, or more populations of different astigmatid mites.
  • the first source of food comprises two populations of different astigmatid mites, more particularly, two populations selected from the group consisting of Acarus, Aleuroglyphus, Suidasia, Tyreophagus, Carpoglyphus and Tyrolichus.
  • the first source of food comprises one of the following combinations of two populations of astigmatid mites: Suidasia and Carpoglyphus, Suidasia and Tyrolichus’, Carpoglyphus and Tyrolichus; Carpoglyphus and Tyreophagus; Suidasia and Tyreophagus; Carpoglyphus and Aleuroglyphus; Suidasia and Aleuroglyphus; Tyrolichus and Tyreophagus; Acarus and Tyrolichus; Acarus and Aleuroglyphus; Acarus and Tyreophagus.
  • the first source of food comprises three populations of different astigmatid mites, more particularly, Suidasia, Carpoglyphus and Tyrolichus, or Carpoglyphus, Suidasia and Tyreophagus, or Carpoglyphus, Tyrolichus and Tyreophagus, or Carpoglyphus, Acarus and Tyreophagus, or Carpoglyphus, Suidasia and Aleuroglyphus.
  • the first source of food is released in the plant or crop at a plurality of ‘spots’.
  • the density of spots of the first source of food in the crop is preferably between 1000 spots /m 2 and 1 spot/25 m 2 . More preferably, the density of spots of first source of food is between 3 spots /m 2 and 1 spot/5 m 2 , most preferably between 1 spot/m 2 and 1 spot/2 m 2 .
  • even higher densities of spots of first source of food in the crop are possible, especially when the material is sprayed by machinery.
  • the astigmatid mites are sprayed onto the crop.
  • a vast number of small spots of astigmatid mites are applied to the crop.
  • these spots of astigmatid mites supply can be located on leaves and other parts of a plant such as stipes, stalks, flowers, etc. These spots can also be located on the ground.
  • the density of the individuals of the mite species belonging to the Astigmata in the first sources of food is between 100.000 and 50 million individuals/litre. Using a density in this range permits the application of a suitable number of individuals per spot on the plant. In a further preferred embodiment, the density of the individuals of the mite species belonging to the Astigmata is between 500.000 and 30 million individuals/litre, most preferably between 1 million and 20 million individuals/litre. At these densities, it is easy to apply a suitable number of individuals to each spot on the plant using known techniques. Independent of the density, in a preferred embodiment of the invention, when a carrier substrate is used, a volume between 0.01 ml and 50 ml of the first sources of food is placed on one spot.
  • These volumes can be located and applied on the plant by known techniques. They include known apparatus such as automatic or manually operated sprayers, syringes, dropping devices or spoons.
  • the breeding population of the predator insect can be provided to the plant or crop together with a second source of food.
  • This second source of food can comprise a natural food, for example pollen, prey arthropods, for example aphids, or a factitious prey or artificial foods, and mixtures thereof.
  • prey arthropods species that are not pests of the target crop are selected. For instance, aphids from cereals that cannot survive and damage a horticultural or an orchard crop, when the last are the target crops, are selected.
  • dead pest species are selected, like frozen dipteran larvae of Ceratitis capitata.
  • Another preferred food are factitious prey, which are arthropods that are not naturally occurring in the living habitat of the predators.
  • pest insects of storage food like eggs of lepidoptera moths, crustacean cysts of Artemia and nematodes.
  • Most preferred factitious prey are the eggs of the moths Ephestia kuehniella and Sitotroga cerealella.
  • artificial food when applied to insects, is defined by several authors as any diet that is not the natural food of the insect. However, the term ‘artificial food’ as used herein excludes the factitious prey.
  • this name encompasses all the various terms such as synthetic, chemically defined, purified, holidic, meridic, and oligidic that are used to describe insect diets, excluding the factitious prey or hosts.
  • Examples are meat paste, cooked chicken eggs, yeast, sugar, water, vegetable oils, vitamins or aminoacids, with or without antimicrobial compounds (Carson Cohen, Insect Diets: Science and Technology (Second Edition) 2015, 439 pp. CRC Press, Boca Raton. ISBN 978-1-4665-9194-3). Therefore, in a particular embodiment, the artificial food is not a factitious prey.
  • artificial foods containing sugars, fatty acids, vitamins and/or aminoacids, combined with or without preservatives can also be supplied with the predator populations.
  • second source of food is selected from the group consisting of eggs of Ephestia kuehniella, cysts of Artemia, eggs of Sitotroga cerealella, pollen, nematodes, larvae of Ceratitis capitata, larvae and/or adults of Drosophila melanogaster, artificial foods, and a combination thereof.
  • Ephestia kuehniella refers to the eggs of this moth, identified in the NCBI database by the Taxonomy ID: 40079.
  • cyst of Artemia refers to the encapsulated eggs of the brine shrimp of the genus Artemia, identified in the NCBI database by the Taxonomy ID: 6660.
  • the cyst of Artemia can be of any species of the genus.
  • eggs of Sitotroga cerealella refers to refers to the eggs of this moth commonly known as Angoumois grain moth, and identified in the NCBI database by the Taxonomy ID: 347735.
  • pollen refers to the fine to coarse powder containing the microgametophytes of seed plants.
  • the pollen can be obtained from any seed plants, including Typha, Ricinnus, corn, Pennisetum, Crotalaria, almond, apple, plum, maize, date palm, maple and Quercus. Pollen can also be a mixture of species when it is bee- collected pollen.
  • nematode refers to a phylum of pluricellular pseudocoelomate organisms of the group Ecdysozoa. I lustrative non-limitative examples of nematodes that can be used as source of food for the predator insects include Panagrellus and Panagrolaimus.
  • levovae or adults of Drosophila melanogaster refers to the larva or adult stage of this fly, and identified in the NCBI database by the Taxonomy ID: 7227.
  • Steps (a) and (b) of the first method of the invention can be performed simultaneously or separately in any order, that is, the plant or crop can be first provided with the breeding population of the predator insect from the family Miridae or Anthocoridae and, after that, the plant can be provided with the first source of food for the predator insect comprising the population of an astigmatid mite; or the plant or crop can be first provided with the first source of food for the predator insect comprising the population or an astigmatid mite and, after that, the plant or crop can be provided with the breeding population of the predator insect from the family Miridae or Anthocoridae; or the plant or crop can be provided with the breeding population of the predator insect from the family Miridae or Anthocoridae and with the first source of food for the predator insect comprising the population or an astigmatid mite simultaneously.
  • the population of the predator insect and the first source of food comprising the population of an astigmatid mite are provided to the plant or crop simultaneously, they can be provided inside a common housing, for example, both populations can be spread by hand into cardboard cages that are hang on the plants.
  • the fist method of the invention comprises allowing the predator insect to prey on the astigmatid mite.
  • the population of the predator insect and the population of the astigmatid mite are provided to the crop simultaneously with any kind of physical separation, this means allowing both populations to be in physical contact.
  • the first method of the invention comprises providing the plant or crop with one or more of the following combinations of predator insects and astigmatid mites:
  • Dicyphus errans as predator and Tyrolichus casei as astigmatid mite Dicyphus errans as predator and Tyrolichus casei as astigmatid mite.
  • Dicyphus errans as predator and Carpoglyphus lactis as astigmatid mite.
  • Dicyphus errans as predator and Aleuroglyphus ovatus as astigmatid mite.
  • Dicyphus errans as predator and Acarus siro as astigmatid mite Dicyphus errans as predator and Acarus siro as astigmatid mite.
  • Dicyphus hesperus as predator and Carpoglyphus lactis as astigmatid mite.
  • Dicyphus hesperus as predator and Suidasia medanensis as astigmatid mite.
  • Dicyphus hesperus as predator and Thyreophagus entomophagus as astigmatid mite.
  • Dicyphus hesperus as predator and Acarus siro as astigmatid mite.
  • Dicyphus hesperus as predator and Aleuroglyphus ovatus as astigmatid mite.
  • Dicyphus hesperus as predator and Tyrolichus casei as astigmatid mite.
  • Macrolophus pygmaeus as predator and Suidasia medanensis as astigmatid mite.
  • Macrolophus pygmaeus as predator and Tyrolichus casei as astigmatid mite.
  • Macrolophus pygmaeus as predator and Thyreophagus entomophagus as astigmatid mite.
  • Macrolophus pygmaeus as predator and Carpoglyphus lactis as astigmatid mite.
  • Macrolophus pygmaeus as predator and Aleuroglyphus ovatus as astigmatid mite
  • Macrolophus pygmaeus as predator and Acarus siro as astigmatid mite.
  • Anthocoris nemoralis as predator and Aleuroglyphus ovatus as astigmatid mite.
  • the invention relates to a method for controlling a pest in a crop comprising:
  • pest refers to plant pest, and is understood to include any species, strain or biotype of plant, animal or pathogenic agent injurious for plant or plant products.
  • the pests that can be controlled by the second method of the invention are animals that are prey for the predator insects from the family Miridae or from the family Anthocoridae.
  • the pest is selected from the group consisting of thrips, whiteflies, aphids and moths.
  • Thrips includes any member of the order Thysanoptera.
  • the order Thysanoptera includes the suborders Terebrantia and Tubulifera, the super families of Aeolothripoidea, Thripoidea, and Merothripoidea, and the families of Aeolothripidae, Heterothripidae, Thripidae, Uzelothripidae, and Phlaeothripidae.
  • thrips include greenhouse thrips ( Heliothrips haemorrhoidalis), banded greenhouse thrips (Hercinothrips femoral is), flower thrips (Frankliniella tritici), Western flower thrips (WFT) (Frankliniella occidental is), onion or tobacco thrips (Thrips tabaci), citrus thrips (Scirtothrips aurantii and Scirtothrips citri), cereals thrips (Limothrips cerealium), pea thrips (Kakothrips robustus), lily bulb thrips (Liothrips), black hunter thrips (Leptothrips mali), coffee thrips (Diarthrothrips), avocado thrips (Scirtothrips perseae), Thrips palmi, fruit tree thrips (Taeniothrips inconsequens), gladiolus thrips (Taen
  • the thrips is from a genus selected from the group consisting of Frankliniella, Thrips and Hoplandrothrips. In a more particular embodiment, the thrips is from a species selected from the group consisting of Frankliniella occidentalis, Thrips tabaci, T. palmi, T. simplex, T. fuscipennis, T. angusticeps and Heliothrips haemorrhoidalis.
  • whiteflies refers to insects of the order Hemiptera that typically feed on the undersides of plant leaves. Whiteflies comprise the family Aleyrodidae, the only family in the superfamily Aleyrodoidea. Main pest species of whiteflies include Aleurocanthus woglumi (citrus blackfly), Aleyrodes proletella (cabbage whitefly), Bemisia tabaci (silverleaf whitefly), Trialeurodes vaporariourum (greenhouse whitefly).
  • aphids refers to plant pest insects belonging to the family Aphididae, including but not limited to Aphis gossypii, A. fabae, A. glycines, A. nerii, A. nasturtii, Myzus persicae, M. cerasi, M. ornatus, Nasonovia especially N. ribisnigri, Macrosiphum especially M. euphorbiae, Aulacorthum especially A. solani and Brevicoryne.
  • moth refers to a group of insects that includes all member of the order Lepidoptera that are not butterflies.
  • the caterpillar of moths feed on leaves or other parts of plants and therefore, moths can be considered as plant pests.
  • the invention relates to the use of an astigmatid mite for rearing a predator insect for the family Miridae or from the family Anthocoridae on a crop.
  • the invention in another aspect, relates to a composition
  • a composition comprising at least a breeding population of a predator insect from the family Miridae or from the family Anthocoridae and a first source of food for said predator insect, wherein said first source of food comprises a population of an astigmatid mite, wherein said population of an astigmatid mite is not a population of Tyrophagus putrescentiae or a population of Carpoglyphus lactis, wherein the breeding population of the predator insect and the first source of food are physically separated so that there is no contact between the population of the predator insect and the population of the astigmatid mite.
  • composition refers to a combination of compounds or ingredients, in this particular case, the breeding population of the predator insect and the first source of food.
  • the astigmatid mite is not Tyrophagus putrescentiae. In a particular embodiment, the astigmatid mite is not Carpoglyphus lactis. In a particular embodiment, the astigmatid mite is not Tyrophagus putrescentiae or Carpoglyphus lactis. In a particular embodiment, the predator anthocorid is not Orius laevigatus. In a particular embodiment, the predator mirid is not Nesidiocoris tenuis. In a particular embodiment, the predator mirid is not Macrolophus pygmaeus.
  • the predator insect is not Orius laevigatus, Nesidiocoris tenuis or Macrolophus pygmaeus.
  • the composition does not comprise Tyrophagus putrescentiae and Orius laevigatus.
  • the composition does not comprise Tyrophagus putrescentiae and Nesidiocoris tenuis.
  • the composition does not comprise Tyrophagus putrescentiae and Macrolophus pygmaeus.
  • the composition does not comprise Carpoglyphus lactis and Orius laevigatus.
  • the composition does not comprise Carpoglyphus lactis and Nesidiocoris tenuis.
  • the composition does not comprise Carpoglyphus lactis and Macrolophus pygmaeus. In a particular embodiment, the composition does not comprise Tyrophagus putrescentiae and Orius laevigatus, or T. putrescentiae and Nesidiocoris tenuis, or T. putrescentiae and Macrolophus pygmaeus, or Carpoglyphus lactis and Orius laevigatus, or Carpoglyphus lactis and Nesidiocoris tenuis, or Carpoglyphus lactis and Macrolophus pygmaeus.
  • the composition comprises one the following combinations of predator and prey:
  • Dicyphus errans as predator and Tyrolichus casei as astigmatid mite Dicyphus errans as predator and Tyrolichus casei as astigmatid mite.
  • Dicyphus errans as predator and Acarus siro as astigmatid mite Dicyphus errans as predator and Acarus siro as astigmatid mite.
  • Dicyphus hesperus as predator and Suidasia medanensis as astigmatid mite.
  • Dicyphus hesperus as predator and Thyreophagus entomophagus as astigmatid mite.
  • Dicyphus hesperus as predator and Aleuroglyphus ovatus as astigmatid mite.
  • Dicyphus hesperus as predator and Tyrolichus casei as astigmatid mite.
  • Dicyphus hesperus as predator and Acarus siro as astigmatid mite.
  • Macrolophus pygmaeus as predator and Suidasia medanensis as astigmatid mite.
  • Macrolophus pygmaeus as predator and Tyrolichus casei as astigmatid mite.
  • Macrolophus pygmaeus as predator and Thyreophagus entomophagus as astigmatid mite.
  • Macrolophus pygmaeus as predator and Aleuroglyphus ovatus as astigmatid mite.
  • Macrolophus pygmaeus as predator and Acarus siro as astigmatid mite.
  • Anthocoris nemoralis as predator and Aleuroglyphus ovatus as astigmatid mite.
  • the composition further comprises a second source of food for the predator insect.
  • second source of food has been previously defined in connection with the first method of the invention. All the particular and preferred embodiments of this term fully apply to the composition of the invention.
  • the wherein the second source of food is selected from the group consisting of eggs of Ephestia kuehniella, cysts of Artemia, eggs of Sitotroga cerealella, pollen, nematodes, larvae of Ceratitis capitata, larvae and/or adults of Drosophila melanogaster, artificial foods, and a combination thereof.
  • the invention refers to the use of the composition of the invention for controlling a pest in a plant, wherein the pest is a prey for the predatory insect.
  • the invention in another aspect, relates to a method of obtaining a strain of a predator insect from the family Miridae or from the family Anthocoridae with an increased tolerance to a diet based on a factitious food, hereinafter third method of the invention, the method comprising artificially selecting a strain of the predator insect for its tolerance to a diet based on pollen under a selective pressure, wherein said selective pressure comprises using pollen as the main source of food.
  • the terms “predator insect”, “family Miridae”, “family Anthocoridae” have been previously defined in connection with the first method of the invention. All the particular and preferred embodiments of said terms defined for the first method of the invention also apply to the third method of the invention.
  • the predator insect from the family Miridae is from a genus selected from the group consisting of Macrolophus, Nesidiocoris, Dicyphus, Deraeocoris, Engytatus, Tupiocoris, Campyloneuropsis, Cyrtopeltis, Pilophorus, Campylomma, Cyrtorhinus, Orthotylus, Eurotas.
  • the predator insect from the family Miridae is from a species selected from the group consisting of Macrolophus pygmaeus, M. costalis, M. basicornis, Nesidiocoris tenuis, N. volucer, N. callani, Dicyphus bolivari., D.
  • the predator insect from the family Miridae is from the genus Nesidiocoris or from the genus Dicyphus or from the genus Macrolophus, more particularly from the species Nesidiocoris tenuis or from the species Dicyphus bolivari or from the species Macrolophus pygmaeus or from the species Dicyphus hesperus.
  • the predator insect from the family Anthocoridae is an insect from a genus selected from the group consisting of Orius, Anthocoris, Blaptostethus, Montandoniola and Xylocoris.
  • the predator insect from the family Anthocoridae is an insect from a species selected from the group consisting of Orius laevigatus, O. insidiosus, O. majusculus, O. niger, O. albidipennis, O. minutus, O. thripoborus, O. naivashae, O. strigicollis, O. sauteri, O. tristicolor, O. nagaii, O. antillus, O. limbatus, O. thripoborus, O. naiashae, O. horvathi, O. vicinus, O. pumilio, Orius laticollis, O.
  • the predator insect from the family Anthocoridae is Orius laevigatus.
  • the term “diet based on a factitious food” means that the factitious food represents the main source of calories of the diet. In a particular embodiment, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or the 100% of the calories of the diet are provided by the factitious food.
  • the term “factitious food”, as used herein, refers to any food different to the one that the animal, in this case the predator insect from the families Miridae or Anthocoridae, eats on its natural habitat.
  • the factitious food comprises a factitious prey, an artificial food or a mixture thereof. In a particular embodiment, the factitious food is not pollen.
  • the term “factitious prey”, as used herein, refers to prey that serve as food for predators although they are not the natural prey for the predator.
  • the factitious prey is selected from the group consisting of an astigmatid mite, eggs of Ephestia kuehniella, cysts of Artemia, eggs of Sitotroga cerealella, nematodes, larvae of Ceratitis capitate and larvae and/or adults of Drosophila melanogaster. All these terms have been previously defined in connection with the first method of the invention.
  • the factitious prey is an astigmatid mite.
  • the astigmatid mite is a storage food mite.
  • the storage food mite is from a family selected from the group consisting of Acaridae, Carpoglyphidae, Glycyphagidae, Pyroglyphidae, Chortoglyphagidae and Suidasiidae.
  • the storage food mite is from a genus selected from the group consisting of Acarus, Tyrophagus, Aleuroglyphus, Lardoglyphus, Caloglyphus, Suidasia, Thyreophagus, Carpoglyphus, Glycyphagus, Tyrolichus, Lepidoglyphus, Blomia and Chortoglyphus.
  • the storage food mite is from a species selected from the group consisting of Suidasia medanensis, Suidasia nesbitti, Lepidoglyphus (Glycyphagus) destructor, Blomia freeman, Carpoglyphus lactis, Carpoglyphus munroi, Carpoglyphus wardleorum, Lardoglyphus konoi, Aleuroglyphus ovatus, Tyrolichus casei, Thyreophagus entomophagus, Chortoglyphus arcuatus and Acarus siro.
  • the storage food mite is from a family selected from the group consisting of Acaridae, Carpoglyphidae and Suidasiidae, more particularly from a genus selected from the group consisting of Acarus, Carpoglyphus, Suidasia, Tyreophagus, Aleuroglyphus and Tyrolichus, even more particularly from a species selected from the group consisting of Acarus siro, Carpoglyphus lactis, Suidasia medanensis, Tyreophagus entomophagus, Aleuroglyphus ovatus and Tyrolichus casei.
  • the term “artificial food” has been previously defined.
  • the third method of the invention allows obtaining a strain of a predator insect from the families Miridae or Anthocoridae with an increased tolerance to a diet based on a factitious food, preferably, a factitious prey, more preferably an astigmatid mite.
  • the term “increased tolerance” refers to an augmented tolerance compared to a wild predator insect from the same species.
  • the term “increased tolerance” refers to an augmented tolerance compared to an insect from the same species that is acclimated to pollen, but that has not been selected for its tolerance to pollen.
  • acclimated means than the insect derives from a population that has been feed with a suboptimal diet based on pollen, for example, providing pollen every 2-3 days and a suboptimal quantity of Ephestia eggs every week.
  • an increased tolerance means increased nymph survival, increased fecundity and increased longevity.
  • the strain of the predator insect obtained by the third method of the invention has increased nymph survival compared to a predator insect of the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • nymph survival refers to the percentage of nymphs that reach adulthood of the total initial number of nymphs emerged from the eggs.
  • the nymph survival is considered increased when it is at least a 1 %, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95% higher or even higher than the nymph survival of a predator insect from the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • the strain of the predator insect obtained by the third method of the invention has increased fecundity compared to a predator insect of the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • the term “fecundity” refers to the ability of the predator insect to lay eggs, being measured as the average number of eggs laid by a female.
  • the fecundity is expressed as a rate of egg or offspring production or as a total number of eggs or offspring produced by the predator insect during a period of time.
  • the fecundity is determined through a period of time of 10 days.
  • the fecundity is “lifetime fecundity”, that is, fecundity throughout the life of the individual.
  • the fecundity is considered increased when it is at least a 1%, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95% higher or even higher than the fecundity of a predator insect from the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • the strain of the predator insect obtained by the third method of the invention has increased longevity compared to a predator insect of the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • the term “longevity” refers to the lifespan of the predator insect as adult.
  • the longevity is considered increased when it is at least a 1 %, at least a 2%, at least a 3%, at least a 4%, at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95% higher or even higher than the longevity of a predator insect from the same species that has not been selected for its tolerance to a diet based on a factitious food.
  • the term “artificially selecting” means that the predator insect is growth under conditions of selective pressure introduced by the human being, in particular, the selective pressure comprises using pollen as the main source of food.
  • the term “main source of food”, as used herein, means that pollen is the main source of calories of the diet. In a particular embodiment, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or the 100% of the calories of the diet are provided by pollen. In a particular embodiment, selective pressure consists on providing pollen as the only source of food.
  • each selected population of the predator insect is maintained before the next selection step with a diet comprising mainly pollen and also other factitious foods, such as Ephestia eggs, before the next cycle of selection.
  • a diet comprising mainly pollen and also other factitious foods, such as Ephestia eggs
  • the selected population is bred with a diet comprising pollen and once a week, other factitious food, such as Ephestia eggs.
  • Artificial selection comprises breeding a population of the predator insect under conditions of selective pressure, in particular, using pollen as the main source of food, and selecting those individuals that better perform under said conditions.
  • the individuals that better performed can be selected based on any of the parameters “nymph survival”, “fecundity” or “longevity”, but are preferably selected based on the parameter “fecundity”.
  • the individuals preferably the female individuals having a nymph survival, fecundity and/or longevity, higher than the mean of any of those parameters in the whole population are selected.
  • the offspring from the individuals previously selected are selected and mixed so that they form a new population.
  • the steps of breeding the population under the selective pressure comprising using pollen as the main source of food, selecting the individuals, preferably the female individuals, with a better performance, preferably with a higher fecundity, selecting the offspring from these individuals and mixing the selected offspring to form a new population is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times at least 11 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times or more.
  • the whole selection cycles are performed at least 11 times.
  • the third method of the invention comprises:
  • step (ii) selecting the female individuals from the population used in step (i) that have a higher fecundity
  • step (iv) mixing the offspring selected in step (iii) to form a new population
  • step (v) repeating steps (i) through (iv) at least once, preferably at least 5 times, more preferably at least 10 times.
  • the invention in another aspect, relates to a method of obtaining a strain of a predator insect from the family Miridae or from the family Anthocoridae with an increased tolerance to a diet based on a factitious food, the method comprising artificially selecting a strain of the predator insect for its tolerance to low temperatures under a selective pressure, wherein said selective pressure comprises rearing the population of the predator insect at a temperature lower than 20°C.
  • low temperature refers to a temperature lower than 20°C, for example, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C or lower, preferably 15°C.
  • the selective pressure comprises rearing the population of the predator insect at a temperature lower than 20°C, preferably a temperature higher than 9°C, for example, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C or 10°C.
  • the third method of the invention comprises:
  • step (ii) selecting the female individuals from the population used in step (i) that have a higher fecundity
  • step (iv) mixing the offspring selected in step (iii) to form a new population
  • the invention relates to a strain of a predator insect obtainable by the methods for obtaining a strain with increased tolerance to a diet based on a factitious food.
  • the predator insect from the family Miridae is from the genus Nesidiocoris, Dicyphus or Macrolophus, more particularly from the species Nesidiocoris tenuis, Macrolophus pygmaeus, Dicyphus bolivari, Dicyphus hesperus, or Dicyphus errans.
  • the predator insect from the family Anthocoridae is an insect from a genus selected from the group consisting of Orius, Anthocoris, Blaptostethus, Montandoniola and Xylocoris.
  • the predator insect from the family Anthocoridae is an insect from a species selected from the group consisting of Orius laevigatus, O. insidiosus, O. majusculus, O. niger, O. albidipennis, O. minutus, O. thripoborus, O. naivashae, O. strigicollis, O. sauteri, O. tristicolor, O. nagaii, O. antillus, O. limbatus, O. thripoborus, O. naiashae, O. horvathi, O. vicinus, O. pumilio, Orius laticollis, O.
  • the predator insect from the family Anthocoridae is Orius laevigatus.
  • the strain of a predator insect obtainable by the methods for obtaining a strain with increased tolerance to a diet based on a factitious food is characterized by a particular amplification pattern when the genome of the strain is amplified by PCR with primers for detecting the SSR (single sequence repetition) Rp02.
  • the strain of a predator insect obtainable by the methods for obtaining a strain with increased tolerance to a diet based on a factitious food is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained in at least 70% of the individuals of the strain:
  • the strain of the predator insect from the species Orius laevigatus obtained by the method based on the selective preasure comprising a diet based on pollen is characterized in that when the genome of the strain is amplified by PCR using the pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2 a fragment of 780-840 bp is obtained in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or the 100% of the individuals of the strain of Orius laevigatus.
  • the strain of the predator insect from the species Orius laevigatus obtained by the method based on the selective preasure based on low temperature is characterized in that when the genome of the strain is amplified by PCR using the pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2 fragment of 1280-1360 base pairs, a fragment of 580-640 base pairs and a fragment of 420-480 base pairs are obtained in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or the 100% of the individuals of the strain of Orius laevigatus.
  • PCR or “polymerase chain reaction”, as used herein, relates to a DNA amplification process by means of polymerase chain reaction, that allows amplifying a single or a few copies of a DNA sequence across several orders of magnitude, generating as a result thousand to millions of copies of a particular DNA sequence.
  • the PCR method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.
  • Primers short DNA fragments
  • Primers short DNA fragments
  • containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification.
  • PCR As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. The PCR process is well known in the art and is thus not described in detail herein.
  • the PCR is performed using the program shown in Figure 10.
  • the PCR is performed under the following conditions: using 1 unit of VWR® Red Taq DNA Polymerase, 1X Key Buffer, 3mM ChMg, 0.5mM of DNTPs, 10pM from each primer, and 5-25ng genomic DNA per reaction, and when the PCR is run in an Eppendorf Mastercycler Pro according to the program shown in Figure 10. Results can be analyzed by electrophoresis in 1.2% agarose gel.
  • the invention relates to a strain of a predator insect of the species Orius laevigatus with increased tolerance to a diet based on a factitious food, wherein said strain is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained:
  • the invention in another aspect, relates to a method for controlling a pest in a crop comprising providing the crop with at least a breeding population of a predator insect from the family Miridae and/or a predator insect from the family Anthocoridae, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by
  • the methods (a) and (b) are the methods for for obtaining a strain with increased tolerance to a diet based on a factitious food of the invention previously defined.
  • the invention relates to the use of a predator insect from the family Miridae and/or a predator insect from the family Anthocoriae, for controlling a pest in a crop, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is obtained by
  • the methods for obtaining the strain with increased tolerance to a diet based on a factitious food is the method previously defined.
  • the method further comprises providing the crop with a first source of food for the predator insect, wherein said first source of food is a factitious food.
  • said factitious food is selected from the group consisting of a factitious prey, an artificial food or a mixture thereof.
  • the factitious prey is selected from the group consisting of an astigmatid mite, eggs of Ephestia kuehniella, cysts of Artemia, eggs of Sitotroga cerealella, nematodes, larvae of Ceratitis capitate and larvae and adults of Drosophila melanogaster.
  • the crop is selected from the group consisting of vegetables, orchards, vineyard, olive trees, berries and ornamental plants, including pot plants and cut flowers.
  • the pest is selected from the group consisting of thrips, whiteflies, aphids and moths.
  • the thrips is from a genus selected from the group consisting of Frankliniella, Thrips and Hoplandrothrips.
  • the thrips is from a species selected from the group consisting of Frankliniella occidentalis, Thrips tabaci, T. palmi, T. simplex, T. fuscipennis, T. angusticeps and Heliothrips haemorrhoidalis.
  • the invention in another aspect, relates to a method for controlling a pest in a crop comprising providing the crop with at least a breeding population of a predator insect from the species Orius laevigatus, wherein the predator insect is from a strain with increased tolerance to a diet based on a factitious food, wherein said strain is characterized in that when the genome of the strain is amplified by PCR using the pair or primers of SEQ ID NO: 1 and SEQ ID NO: 2 the following fragments are obtained in at least 70% of the individuals of the strain:
  • the strain of the predator insect is obtained by the methods for obtaining a strain of a predator insect with increased tolerance to a diet based on a factitious food of the invention previously described.
  • a method of obtaining a strain of a predator insect from the family Miridae or from the family Anthocoridae with an increased tolerance to a diet based on a factitious food comprising artificially selecting a strain of the predator insect for its tolerance to a diet based on pollen under a selective pressure, wherein said selective pressure comprises using pollen as the main source of food.
  • step (ii) selecting the female individuals from the population used in step (i) that have a higher fecundity
  • step (iv) mixing the offspring selected in step (iii) to form a new population
  • the predator insect from the family Miridae is from a genus selected from the group consisting of Macrolophus, Nesidiocoris, Dicyphus, Deraeocoris Engytatus, Tupiocoris, Campyloneuropsis, Cyrtopeltis, Pilophorus, Campylomma, Cyrtorhinus, Orthotylus, Eurotas, or wherein the predator insect from the family Anthocoridae is from a genus selected from the group consisting of Orius, Anthocoris, Wollastoniella, Blaptostethus, Montandoniola, Macrotracheliella and Xylocoris. 4.
  • the predator insect from the family Miridae is an insect from a species selected from the group consisting of Macrolophus pygmaeus, M. costalis, M. basicornis, Nesidiocoris tenuis, N. volucer, N. callani, Dicyphus bolivari., D. errans, D. hesperus, D. marrocanus, D. geniculatus, D. tamaninii, Engytatus varians, E. modestus, Tupiocoris cucurbitaceus, Campyloneuropsis infumatus, Cyrtopeltis callosus, Deraeocoris brevis, D.
  • the predator insect from the family Anthocoridae is an insect from a species selected from the group consisting of Orius laevigatus, O. insidiosus, O. majusculus, O. niger, O. albidipennis, O. minutus, O. thripoborus, O. naivashae, O. strigicollis, O. sauteri, O. tristicolor, O. nagaii, O. antillus, O. limbatus, O. thripoborus, O. naiashae, O. horvathi, O. vicinus, O. pumilio, Orius laticollis, O.
  • a strain of a predator insect obtainable by the method according to any one of aspects 1 to 7.
  • the trial was conducted in a commercial greenhouse in the south of Spain (Almeria) in the spring-summer of 2019.
  • the greenhouse was a typical ‘parral’ structure used in the Mediterranean area with 2,400 m 2 .
  • a pepper crop of a mini Capia type variety Pepperito was transplanted in May the 10 th .
  • Two introductions of O. laevigatus were performed, one with a dosage of 5 individuals/m 2 in June the 21 st , and another one with 3 individuals/m 2 , in July the 5 th .
  • Four treatments were evaluated applying different in-crop factitious prey for the predator:
  • Carpoglyphus lactis (Acari: Astigmata)
  • Treatments 1 , 2 and 3 were applied in approximately 750 m 2
  • treatment 4 control was applied in a surface of approximately 150 m 2 .
  • the grower did not want to establish a bigger area as a control plot, without a feeding system, since he had already released high amounts of O. laevigatus in two previous crops (same variety of peppers) without success.
  • This control plot was separated with a plastic screen 2.5 m high to avoid movement of predators from/to the area where foods were introduced.
  • ⁇ SE Mean number ( ⁇ SE) of nymphs and adults of O. laevigatus counted in the four treatments in each sampling date are represented in figure 1.
  • the populations were well established in 100% of the plants in the 3 treatments with in-crop factitious prey added, while only 10-30% of the plants had predators in the control plot.
  • the total mean number of individuals per two flowers was 1.26, 1 .26, 1 .20 and 0.26 in treatments 1 , 2, 3 and 4, respectively, so it was about 5 times higher in the treatments with factitious prey compared to the control.
  • Nesidiocoris tenuis Heteroptera: Miridae
  • the strategy was compared to the standard procedure in the South of Spain, where commonly only one (occasionally two) additions of eggs of the moth Ephestia kuehniella, as a factitious prey, are introduced at the top of the plants, at the same time when the predators are released. In most of the cases the predators are released in the seedlings, before transplanting the crop, but sometimes, they are introduced during the first weeks after transplanting.
  • the plastic greenhouse had 4.500 m 2 , with the typical ‘parral’ structure of the area, with a flat roof, with zenithal and lateral windows to offer ventilation, all covered with insect proof nets, and without active climate control, which are the structures comprising 80% Almeria’s greenhouses.
  • a tomato crop of the variety Genio (Clause) was transplanted on August the 23 th , 2017, with a density of 1.5 plants I m 2 .
  • the predator N. tenuis was released in the crop with a rate of 0.88 individuals/plant on September the 7th.
  • the same day when predators were released a feeding was performed, following the standard procedure by the growers, introducing 10 g of eggs of the moth E. kuehniella every 1 ,000 predators.
  • Treatments 1 and 2 were applied in approximately 600 m 2 and were separated by the central corridor of the greenhouse.
  • Treatment 3 (control) was applied in a surface of aprox. 1 .300 m 2 .
  • An insect proof net was established as a barrier to separate physically the control treatment. The height of the barrier was about 2.5 meters, and it was considered that it was enough to minimize flying adults from/to the area where foods were introduced.
  • the populations of the mite S. medanensis were produced and packaged in the facilities of Agrobio, with bran and vermiculite as a substrate. It is a product commercially sold by Agrobio with the trade name Powerfood, packaged in cardboard bottles of 1 liter or paper bags of 5 liters, with a density of about 2 million of mites per liter. Mites were introduced in treatments 1 , every two to three weeks, in the following dates: 21/9, 4/10, 16/10, 31/10, 17/11 , 8/12, 27/12, 17/01 , 30/01 , 21/02, 14/03, 28/03 and 18/04. The dosage introduced each feeding day was 27 liters/ ha. To distribute the material a full spoon was released by hand (aprox 1.8 mis) in each plant.
  • treatment 2 Artemia was introduced at a commercial dosage of 200 grams/ha (250,000 cysts per gram) in the same feeding dates than treatment 1. The material was spread applying a small amount (aprox. 0.07 g) at the top of a leave (on a medium-higher position) every 5 plants.
  • ⁇ SE Mean number ( ⁇ SE) of nymphs and adults of N. tenuis counted in the three treatments in each sampling date are represented in figure 2.
  • the populations were well established in 100% of the plants in the 3 treatments.
  • the total mean number of individuals was 3.1 , 2.1 and 1.2 in treatments 1 , 2 and 3, respectively. No differences among treatments were observed the first two months, when temperatures were low (no heating system in the greenhouse), but from January, when temperatures slowly increased, N. tenuis developed higher populations in the treatments with in-crop food added, compared to the control.
  • the aim is to compare these strategies with a standard procedure, without a feeding program, and also to compare it with the introduction of the eggs of the moth Ephestia kuehniella, which is a well-known factitious food with a high nutritive quality, used in most of the laboratory and industrial mass-rearing systems of predatory bugs produced in climatic chambers by biomanufacturers.
  • the trial was conducted in an experimental greenhouse in the south of Spain (Almeria) in the autumn-spring of 2018/19.
  • the plastic greenhouse had 800 m 2 , with the typical ‘parral’ structure of the area, with a flat roof, with zenithal and lateral windows to offer ventilation, all covered with insect proof nets, and without active climate control, which are the structures comprising 80% Almeria’s greenhouses.
  • a tomato crop of the variety Caniles was transplanted on October the 31 th , 2018, with a density of 1.5 plants/m 2 .
  • the predator D. bolivari was released in the seedlings two weeks before transplanting, with a rate of 1.5 individuals/plant. The same day when the predators were released a feeding was performed, introducing 10 gr of eggs of the moth E.
  • T2 T. casei complemented with cysts of the brine shrimp Artemia (Branchiopoda: Artemiidae)
  • the populations of the mite T. casei were produced and packaged in the facilities of Agrobio, with bran and vermiculite as a substrate, with a density between 1 and 2 million of mites per liter. Mites were introduced in treatments 1 and 2, every two to three weeks, in the following dates: 21/9, 4/10, 16/10, 31/10, 17/11 , 8/12, 27/12, 17/01 , 30/01 , 21/02, 14/03, 28/03 and 18/04.
  • the dosage introduced each feeding day was 33 liters/ ha. To distribute the material a full spoon was released by hand (approximately 2.2 mL) in each plant.
  • ⁇ SE Mean number ( ⁇ SE) of nymphs and adults of D. bolivari counted in the four treatments in each sampling date are represented in figure 3.
  • the populations were well established in 100% of the plants in the 4 treatments. However, the numbers were very different, with a total mean number of individuals (every 3 leaves) of 1 .8, 2.4, 2.0 and 0.5 in treatments 1 , 2, 3 and 4, respectively.
  • the populations of predators were kept at high numbers during the winter in all the treatments with a feeding program, while the populations were lower in the control plot, as expected.
  • the same low numbers of another miridae (/V. tenuis) are counted in most of the tomato greenhouses in winter in the south of Spain, where no heating system is available, and no feeding program is applied.
  • Wild populations of Orius laevigatus were collected from natural flowering plants in different non-cultivated areas all over the Mediterranean basin. For each population, early fecundity (10 days) was assessed on an alternative diet consisting of finely ground dehydrated commercial honey bee pollen (hereafter pollen). Freshly emerged adults ( ⁇ 24 h old) from each population were separated. After 4 days during which mating and pre-oviposition period took place, adults were sexed and females isolated to test fecundity. At least 50 females were placed individually into small polypropylene recipients (45 mL) with ventilated lids with a piece of green bean pod end-sealed with paraffin wax as an egg-laying substrate, and pollen as food.
  • pollen finely ground dehydrated commercial honey bee pollen
  • the selected strain was reared on a suboptimal diet, providing with pollen every 2-3 days, and with restricted (half the optimum) Ephestia eggs every week, in order to maintain zoophytophagy in balance.
  • each selection cycle consisted on evaluating at least 200 females for their Fw feeding on pollen.
  • Adults were changed from the suboptimal diet to pollen within 24 h after emergence.
  • Three days after, females were isolated and tested for fecundity during 10 days.
  • the offspring from those females with a Fw value above the mean rate in the previous selection cycle was mixed together to initiate the next selection cycle.
  • This procedure was followed for every subsequent selection cycle up to the sixth one (POL06). From then on, solely for the purpose of accelerating genetic gaining, a full-sibling family selection was carried out up to reach POL11.
  • the offspring of the most fecund females was selected separately and then these second-generation families were assayed in the same way, isolating females to evaluate Fw, keeping bean pods with eggs at low temperature (15 °C). Finally, the best FI families feeding on pollen were selected, mixing their offspring together to obtain the next selection step.
  • the program r m 2.0 was used (Taberner A, Castahera P, Silvestre E, Bopazo J (1993) Comput Appl Biosci 9:535-5409), applied to the surviving data and the fecundity rates at different age classes, previously calculated, and considering a sex ratio of 1 :1 based on the development observed during the bioassay.
  • the data analysis was carried out using the bootstrap technique, performing 1000 repetitions, as suggested (Aragon-Sanchez M, Roman-Fernandez LR, Martinez- Garcia H et al (2016). BioControl 63: 785-794). Survival by ages (/ x ), fecundity by ages (m x ), as well as the following parameters were estimated from the life tables: intrinsic rate of natural increase (r m ) and net reproduction rate (Ro).
  • Table 1 shows results of immature survival, longevity and lifetime fecundity for both nutritious treatments, Ephestia eggs and pollen. Immature survival on pollen was considerably higher in the selected strain (28%) compared to the reference populations (2-8%). By contrast, in the Ephestia treatment, survival was very similar among the Agrobio commercial population and the selected strain (71-76%), and faintly lower for the pollen-acclimated and Koppert commercial populations (52-57%).
  • the new strain showed enhanced reproductive fitness feeding on pollen compared to commercial populations.
  • This strain selected for better performance when fed a plant-produced material like pollen showed no trade-offs when feeding on prey, with no differences among the new strain and the commercially available populations in life-history values when feeding on the factitious prey.
  • the new selected strain also exhibited better performance when fed pollen than the acclimated strain. Therefore, the improvement was due to genetic gain rather than to phenotypic plasticity to tolerate nutritious restriction.
  • Example 5 Characterization of the ability of the new strain of Orius laevigatus to feed on a factitious prey
  • a new strain of Orius laevigatus was selected for better fitness when fed a plant- produced material, showing a superior ability to exploit non-prey food. This selection might involve a loss in the ability to reproduce on prey.
  • This strain showed similar fitness than commercial populations when feeding on a highly nutritious prey, like Ephestia eggs. However, it was necessary to know whether the reproductive performance was maintained when fed a suboptimal factitious prey of poor nutritional quality.
  • the objective was to compare the longevity and fecundity of the new strain, selected for the trait ‘better performance when fed pollen’, with a standard commercial population of O. laevigatus, when provided with a suboptimal factitious prey, Astigmatid mites, as the main food source.
  • Biological characteristics of adults were evaluated on the diet based on Astigmatid mites, Carpoglyphus lactis produced and commercially sold by Agrobio (Powermite), which are packaged with bran and vermiculite as a substrate in cardboard bottles (density between 1-5 million mites per liter).
  • the selected strain (POL11) was compared with one commercial population (Agrobio).
  • the new strain exhibited better performance than the standard population when fed Astigmatid mites.
  • This new strain of O. laevigatus was selected for the trait ‘better performance with pollen as food’. Pollen being a plant material, the selection might involve a loss in the ability to exploit animal prey. Our aim was to check whether this loss was not too large, and the new strain was capable to maintain reproductive fitness when fed a factitious prey of poor nutritional quality like Astigmatid mites. Unexpectedly, not only was the fitness maintained, but the new strain performed much better when fed this suboptimal animal prey, compared to the standard commercially available O. laevigatus.
  • the trial was conducted in a commercial greenhouse in the south of Spain (Almeria) in the spring of 2020.
  • the greenhouse was a typical ‘parral’ structure used in the Mediterranean area with aprox. 2,000 m 2 .
  • a pepper crop of the mini Capia variety ‘Pepperito’ was transplanted in February the 2 on .
  • Two introductions of O. laevigatus were performed, each one with a dosage of 4 individuals/m 2 , in March the 18 th and the 23 rd . Two treatments were evaluated:
  • the treatments were separated by a net that was placed in the middle of the greenhouse from the ground to the roof.
  • the predators of both treatments were fed with a regular program of introductions of factitious prey.
  • Example 7 Introduction of astigmatid mites as food for the new strain of Orius laevigatus in a cucumber crop
  • the trial was conducted in a commercial greenhouse in the south of Spain (Almeria) in the spring of 2020.
  • the greenhouse was a typical ‘parral’ structure used in the Mediterranean area with aprox. 3,000 m 2 .
  • a cucumber crop of a Dutch type variety Litoral (Rijk Zwaan) was transplanted in February the 27 th .
  • Five introductions of O. laevigatus were performed, each one with a dosage of 2 individuals/m 2 , in March the 18 th and the 25 th , and in April the 1 st , the 8 th and the 15 th .
  • Two treatments were evaluated:
  • the treatments were separated by a net that was placed in the middle of the greenhouse from the ground to the roof.
  • the predators of both treatments were fed with a regular program of introductions of factitious prey.
  • the cysts of brine shrimps were homogeneously mixed with the populations of mites and distributed together with the mites. To distribute the material a small spoon was released by hand (aprox 0.33 mis) in every two plants.
  • the populations of thrips were successfully controlled in both treatments but were lower in the treatment with the new strain of the predator in the last two sampling dates. A decrease of the populations of thrips was obtained with the new predator strain from mid-April, where the pest populations decreased to half the numbers compared to the control in mid- May.
  • the trial was conducted in a commercial greenhouse situated in the Dutch province of Gelderland, between January and March 2020.
  • the greenhouse was a typical high-tech Dutch glasshouse with artificial light and heating system with aprox. 3 hectares.
  • Chrysanthemums are planted all year round (5 cycles per year), and following the common practice of the producers, the greenhouse was divided in plots of approx. 1 ,000 m 2 , and different varieties of Chrysanthemums were planted in different plots with different starting dates, with a duration of each crop of 10 weeks.
  • the trial was performed on two different plots of 1 ,000 m 2 with the same variety, both planted on January the 28 th .
  • O. laevigatus was performed the first week after transplanting with a dosage of 10 individuals/m 2 . Two treatments were evaluated (one in each plot) based on releases of different predator populations:
  • the predators of both treatments were fed with a regular program of introductions of factitious prey.
  • 2.5 liters of a populations of the astigmatid mite Suidasia medanensis, commercially sold by Agrobio (Powerfood) as in-crop food for predatory mites were introduced once per week during all the trial.
  • This prey mite populations which are packaged with bran and vermiculite as a substrate in paper bags (aprox. 1 million mites per liter), were complemented in each release with 15 grams of cysts of the brine shrimps Artemia (250,000 cysts/gram).
  • the cysts of brine shrimps were homogeneously mixed with the 2.5 I populations of mites and distributed together with the mites.
  • Biospreader designed to distribute mites was used. This is based on a modified spin disc apparatus that is hang in the irrigation system and offers an even distribution of the material on the top of all the plants, so that a ratio of >100 factitious prey mites/plant were introduced each time.
  • Example 10 Characterization of the ability of the new strain of Orius laevigatus to develop feeding on two astigmatid mites
  • a new strain of Orius laevigatus was selected for better fitness when fed a plant- produced material, showing a superior ability to exploit non-prey food. This selection might involve a loss in the ability to reproduce on prey.
  • This strain showed similar fitness than commercial populations when feeding on a highly nutritious prey, like Ephestia eggs.
  • it was tested that the new strain exhibited better performance than the standard population when fed the Astigmatid mite species Carpoglyphus lactis (Example 5). However, it was necessary to know whether the performance was maintained when fed on other Astigmatid mites species.
  • the survival from egg to adult of the new strain of Orius laevigatus feeding on two Astigmatid mite species, Tyrolichus casei and Aleuroglyphus ovatus was assessed.
  • the objective was to compare the survival of the new strain, selected for the trait ‘better performance when fed pollen’, with a standard commercial population of O. laevigatus, when provided with a suboptimal factitious prey, Astigmatid mites, as the main food source.
  • Immature survival was evaluated on the diet based on Astigmatid mites, Tyrolichus casei and Aleuroglyphus ovatus, produced by Agrobio, which are packaged with bran and vermiculite as a substrate in cardboard bottles (density between 1-5 million mites per liter).
  • the selected strain (POL11) was compared with one commercial population (Agrobio).
  • the new strain exhibited better performance than the standard population when fed both Astigmatid mites.
  • This new strain of O. laevigatus was selected for the trait ‘better performance with pollen as food’. Pollen being a plant material, the selection might involve a loss in the ability to exploit animal prey.
  • a previous experiment (example 5) we checked that not only was this loss not too large, but the new strain outperformed the standard population when fed a factitious prey of poor nutritional quality like the Astigmatid mite Carpoglyphus lactis.
  • Example 11 Characterization of the ability of Orius laevigatus to reproduce feeding on other prey mites
  • the standard Orius laevigatus strain was able to reproduce feeding on the Astigmatid mite Carpoglyphus lactis (see Example 5), but there is no information about whether is able to reproduce feeding on other Astigmatid mites, such as Tyrolichus casei, Aleuroglyphus ovatus and Tyreophagus entomophagus.
  • Lifetime fecundity was evaluated on the diet based on the Astigmatid mites, Tyrolichus casei, Aleuroglyphus ovatus and Tyreophagus entomophagus produced by Agrobio, which are packaged with bran and vermiculite as a substrate in cardboard bottles (density between 1-5 million mites per liter).
  • O. laevigatus was able to reproduce feeding on the Astigmatid mites, Tyrolichus casei, Aleuroglyphus ovatus and Tyreophagus entomophagus.
  • Example 12 Selection of a strain of Orius laevigatus tolerant to low temperatures
  • BCAs Biological control agents
  • O. laevigatus 11°C
  • F. occidentalis 8°C
  • Wild populations of Orius laevigatus were collected from natural flowering plants in different non-cultivated areas all over the Mediterranean basin. For each population, early fecundity (10 days) was assessed at 15°C. Freshly emerged adults ( ⁇ 24 h old) from each population were separated. After 4 days during which mating and pre-oviposition period took place, adults were sexed and females isolated to test fecundity. At least 50 females were placed individually into small polypropylene recipients (45 mL) with ventilated lids with a piece of green bean pod end-sealed with paraffin wax as an egglaying substrate, and Ephestia eggs as food. Eggs counting was carried out every 7 days, switching females to a clean piece of bean pod and adding fresh food.
  • each selection cycle consisted on evaluating at least 200 females for their Fvsat 15°C. Females were isolated and tested for fecundity during 15 days. Then, the offspring from those females with a F15 value above the mean rate in the previous selection cycle was mixed together to initiate the next selection cycle. This procedure was followed for every subsequent selection cycle, eventually obtaining the strain COLD PLUS.
  • Table 3 shows results of early fecundity (F15) on Ephestia eggs at low temperature (15°C) for the selected population (COLD PLUS) and commercial populations as reference.
  • the new strain showed enhanced reproductive fitness at temperatures close to its lowest thermal threshold ( ⁇ 15°C) compared to commercial populations.
  • Example 13 Genetical characterization of two selected strains that perform better when eating astigmatid mites as a factitious prey
  • Genomic DNA from individuals of the two selected strains, Orius PLUS and Orius COLD-PLUS and from individuals of a wild-type (WT) of Orius laevigatus population (used as a control) was isolated by E.Z.N.A.® insect DNA kit (Omega Bio-Tek) following the manufacturer’s instructions from individuals at adult stage, which were previously grinded during 5 minutes at 35Hz by a laboratory ball mill (Retsch MM400) with a 3mm of diameter tungsten sphere.
  • the DNA of three single individuals of each population of O. laevigatus was tested with approximately 200 SSR markers designed by us.
  • RP02_F (5'-TACACACTTTGCCGTAAATCAGA-3') (SEQ ID NO: 1) and RP02_R (5' -CGTTGTATTGGCATGATATAGG-3') (SEQ ID NO: 2) drove a polymorphic amplification pattern, which was recognizable among improved strains.
  • the RP02 SSR marker was first described by Duan et al. (PLOS One, 2017, 12(2): e0172513). In order to ensure this preliminary result, at least 250 single individuals from each O.
  • laevigatus population were tested by PCR using 1 unit of VWR® Red Taq DNA Polymerase, 1X Key Buffer, 3mM ChMg, 0.5mM of DNTPs, 10pM from each oligonucleotide, and 5-25ng genomic DNA per reaction.
  • the PCR was running in an Eppendorf Mastercycler Pro according to the program shown in Figure 10.
  • SSR markers in combination with RAPD markers have become an important tool to understand the molecular diversity and the genetic relationships within species, including inter- and intraspecific genetic variability. More than 200 primers were analyzed in the three O. laevigatus genotypes (3 individuals of each one). However, only 10 SSR makers were found polymorphic and associated to phenotypes. After that, 16 adults of each O. laevigatus population were utilized to test the 10 SSR markers chosen in the first analysis, and only one of them showed a polymorphic pattern associated to the phenotype under study. Finally, at least 250 individuals were assayed with the RP02 SSR marker, in order to study the genetic linkage between the RP02 SSR marker and the selected character in both improved strains of O. laevigatus.

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