Insect primary cell isolation methods

Abstract

The present invention generally relates to the field of plant genetics and molecular biology. More particularly, the compositions and methods described herein are directed to methods of isolating insect primary cells, insect primary cells so generated, and methods of use thereof.

Description

INSECT PRIMARY CELL ISOLATION METHODSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 582,660, filed on September 14, 2023, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention is drawn to the field of plant genetics and molecular biology. More particularly, the compositions and methods are directed to methods of isolating insect primary cells, insect primary cells so generated, and methods of use thereof.BACKGROUND OF THE INVENTION

[0003] Insect pests are a major factor in the loss of the world’s agricultural crops. The feeding damage caused by lepidopteran insect species such as com earworm (CEW, Helicoverpa zed) and fall armyworm (FAW, Spodoptera frugiperda), which are primary insect pests of corn, and soybean looper (SBL, Chrysodeixis inchidens), velvetbean caterpillar (VBC, Anticarsia gemmatalis) and CEW which are primary insect pests of soybean, results in reduced crop yield and seed quality. Likewise, the coleopteran insect species western corn rootworm, (WCR) (Diabrotica virgifera virgifera LeConte), is one of the most significant and influential pests of corn in both North America and Europe.

[0004] Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and a commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.

[0005] Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemipteraand others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. Additionally, insecticidal proteins have been discovered from other microorganisms, fungi, and plants that show agricultural promise as transgenic insect control traits.

[0006] Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus and other sources. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bacillus thuringiensis. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods.

[0007] The use of insect cell culture is an important tool in evaluating the insecticidal activity and exploring the toxic mechanisms of candidate insecticidal proteins prior to development of transgenic crop plants. Enzyme mediated tissue dissociation is important for use of insect primary cells in cytotoxicity assays, as well as use in other downstream applications such as generating transcriptome and proteome data, in binding assays, and in cell patch clamp assays to assist in mode of action studies.

[0008] Tissue digestion with certain enzymes can lead to undesirable cleavage of endogenous insect cell proteins. Additionally, high background noise can completely cover the toxicity signal in cytotoxicity assays. Therefore, it is desirable to have new methods of enzyme mediated tissue dissociation for insect primary cell isolation that preserve toxin and membrane receptors and improve the signal to noise ratio for downstream applications.SUMMARY

[0009] The present invention offers methods for isolating insect primary cells, insect primary cells-comprising compositions produced using isolating methods, and uses of said insect primary cell s-compri sing compositions.

[0010] Methods are provided for isolating insect primary cells. In one embodiment, methods are provided for isolating primary cells from an insect comprising dissecting insect midguts,enzymatically dissociating the insect midgut tissue, mechanically dissociating the insect midgut tissue, and filtering in order to isolate the primary cells. In a further embodiment, the isolated insect primary cells are assayed for viability.

[0011] In another embodiment, the methods described herein may be used to isolate primary cells from a number of different insects, including insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera and Coleoptera, as well as the Nematoda phylum.

[0012] In a further embodiment, insects include Lepidoptera species including but not limited to: Fall Armyworm (FAW) (Spodoptera frugiperda), Com Earworm, (CEW) (Helicoverpa zea), European Corn Borer (ECB) (Ostrinia nubialis), diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker; and velvet bean caterpillar e.g., Anticarsia gemmatalis Hiibner and Coleoptera species including but not limited to Western corn rootworm (WCR or WCRW) (Diabrotica virgifera). Southern com rootworm (SCRW) (Diabrotica undecimpunctata howardi), and Northern corn rootworm (NCRW) (Diabrotica barberi) .

[0013] In one embodiment, insect midgut tissue is obtained by dissecting an insect. Dissecting the insect midgut comprises sedating and sanitizing the insect followed by removing the midguts. In a further embodiment, the food bolus is removed from the dissected midguts and / or the dissected midguts are cut or minced after removal from the insect.

[0014] In one embodiment, the invention provides a cell culture medium supplemented with the enzymes trypsin and chymotrypsin (such enzymes can be found at, for example, sigmaaldrich.com / US / en / products / protein-biology / proteins-and-enzymes / tissue-dissociation which can be accessed on the world-wide web using the "www" prefix) combined with dissected insect midgut tissue. In another embodiment, methods are provided for digesting the insect midgut tissue with enzymes comprising preparing cell culture medium supplemented with the enzymes trypsin and chymotrypsin and combining the supplemented medium with dissected insect midgut tissue. In another embodiment, the enzyme digestion occurs in two or more steps with one step being the digestion in an inert carrier fluid and another step of substantially pure culture medium. The supplemented medium and the dissected insect midgut tissue are combinedfor a length of time and at a temperature sufficient to allow for digestion. The working concentration of each enzyme and incubation time is adjusted based on the mass of the tissue.

[0015] In one embodiment, the invention provides a cell culture medium comprising, or alternatively consisting essentially of or consisting of a culture medium supplemented with the enzymes trypsin and chymotrypsin combined with dissected insect midgut tissue, which has been mechanically dissociated. In another embodiment, methods are provided for mechanically dissociating the insect midgut tissue comprising grinding the midgut tissue. The methods disclosed herein contemplate that the steps may be conducted manually, the steps may be automated, or some combination thereof. Methods are provided for grinding the tissue using an automated system. In another embodiment, the tissue grinding method uses manual hand grinding.

[0016] In another embodiment, methods are provided for mechanically dissociating the combined supplemented medium and dissected insect midgut tissue using an automated tissue dissociator. The combined supplemented medium and dissected insect midgut tissues are mechanically dissociated using an automated tissue dissociator for a time and at a temperature sufficient to allow for complete digestion and dissociation of the tissues. The resulting digested dissociated tissue is filtered, for example using cell strainers, to remove any undigested tissue pieces or aggregates from the prepared insect primary cells and washed to remove excess enzyme. In a further aspect, the isolated insect primary cells are assayed for viability.

[0017] In one aspect, the methods of combined enzymatic and mechanical dissociation of insect midgut tissue described herein increases the yield and viability of the primary cells as compared to grinding or enzyme digestion alone. In another aspect, the methods described herein are used to remove high background noise in primary cell viability assays.

[0018] In another embodiment, the method of combined enzymatic and mechanical dissociation of insect midgut tissue described herein preserves certain endogenous insecticidal cell proteins such as toxin protein receptors and membrane receptors of the midgut tissue. The isolated insect primary cells are assayed to determine the intactness of certain toxin and / or membrane receptors.

[0019] The isolated insect primary cells may be used in a number of processes and methods, including but not limited to cytotoxicity assays with insecticidal toxin proteins, generating transcriptome, proteome, and lipidome data, and in functional assays, such as binding assays or cell patch clamp assays.BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1A shows the results of an ATP viability assay using FAW primary cells isolated by hand grinding alone and FAW primary cells isolated using enzyme digestion with either pepsin or papain combined with automated mechanic tissue dissociation in the presence or absence of insecticidal toxin protein 1.

[0021] FIG. IB shows the results of an ATP viability assay using FAW primary cells isolated by the method(s) described herein in the presence or absence of insecticidal toxin protein 1.

[0022] FIG. 2 shows the results of an ATP viability assay using FAW primary cells isolated by hand grinding alone and FAW primary cells isolated by the method described herein in the presence or absence of insecticidal toxin protein 2 or insecticidal toxin protein 3.

[0023] FIG. 3 shows the results of an ATP viability assay using WCR primary cells isolated by the method described herein in the presence or absence of insecticidal toxin protein 4.

[0024] FIG. 4 shows the results of an ATP viability assay using WCR primary cells isolated by the method described herein in the presence or absence of insecticidal toxin protein 5.DETAILED DESCRIPTION

[0025] Compositions and methods are provided for the isolation and production of insect primary cells. Methods for producing insect primary cells include the use of enzymatic and mechanical tissue dissociation. Further provided are insect primary cells produced by the methods, and compositions comprising the same. Methods for the use of insect primary cells are provided, including cytotoxicity assays using isolated insect primary cells and candidate insecticidal proteins.

[0026] Primary cells are directly isolated from fresh tissue of a living organism and can be obtained from tissue biopsies or from organ removal. Primary cells are considered better representations of functional components of tissue compared to immortalized cells as they are a closer match to an in vivo model. Tissue dissociation is an important technique for primary cell isolation.

[0027] Tissue dissociation can utilize mechanical dissociation, enzymatic dissociation, or a mixture of both methods to obtain primary cells from tissue. Mechanical dissociation is when a tissue sample is cut, crushed, pulverized, minced, or homogenized using an instrument to createsmaller, digestible pieces of tissue. Mechanical dissociation is often required for tissues that have strong adhesions that need to be broken down before single cells can be extracted. However, tissues that are subjected to harsh mechanical dissociation may result in low viability and low yield of primary cells. Enzyme dissociation, or enzymatic digestion, is important for cell harvesting of primary cells and is often used to isolate cells from tissue. However, tissue dissociation enzymes can cleave cell surface markers to varying degrees, which can affect the detection levels of markers in downstream analyses.

[0028] Dissociation of mammalian intestinal cells using EDTA with the enzymes collagenase, dispase and trypsin has been demonstrated with a focus on culturing the primary cells and inducing stem cell differentiation, (see, for example Chopra, et al., (2010) Stem Cells and Development, 19(1): 131-141) Methods of preparing insect cell primary cells and cell lines include using the enzyme collagenase or using trypsin alone at low concentration and incubation time (see for example Garcia, et al., (2001) In Vitro Cell Dev Biol Anim., 37:353-359 and Zhou, et al., (2020) In Vitro Cell Dev Biol Anim., 56: 10-14). The present invention differs from and is an improvement over known methods of isolating insect primary cells in the use of enzymes comprising, or alternatively consisting essentially of or consisting of, both trypsin and chymotrypsin for the enzymatic dissociation combined with the automated mechanic tissue dissociation, which improves yield and viability, decreases background noise, and preserves certain insecticidal toxin protein receptors.Methods for isolating insect primary cells

[0029] The methods for isolating primary cells from an insect according to the present invention comprise dissecting insect midguts, enzymatically dissociating the insect midgut tissue, and mechanically dissociating the insect midgut tissue. The method may further comprise filtering using cell strainers in order to isolate the primary cells.

[0030] Examples of insects from which primary cells may be isolated include a number of different insect pests, including members of the Lepidoptera, Coleoptera, Diptera, and Hemiptera orders and the Nematoda phylum.

[0031] Larvae of the order Lepidoptera include, but are not limited to, army worms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Htibner (beet armyworm); S. litura Fabricius (tobacco cutworm, clustercaterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Htibner (cotton leaf worm);Trichoplusia ni Htibner (cabbage looper); Pseudoplusia inchidens Walker (soybean looper); Anticarsia gemmatalis Htibner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (army worm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vitlella Fabricius (spotted bollworm); Helicoverpa armigera Htibner (American bollworm); H. zea Boddie (com earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Htibner (European com borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Htibner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Htibner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Htibner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernaid (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham(filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermiiller (European grape vine moth); Spilonota ocellana Denis & Schiffermtiller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hubner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp. Choristoneura spp..

[0032] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatelia Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Elemileuca oliviae Cockrell (range caterpillar); Elyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tulaabsoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

[0033] Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae and Curculionidae (including, but not limited to: Anthonormis grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); 5. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith and Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (com flea beetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle); Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from the family Coccinellidae (including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle)); chafers and other beetles from the family Scarabaeidae (including, but not limited to: Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grubj: Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; and beetles from the family Tenebrionidae.

[0034] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot).

[0035] Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae and red bugs and cotton stainers from the family Pyrrhocoridae.

[0036] Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov / Mordvilko (Russian wheat aphid); Dysciphis planlaginea Paaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbias Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (com leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T.vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcif ra Horvath (white-backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Pianococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead (persimmon psylla).

[0037] Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herri ch- Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one- spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaffooted pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

[0038] Furthermore, embodiments may be used with Hemiptera such as, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygasterspp. ; Coreidae spp. ; Pyrrhocoridae spp. ; Tinidae spp. ; Blostomatidae spp. ; Reduviidae spp. and Cimicidae spp.

[0039] Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Muller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Pcinonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health.

[0040] Insect pest of interest include the superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid) and the family Cydnidae (Scaptocoris castanea - Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Hiibner.

[0041] Nematodes include parasitic nematodes such as root-knot, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode) and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

[0042] Dissection of insect midgut tissue may be performed according to any known methodology, (see, for example Garcia, et al., (2001) In Vitro Cell Dev Biol Anim., 37:353-359 and Zhou, et al., (2020) In Vitro Cell Dev Biol Anim., 56: 10-14) The insects are typically sedated and surface sanitized prior to the dissection. In one embodiment, a combination of acetone and ethanol is used to sedate and sanitize the surface of the insect. Dissection comprises making one or more cuts on the insect body followed by pulling out the midgut. The food bolusis removed, if possible. The dissected insect midguts are then soaked in the medium supplemented with enzymes to start the digestion, or they are soaked in the medium and mechanical dissociation such as cutting, mincing, or the like is started. In a further embodiment, the dissected insect midguts are placed on ice or left at room temperature prior to starting the digestion.

[0043] Enzymatic dissociation of the insect midgut tissue comprises combining the dissected midguts with a solution comprising enzymes and insect cell culture medium. Use of enzymes for tissue dissociation are necessary for removing the scaffolding and structural support of tissues that are essential for cell viability and the success of many downstream applications. Enzymes used typically include trypsin, pepsin, papain, collagenase D, other types of collagenases, nucleases, hyaluronidase, elastase, protease XIV, proteases isolated from bovine pancreas, Dispase®, and Liberase™. Temperature is an important factor to consider in enzymatic digestion as it can affect the stability and activity of enzymes. Additionally, concentration of the enzyme is also important as adding too much enzyme may facilitate the tissue dissociation, however, it increases the risk of protein receptor cleavage.

[0044] In a preferred embodiment of the present invention, the enzymes comprise, alternatively consist essentially of, or consist of trypsin and chymotrypsin. The working concentration of each enzyme is about 0.5 mg / ml to about 4 mg / ml. The enzymes are added to the cell culture medium at the appropriate concentration and about 10 ml of the supplemented medium is added into an appropriate tube for use in an automated tissue dissociator, for example a C-tube (Miltenyi Biotec, Germany), along with about 200 mg (wet weight) of the dissected insect midguts. The amount of dissected insect midguts may be smaller or larger than 200 mg. The total amount of enzymes used is proportional to the total amount of midguts, with the enzyme to insect midgut tissue mass ratio ranging from about 1 / 200 to about 1 / 5.

[0045] Trypsin has been reported to have a very harsh effect on cell membrane proteins and has been shown to lead to free-DNA induced aggregation of cells, indicating cell lysis is occurring. As a result, use of trypsin for tissue dissociation is often avoided, (see Reichard, et al., (2019) Cytometry Part A, 95A:219-226) Surprisingly, it is found that insect primary cell viability remains high and certain insecticidal toxin receptors and membrane receptors are preserved when trypsin and chymotrypsin are used together according to the methods of the present invention.

[0046] Mechanical dissociation uses physical processes to break down the tissue into smaller pieces. Mechanical dissociation of the insect midgut tissue comprises hand grinding tissue with a cell strainer pestle, using mincing scissors or blade cutting or the like, or grinding the tissue using an automated tissue dissociator. Examples of automated tissue dissociators include but are not limited to the gentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) and the DSC-400 single cell suspension dissociator (RWD, Guangdong, China). In a further embodiment, mechanical dissociation of the insect midgut tissue comprises a manual system, including but not limited to mincing, cutting, and / or hand grinding. In another embodiment, both automated and manual systems are employed for mechanical dissociation of the insect midgut tissue.

[0047] In one embodiment the tube containing the supplemented medium and dissected insect midgut tissue is placed into an automated tissue dissociator. The mechanical dissociation involves running a gentle grinding program, for example the “Brain” program on the gentleMACS™ dissociator once, rotating the tube for approximately 30 minutes at about 28° C, again running a gentle grinding program once, rotating the tube for approximately 30 minutes at about 28° C, and running a gentle grinding program once again at the end to completely dissociate the tissues. In some embodiments, the digestion temperature ranges from about 20° C to about 37° C. In other embodiments, the incubation time is between about 20 minutes to about one hour. The primary cell preparation is then filtered using cell strainers to sift out debris. At the end of the enzyme digestion, the cells are spun down to the bottom of a tube and washed to remove leftover enzymes. The isolated insect primary cells can be stored at about 28° C for up to about 48 hours, after which the primary cells will gradually die out.

[0048] In a further embodiment, the viability of the isolated insect primary cells is tested. Any means of testing cell viability may be used. Examples include but are not limited to a luminescent cell viability assay such as CellTiter-Glo® 2.0 cell viability assay (Promega, Madison, WI), which is an ATP assay for detection of viable cells, LDH-Glo™ cytotoxicity assay (Promega, Madison, WI), MultiTox-Fluor Multiplex cytotoxicity assay (Promega, Madison, WI), or calcium influx assays (Abeam, Cambridge, UK). Surprisingly, it was found that insect primary cells isolated by the methods provided herein resulted in high cell viability.Insect Primary Cells and Compositions Comprising

[0049] In one embodiment, a composition comprises dissected insect midgut tissue and cell media supplemented with enzymes comprising, or alternatively consisting essentially of or consisting of trypsin and chymotrypsin, which together have been mechanically dissociated. In a further embodiment, a composition comprises dissected insect midgut tissue and cell media supplemented with enzymes comprising, or alternatively consisting essentially of or consisting of trypsin and chymotrypsin, which together have been mechanically dissociated and fdtered.Methods of Use of Isolated Insect Primary Cells and Insect Primary Cell Compositions

[0050] Isolated insect primary cells may be used in a number of processes and methods, including but not limited to cytotoxicity assays with insecticidal toxin proteins, generating transcriptome, proteome, and lipidome data, and in functional assays, such as binding assays or cell patch clamp assays.

[0051] In one embodiment is contemplated a method of screening candidate actives or known insecticidal toxin proteins for insecticidal activity comprising running cytotoxicity assays with the isolated insect primary cells and candidate insecticidal proteins. Methods for performing cytotoxicity assays are well known in the art, and examples include but are not limited to commercial cytotoxicity assays such as LDH-Glo™ cytotoxicity assay (Promega, Madison, WI), MultiTox-Fluor Multiplex cytotoxicity assay (Promega, Madison, WI), or calcium influx assays (Abeam, Cambridge, UK). The candidate insect control active may be a polypeptide having insect inhibitory or insecticidal activity.

[0052] In one embodiment, the cytotoxicity assay is combined with the viability assay. For example, the candidate insecticidal toxin or known insecticidal toxin is incubated with the ATP assay testing cell viability compared to the ATP assay components alone.

[0053] In a further embodiment, the isolated insect primary cells are used in generating transcriptome and / or proteome data, (see for example, Yu, et al., (2019) Nat. Commun., 10:3574 and Pan, et al., (2009) Mol. Cell Proteomics, 8(3):443-450). In other embodiments, the isolated insect primary cells are used in lipidomic analysis, (see for example, Hancock, et al., (2023) J. Lipid Res., 64(3): 100341).

[0054] In another embodiment, the isolated insect primary cells are used in functional assays, for example in binding assays or cell patch clamp electrophysiology assays to assist a mode of action study.

[0055] Insect resistance genes may encode resistance to pests such as rootworm, cutworm, armyworm, European Corn Borer, and the like. Such genes include, for example, insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1- 13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58: 12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3: 101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US 9,688,730; an AflP-lA and / or AfTP-lB polypeptide of US 9,475,847; a PIP-47 polypeptide of US Patent Application Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of International Patent Application Publication Number WO2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number W02018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Application Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Patent Application Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO2018 / 232072; a PtIP-83 polypeptide of US Patent Application Publication Number US20160347799; a PtIP-96 polypeptide of US Patent Application Publication Number US20170233440; an IPD079 polypeptide of International Patent Application Publication Number WO2017 / 23486; an IPD082 polypeptide of International Patent Application Publication Number WO2017 / 105987; an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560; an IPD093 polypeptide of International PatentApplication Publication Number WO2018 / 11 1551 ; an IPD103 polypeptide of International Patent Application Publication Number W02018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882; and 8-endotoxins including but not limited to a Cryl, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, CrylO, Cryl l, Cry 12, Cry 13, Cry 14, Cry 15, Cryl6, Cryl7, Cryl8, Cryl9, Cry20, Cry21, Cry 22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of 6-endotoxin polypeptides and the B. thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0056] Examples of 8-endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275, 7,858,849, and 8,878,007; a CrylAc mutant of US9, 512, 187; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1 and / or a-helix 2 variants of cry proteins such as CrylA, Cry3A) of US Patent Numbers 8,304,604, 8.304,605 and 8,476,226; Cry IB of US Patent Application Publication Number US20060112447, US Patent Application Publication Number US20160194364, and US Patent Numbers 9,404,121 and 8,772,577; Cry IB variants of PCT Publication Number WO2016 / 61197 and Serial Number PCT / US 17 / 27160; CrylC of US Patent Number 6,033,874; CrylD protein of US20170233759; a CrylE protein of PCT Serial Number PCT / US 17 / 53178; a CrylF protein of US Patent Numbers 5,188,960 and 6,218,188; CrylA / F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063; a Cryll protein of PCT Publication number WO 2017 / 0233759; a Cryl J variant of US Publication US20170240603; a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249 and Cry2A.127 protein of US 7208474; a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010 / 0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US PatentNumbers 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,339,092, 7,378,499, 7,462,760, and 9,593,345; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families including the Cry9 protein of US Patent 9,000,261 and 8,802,933, and US Serial Number WO 2017 / 132188; a Cryl5 protein of Naimov, et al., (2008) Applied and Environmental Microbiology, 74:7145-7151; a Cryl4 protein of US Patent Number US8,933,299; a Cry22, a Cry34Abl protein ofUS Patent Numbers 6,127, 180, 6,624,145 and 6,340,593; a truncated Cry34 protein of US Patent Number US8, 816, 157; a CryET33 and cryET34 protein of US Patent Numbers 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006 / 0191034, 2012 / 0278954, and PCT Publication Number WO 2012 / 139004; a Cry35Abl protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of US Patent Number 9,403,881, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US Patent Application Publication Number 2008 / 0295207; TIC853 of US Patent US8,513,493; ET29, ET37, TIC809, TIC810, TIC812, TIC 127, TIC 128 of PCT US 2006 / 033867; engineered Hemipteran toxic proteins of US Patent Application Publication Number US20160150795; AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031, AXMI- 039, AXMI-040, AXMI-049 of US Patent Number 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006 / 083891; AXMI-010 of WO 2005 / 038032; AXMI-003 of WO 2005 / 021585; AXMI-008 of US Patent Application Publication Number 2004 / 0250311; AXMI- 006 of US Patent Application Publication Number 2004 / 0216186; AXMI-007 of US Patent Application Publication Number 2004 / 0210965; AXMI-009 of US Patent Application Number 2004 / 0210964; AXMI-014 of US Patent Application Publication Number 2004 / 0197917; AXMI-004 of US Patent Application Publication Number 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI- 014 and AXMI-004 of WO 2004 / 074462; AXMI-150 of US Patent Number 8,084,416; AXMI- 205 of US Patent Application Publication Number 2011 / 0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI- 034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011 / 0263488; AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMIO55, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075,AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132, AXMI137, AXMI138 of US Patent US8461421 and US8,461,422; AXMI-R1 and related proteins of US Patent Application Publication Number 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011 / 103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011 / 103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI- 035 and AXMI-045 of US Patent Application Publication Number 2010 / 0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMIl 11, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMU61, AXMI183, AXMI132, AXMI138, AXMI137 of US Patent US8461421; AXMI192 of US Patent US8,461,415; AXMI281 of US Patent Application Publication Number US20160177332; AXMI422 of US Patent Number US8, 252, 872; cry proteins such as Cry 1A and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; a CrylAc, Cry2Aa and Cry ICa toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011 / 0064710. The Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S, MP214S,MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S, MP319S, MP325S, MP326S, MP327S, MP328S, MP334S, MP337S, MP342S, MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S, MP564S, MP566S, MP567S, MP569S, MP573S, MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S, MP632S, MP633S, MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S, MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S, MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S, MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of US Serial Number 62 / 607372. The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101 : 1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry -transgenic plants including but not limited to plants expressing Cry 1 Ac, CrylAc+Cry2Ab, Cry 1 Ab, Cry 1 A.105, CrylF, CrylFa2, CrylF+CrylAc, Cry2Ab, Cry3A, mCry3A, Cry3Bbl, Cry34Abl, Cry35Abl, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA. (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org / index. php?action=gm crop database which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & CrylFa (US2012 / 0317682); CrylBE & CrylF (US2012 / 0311746); CrylCA & CrylAB (US2012 / 0311745); CrylF & CryCa (US2012 / 0317681); CrylDA & CrylBE (US2012 / 0331590); CrylDA & CrylFa (US2012 / 0331589); CrylAB & CrylBE(US2012 / 0324606); CrylFa & Cry2Aa and Cryll & CrylE (US2012 / 0324605); Cry34Ab / 35Ab & Cry6Aa (US20130167269); Cry34Ab / VCry35Ab & Cry3Aa (US20130167268); CrylDa & Cry ICa (US 9796982); Cry3Aa & Cry6Aa (US 9798963); and Cry3A & Cry lAb or Vip3Aa (US9,045,766). Pesticidal proteins also include insecticidal lipases including lipid acylhydrolases of US Patent Number 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15: 1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html which can be accessed on the world-wide web using the "www" prefix). Pesticidal proteins also include Cyt proteins including CytlA variants of PCT Serial Number PCT / US2017 / 000510; Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptAl and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB IXb and XptCIWi. Examples of Class C proteins are TccC, XptClXb and XptBIWi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but not limited to ly cotoxin- 1 peptides and mutants thereof (US Patent Number 8,334,366).

[0057] One embodiment includes a method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells.

[0058] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the insect is a Lepidopteran or a Coleopteran insect species.

[0059] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the dissecting insect midguts comprises sanitizing and sedating the insect with a combination of acetone and ethanol followed by cutting the insect and removing the insect midguts.

[0060] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the working concentration of trypsin and chymotrypsin is between about 0.5 mg / ml to about 4.0 mg / ml each.

[0061] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the enzyme to midgut tissue mass ratio is between about 1 / 200 to about 1 / 5.

[0062] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the mechanic tissue dissociation of the insect midgut tissue comprises hand grinding tissue with a cell strainer pestle, using scissors mincing, using blade cutting, or grinding the tissue with an automated tissue dissociator.

[0063] A further embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain theprimary cells, wherein the mechanic tissue dissociation comprises using an automated tissue dissociator.

[0064] A further embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the mechanic tissue dissociation comprises using an automated tissue dissociator, wherein the automated tissue dissociator performs at least two mechanical dissociation steps.

[0065] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the mechanic tissue dissociation comprises using an automated tissue dissociator, wherein the automated tissue dissociator performs at least two mechanical dissociation steps, wherein the dissected midguts in a solution of enzymes are rotated in between each mechanical dissociation step.

[0066] Another embodiment includes the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, wherein the total digesting and mechanical tissue dissociation time is between about 20 to about 60 minutes.

[0067] One embodiment includes a method of sanitizing and sedating an insect prior to dissection comprising exposing the insect to a combination of acetone and ethanol.

[0068] Another embodiment includes a method of performing cytotoxicity assays comprising subjecting the insect primary cells isolated by the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, to insecticidally active proteins.

[0069] Another embodiment includes a method of generating transcriptome, proteome, or lipidome data comprising using the insect primary cells isolated by the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, in transcriptome, proteome, or lipidome analyses.

[0070] Another embodiment includes a method of generating functional data comprising using the insect primary cells isolated by the method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells, in binding assays or cell patch clamp electrophysiology assays.

[0071] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

[0072] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0073] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1. Dissection of insect midguts

[0074] Dissection of insect midgut tissue may be performed according to any known methodology. The insects are typically sedated and surface sanitized prior to the dissection. A mixture of 20% acetone in 70% ethanol was used to sedate and sanitize the surface of the insects. Dissection comprised making cuts on the insect body to remove the head and tail ends, followed by pulling out the midgut. The food bolus was removed, when possible.

[0075] Insects used in the method disclosed herein include Lepidoptera species Fall Armyworm (FAW) (Spodoptera frugiperda) Southern Armyworm (SAW) (Spodoptera eridania), Corn Earworm (CEW) (Helicoverpa zea), European Com Borer (ECB) (Ostrinia nubialis), soybean looper, e.g., Pseudoplusia includens Walker and Coleoptera species Western corn rootworm (WCR) (Diabrotica virgifera).Example 2. Enzymatic and mechanical mediated tissue dissociation and primary cell preparation

[0076] To isolate the primary cells, a combination of enzymatic and automated mechanical tissue dissociation was used with the dissected insect midguts. Cell culture medium was prepared and supplemented with 1 mg / ml of trypsin and 1 mg / ml of chymotrypsin for the enzymatic digestion. The working concentration of each enzyme was 0.5 - 2 mg / ml with the total enzyme concentration below 4 mg / ml. Ten milliliters of the supplemented medium was dispensed into a C-tube (Miltenyi Biotec, Bergisch Gladbach, Germany) and 200 mg (wet weight) of the dissected insect midguts were added. A gentleMACS™ tissue dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to grind the tissue for the mechanic tissue dissociation using the gentleMACS™ brain program once, followed by rotating the C-tube for 1 hour at 28° C, grinding with the brain program once halfway through and once at the end to completely dissociate the tissues. The total digesting time was approximately 30-60 minutes. The primary cell prep was then filtered using cell strainers to sift out debris. At the end of the enzyme digestion, the cells were spun down to the bottom of a tube and washed twice to remove leftover enzymes.Example 3. Primary cell viability assay

[0077] To verify the viability of the isolated primary cells, the CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI) protocol was followed. The CellTiter-Glo® 2.0 Cell Viability Assay determines the number of viable cells in culture by quantifying ATP, which indicates the presence of metabolically active cells. The cells were also incubated with insecticidal proteins at various doses overnight. The cells were lysed, and the luminescent signal was read. The luminescence readout is directly proportional to the number of viable cells in culture.Example 4. Preservation of toxin and membrane receptors

[0078] Binding of an insecticidal toxin to its target receptor in the insect cell is the first step of toxin induced cytotoxicity. For example, Cry protein toxins, when ingested by susceptible insect larvae, bind to specific receptors on the midgut epithelial cell brush border membrane and lead to insect death. Therefore, maintaining the integrity of target receptors is important when preparing primary cells for downstream applications.

[0079] A number of different insects have been used to evaluate the viability and receptor preservation of insect primary cells isolated by the methods described herein using enzyme digestion with trypsin and chymotrypsin combined with mechanical tissue dissociation using an automated tissue dissociator.Preservation of receptors in Lepidopteran insects

[0080] Fall armyworm (FAW) was used to evaluate the viability and receptor preservation of insect primary cells isolated by the method of the invention in modified cytotoxicity assays with insecticidal toxin proteins. Numerous proteins insecticidal to Lepidopteran insects have been tested.Preservation of receptor A

[0081] FAW primary cells isolated by the method described herein were compared to FAW primary cells isolated using enzyme digestion with either pepsin or papain combined with automated mechanic tissue dissociation or to FAW primary cells isolated using hand grinding alone. The hand grinding method refers to using strong mechanic force only to dissociate the tissue. Equal weights (wet) of FAW midguts were dissociated and cell viability was tested usinga CellTiter-Glo® Cell Viability Assay with the luminescent signal normalized to the control groups. Primary cells were suspended in equal amount of culture medium, and equal volume of cell suspensions were seeded into 96 well culture plates. Primary cells isolated using hand grinding alone or with pepsin or papain were incubated with insecticidal toxin protein 1 at 60 ppm overnight at 28° C, with equal volume of vehicle added to the control group. Primary cells isolated according to the method described herein were incubated with insecticidal toxin protein 1 at 5 ppm overnight at 28° C and equal volume of vehicle was added to the control group. Insecticidal toxin protein 1 targets receptor A in insect cells. If receptor A is cleaved or rendered nonfunctional during the combined enzymatic and mechanic tissue dissociation, the cells will not respond to insecticidal toxin protein 1.

[0082] FIG. 1 A shows the results for the assay using FAW primary cells isolated by hand grinding alone and FAW primary cells isolated using enzyme digestion with either pepsin or papain combined with automated mechanic tissue dissociation. FIG. IB shows the results for the assay using FAW primary cells isolated by the method described herein. The results indicate that pepsin and papain cleaved receptor A and / or rendered receptor A nonfunctional, whereas trypsin plus chymotrypsin preserved receptor A.Preservation of receptor B

[0083] FAW primary cells isolated by the method described herein were compared to FAW primary cells isolated using hand grinding alone and assayed for viability as described above. One group of cells were incubated overnight at 28° C with 10 ppm of insecticidal toxin protein 2 and the other group of cells were incubated overnight at 28° C with 100 ppm of insecticidal toxin protein 3. Equal volume of vehicle was used to treat control group of cells. Insecticidal toxin protein 2 targets receptor B in insect cells. If receptor B is cleaved or rendered nonfunctional during the combined enzymatic and mechanic tissue dissociation, the cells will not respond to insecticidal toxin protein 2. Insecticidal toxin protein 3 has insecticidal activity against Coleopteran insect pests.

[0084] As shown in FIG. 2, trypsin and chymotrypsin treatment increased the yield and viability of the primary cells and also preserved receptor B, as compared to hand grinding alone.Preservation of receptors in Coleopteran insects

[0085] Western corn rootworm (WCR) was used to evaluate the viability and receptor preservation of insect primary cells isolated by the methods in modified cytotoxicity assays with insecticidal toxin proteins. Numerous proteins insecticidal to Coleopteran insects have been tested.Preservation of receptor C

[0086] WCR primary cells were isolated by the method(s) and assayed for viability as described above. Primary cells were incubated with 100 ppm of insecticidal toxin protein 4 overnight and equal volume of vehicle was added to the control group. Receptor C is the receptor of insecticidal toxin protein 4. WCR primary cells that respond well to insecticidal toxin protein 4 are believed to have their receptor C preserved.

[0087] As shown in FIG. 3, isolation of WCR primary cells according to the method of the invention preserved receptor C.Preservation of receptor D

[0088] WCR primary cells were isolated by the method of the invention and assayed for viability as described above. Primary cells were incubated with 50 ppm of insecticidal toxin protein 5 overnight and equal volume of vehicle was added to the control group. Insecticidal toxin protein 5 is a potential binder of receptor D. WCR primary cells that respond well to insecticidal toxin protein 5 are believed to have their receptor D preserved.

[0089] As shown in FIG. 4, isolation of WCR primary cells according to the method of the invention preserved the potential receptor D.Example 5. Downstream applications for isolated primary cells

[0090] The isolated primary cells can be used in a number of downstream applications, including but not limited to cytotoxicity screening, generating transcriptome, proteome, and lipidome data, and functional assays, such as binding assays and cell Patch Clamp assays. Methods for performing cytotoxicity assays are well known in the art, and examples include but are not limited to commercial cytotoxicity assays such as LDH-Glo™ cytotoxicity assay (Promega, Madison, WI), MultiTox-Fluor Multiplex cytotoxicity assay (Promega, Madison, WI), or calcium influx assays (Abeam, Cambridge, UK). The candidate insect control active may be a polypeptide having insect inhibitory or insecticidal activity.

[0091] The cytotoxicity assay may be combined with the viability assay. For example, the candidate insecticidal toxin or known insecticidal toxin is incubated with the ATP assay testing cell viability compared to the ATP assay components alone.

[0092] The isolated insect primary cells may be used in generating transcriptome and / or proteome data, (see for example, Yu, et al., (2019) Nat. Commun., 10:3574 and Pan, et al., (2009) Mol. Cell Proteomics, 8(3):443-450). Additionally, the isolated insect primary cells may be used in lipidomic analysis, (see for example, Hancock, et al., (2023) J. Lipid Res., 64(3): 100341).

[0093] The isolated insect primary cells may be used in functional assays, for example in binding assays or cell patch clamp electrophysiology assays to assist a mode of action study.

Claims

CLAIMSWhat is claimed is:

1. A method of isolating primary cells from an insect comprising dissecting insect midguts, enzymatically digesting the dissected midguts in a solution comprising enzymes and cell culture medium, the enzymes consisting essentially of trypsin and chymotrypsin, mechanically dissociating the insect midgut tissue, and filtering to obtain the primary cells.

2. The method of claim 1, wherein the insect is a Lepidopteran or a Coleopteran insect species.

3. The method of claim 1, wherein the dissecting insect midguts comprises sanitizing and sedating the insect with a combination of acetone and ethanol followed by cutting the insect and removing the insect midguts.

4. The method of claim 1, wherein the working concentration of trypsin and chymotrypsin is between about 0.5 mg / ml to about 4.0 mg / ml each.

5. The method of claim 1, wherein the enzyme to midgut tissue mass ratio is between about 1 / 200 to about 1 / 5.

6. The method of claim 1, wherein the mechanic tissue dissociation of the insect midgut tissue comprises hand grinding tissue with a cell strainer pestle, using scissors mincing, using blade cutting, or grinding the tissue with an automated tissue dissociator.

7. The method of claim 6, wherein the mechanic tissue dissociation comprises using an automated tissue dissociator .

8. The method of claim 7, wherein the automated tissue dissociator performs at least two mechanical dissociation steps.

9. The method of claim 8, wherein the dissected midguts in a solution of enzymes are rotated in between each mechanical dissociation step.

10. The method of claim 1, wherein the total digesting and mechanical tissue dissociation time is between about 20 to about 60 minutes.

11. A method of sanitizing and sedating an insect prior to dissection comprising exposing the insect to a combination of acetone and ethanol.

12. A method of performing cytotoxicity assays comprising subjecting the insect primary cells isolated by the method of claim 1 to insecticidally active proteins.

13. A method of generating transcriptome, proteome, or lipidome data comprising using the insect primary cells isolated by the method of claim 1 in transcriptome, proteome, or lipidome analyses.

14. A method of generating functional data comprising using the insect primary cells isolated by the method of claim 1 in binding assays or cell patch clamp electrophysiology assays.

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