Use of real-time PCR in insect nematology

Entomopathogenic nematodes and RT-PCR- nematodeinformation

Read following papers on the real-time PCR and Insect Nematology

Bae, S. and Kim, Y. 2003.   Lysozyme of the beet armyworm, Spodoptera exigua: activity induction and cDNA structure. Comparative Biochemistry and Physiology B-Biochemistry and Molecular Biology 135: 511-519.

Campos-Herrera R, El-Borai F.E., Stuart R.J., Graham J.H., DuncanL.W. 2011. Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs inFlorida citrus groves. Journal Invertebrate Pathology 108:30-9.

Campos-Herrera, R., Johnson, E. G, El-Borai, F. E., Stuart, R. J., Graham, J. H. and Duncan, L. W.2011. Long-term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real-time PCR. Annals of Applied Biology 158: 55-68.

Ciche, T.A. and Sternberg, P.W. 2007.  Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts. BMC Developmental Biology 7, Article Number: 101.

Ji, D.J. and Kim, Y. 2004.   An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua. Journal of Insect Physiology 50: 489-496.

Park, D., Ciezki, K., van der Hoeven, R., Singh, S., Reimer, D., Bode, H.B. and Forst, S. 2009. Genetic analysis of xenocoumacin antibiotic production in the mutualistic bacterium Xenorhabdus nematophila. Molecular Microbiology 73: 938-949.

Pathak, E., El-Borai, F.E., Campos-Herrera, R., Johnson, E.G., Stuart, R.J., Graham, J.H. and Duncan, L.W. 2012.  Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi.  Fungal Biology 116: 563-573.

Shrestha, Y.K. and Lee, K.Y. 2012. Oral toxicity of Photorhabdus culture media on gene expression of the adult sweetpotato whitefly, Bemisia tabaci. Journal of Invertebrate Pathology 109: 91-96.

Son, Y. and Kim, Y. 2011.  Immunosuppression induced by entomopathogens is rescued by addition of apolipophorin III in the diamondback moth, Plutella xylostella. Journal of Invertebrate Pathology 106: 217-222.

Song, C.J., Seo, S., Shrestha, S. and Kim, Y.  2011. Bacterial Metabolites of an Entomopathogenic bacterium, Xenorhabdus nematophila, inhibit a catalytic activity of phenoloxidase of the diamondback moth, Plutella xylostella. Journal of Microbiology and Biotechnology 21: 317-322.

Torr, P., Spiridonov, S.E., Heritage, S. and Wilson, M.J. 2007. Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions. Journal of Animal Ecology 76: 238-245.

How and when to apply insect-parasitic nematodes

How to apply nematodes Insect-parasitic nematodes can be easily applied using conventional pesticide and fertilizer sprayers that have up to 300 PSI pressures.  However, nematodes will be easily damaged, if they are agitated through excessive recirculation of spray mix or if the temperature in the tank increases beyond 86 degrees F. Nematodes can also be applied through different types of irrigation systems but pumps should have proper pressure to avoid damage to nematodes and screen sizes should be larger than 50 mesh so that nematodes will pass through them live. Watering cans are used to apply nematodes in small areas including vegetable and ornamental gardens.

How many nematodes should be applied

For the suscessful control most of the soil dweling insect pests, the optimal rate of 1 billion infective juvenile nematodes in 100 to 260 gallons of water per acre is generally recommended.

Optimal soil and environmental condtions to apply nematodes

All nematodes require proper soil moisture for their optimal movement and infectivity. The activity and infectivity of nematodes can be enhanced by maintaining optimum moisture levels in the soil before and after their application.  In case of nematode application in turf, turf should be irrigated immediately after applicationwith at least 1/2 inch of water to rinse off nematodes from the folliage and move them into the soil and thatch. As nematodes are very sensitiv to heat and cold, their infectivity will be reduced if soil temperature is below 4 degrees C and above 35 degrees C. Soil temperatures between 20 to 30 degrees C are considered favourable for application of majority of nematode species and their virulence.  Nematode survival and activity also influenced by soil type.  Both survival and activity of nematodes is higher in sandy-loam soils than in heavy clay soils.

When to apply nematodes

Since nematodes are very sensitive to UV light, they will die within a minute or two when exposed to full sun. Therefore, nematodes should be applied early in the morning or late in the evening to avoid exposure to UV light.

Use insect-parasitic nematodes to control citrus root weevils

The citrus root weevil also called as Diaprepes root weevil (Diaprepes abbreviatus) is one of the major insect pests of citrus and many ornamental plants in Florida and California. Several researchers have demonstrated that the application of an insect-parasitic nematode can supress the populations of root weevils in citrus orchards. For example, Steinernema riobrave infective juveniles when applied in citrus orchards or greenhouses can provide 50 to 90% reduction in populations of D. abbreviatus (Bullock et al., 1999; Duncan and McCoy, 1996; Duncan et al., 1996; Shapiro and McCoy, 2000ab).  Applications of S. carpocapsae (All strain), Heterorhabditis bacteriophora (HP-88 strain) or H. bacteriophora (Florida strain) in the citrus grove can also reduce 50-70% adult emergence of D. abbreviatus (Duncan et al., 1996; Schroeder, 1992).  According to Shapiro et al. (1999), S. riobrave, H. bacteriophora and H. indica were highly virulent against younger (50-day-old) than older (100-day-old) D. abbreviatus larvae at 24 or 27 degrees C temperature. Heterorhabditis indica was more virulent than H. bacteriophora in 50-day-old D. abbreviatus larvae at all temperatures. However, H. bacteriophora was more virulent than S. riobrave in 20-day-old larvae at 24 degrees C but it was less virulent than S. riobrave in 50-day-old larvae at 21 degrees C.

Please Read following literature for detailed information on interaction between insect-parasitic nematodes and citrus root weevil.

Bullock, R.C., Pelosi, R.R. and Killer, E.E. 1999. Management of citrus root weevils (Coleoptera : Curculionidae) on Florida citrus with soil-applied entomopathogenic nematodes (Nematoda : Rhabditida). Florida Entomologist. 82: 1-7.

Duncan, L.W and McCoy, C.W. 1996 Vertical distribution in soil, persistence, and efficacy against citrus root weevil (Coleoptera: Curculionidae) of two species of entomogenous nematodes (Rhabditida: Steinernematidae; Heterorhabditidae). Environmental Entomology. 25: 174-178.

Duncan, L.W. McCoy, C.W. and Terranova, A.C. 1996. Estimating sample size and persistence of entomogenous nematodes in sandy soils and their efficacy against the larvae of Diaprepes abbreviatus in Florida. Journal of Nematology. 28: 56-67.

Schroeder, W.J. 1992. Entomopathogenic nematodes for control of root weevils of citrus. Florida Entomologist 75: 563-567.

Shapiro, D.I. and McCoy, C.W. 2000a. Susceptibility of Diaprepes abbreviatus (Coleoptera : Curculionidae) larvae to different rates of entomopathogenic nematodes in the greenhouse. Florida Entomologist. 83: 1-9.

Shapiro, D.I. and McCoy, C.W. 2000b. Effects of culture method and formulation on the virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae). Journal of Nematology 32: 281-288.

Shapiro, D.I., Cate, J. R., Pena, J., Hunsberger, A. and McCoy, C.W. 1999. Effects of temperature and host age on suppression of Diaprepes abbreviatus (Coleoptera : Curculionidae) by entomopathogenic nematodes. Journal of Economic Entomology. 92: 1086-1092.

Use Good Bugs to Control Bad Bugs: Predatory insects

Before starting to write about this topic, I would like to make it clear that taxonomically all bugs are insects but all the insects are not bugs. As far as I know, both in the USA and Canada, almost all people except entomologists call each and every insect as a bug.  Even extension entomologists when they are giving extension seminars to farmers/growers about insect pests of different crops, they often refer them as bad bugs for the understanding of growers. "True" bugs are mainly belong to two insect orders including Hemiptera and Homoptera. All natural enemies of insect pests are considered as good bugs because they can kill and feed on insect pests that cause tremendous yield losses to many economically important crops. Since many of these natural enemies are commercially produced and used in the integrated pest management program (IPM), they are called as biological control agents. These biological control agents can be parasitic or predatory insects.  In addition to these predators and parasites (good bugs), there are some microorganisms such as bacteria, fungi, protozoa and viruses that can cause diseases and kill insect pests.  These microorganisms are termed as insect pathogens and also considered as biological control agents. Nematodes belonging to two families, Steinernematidae and Heterorhabditidae are also considered as insect parasites or pathogens and used as biological agents in controlling many soil dwelling insect pests of many economically important crops (in this blog, please read several posts that are devoted to insect- parasitic nematodes). Furthermore, mites are closely related to spiders but not considered as insects. Some species of mites are predatory in nature but others are serious pests of many plant species.

Predators: Although, there are many kinds of vertebrate predators including birds, amphibians, reptiles, fish and mammals that feed on insects, in this blog I am going to focus on the predatory insects that are generally used in biological control programs. These insects are called predators because they feed and complete their entire life cycle by remaining outside of their prey host as opposed to parasites that complete at least part of their life cycle inside their hosts.  Predators are generally larger than their prey, they kill and feed on both immature and adult stages of many different kinds of hosts.

Following are the examples of insect predators that can be used as biological control agents against many kinds of insect pests.

Aphid midge (Aphidoletes aphidimyza): This predatory midge fly often found in many vegetable crops (potatoes, cabbage and cauliflower), fruit orchards (apple, blueberries and peaches) and many ornamental plants throughout North America. The larval stages of this midge fly are mainly predators of aphids. This midge fly is commercially available and widely used as biocontrol agents in the greenhouses against over 60 species of aphids infesting both vegetable and ornamental plants.

Bigeyed bug (Geocoris spp.): There are four most common species of bigeyed bug (G. punctipes, G. pallens, G. bullatus and G. uliginosus) found in almost all cropping systems in North America.  Bigeyed bugs generally feed on many small insects including aphids, mites and whiteflies, eggs and nymphs of many plant bugs. They can also feed on eggs and small larval stages of cotton ballworms, pink ballworms and tobacco budworms. Since this bug is very susceptible to broad spectrum pesticides, care should be taken to avoid killing of this important biocontrol agent.  This predator is commercially available from insectories in the USA.

Brown lacewings (Hemerobius stigma): These lacewings found throughout North American forests and are mainly predators of aphids and many other soft-bodied small insects including balsam woolly adelgis (Adelges piceae), pine bark adelgid (Pineus strobi) and Cooley's spruce gall adelgid (Adelges cooleyi). These lacewings are not commercially available.

Deraeocoris bug (Deraeocoris nebulosus): This is a very important predator of many insect and mite pests different agricultural, horticultural and landscape plants in the Canada and USA. This is a true predatory bug, which is generally found in many fruit orchards including apple, peach and pecan.  They also found in cotton fields and many landscape settings.  These bugs are natural enemies of many small insects including aphids, lace bugs, psyllids, scales and whiteflies. They also feed on mites. These bugs are not commercially available.

Dragon and damselflies: Adult dragon and damsel flies generally feed on small flying small adult insects including midge flies, mayflies, mosquitoes, ants and termites in the air where as dragon/damsel fly nymphs feed on mosquito larvae in the water.

Green lacewing (Chrysoperla carnea, C. rufilabris): Lacewings adults are not predatory in nature but mainly feed on nectar, honeydew and pollens.  However, larvae of lacewings are predatory in nature and feed on insect pests of many crops including apples, asparagus, cotton, corn, cole crops, eggplants, leafy vegetables, potatoes, tomatoes, peppers and strawberries. Lacewing larvae generally prey on aphids, leafhopper eggs, eggs of butterflies and moths, mealybugs, mites, thrips, small larvae of beetles and moths. Both species of lacewings are commercially available and sold in all stages (eggs, larvae and adults).

Ladybird beetles (Hippodamia parenthesis and Harmonia axyridis): These beetles are also recognized as lady beetles or ladybugs and more than 450 of this beetles have been reported from North America. Both larval and adult stages of this predator found on many agricultural and ornamental plants and they primarily feed on aphids. In addition, they can feed on small insects, mites, scales, thrips and eggs of many moths and beetles. they can eat nectar or pollen if insect hosts are not around. These predators are now commercially available to use against many crop pests, especially aphids.

Lebia beetles (Lebia grandis): These beetles are natural enemies of Colorado potato beetle, Leptinotarsa decemlineata. Adults of the predatory insect can feed on all immature stages of colorado potato beetle. Larval stages of Labia beetles are generally parasitic in nature and therefore, they are considered as ectoparasites of larval and pupal stages of colorado potato beetles. These predators are not commercially produced.

Pirate bugs (Orius spp.): Both adults and nymphs of these predatory insects have a sharp, needle-like beak that they use to suck body content of their prey. These insects found in many crops including alfalfa, corn, cotton, pea, peanuts, and strawberries. These are predators of aphids, mites, thrips, small larval stages of many insects, eggs of many different kinds of insects. These insect predators are commercially available in the USA and most often suscessfully used as biocontrol agents in controlling greenhouse pests.

Rove beetles (Aleochara bilineata): These beetles naturally found in many vegetable crops including onions, different cole crops, turnip, radish and sweet corn.  Rove beetle adults are predatory in nature but their larval stages are parasitic in nature. Rove beetles generally feed on egg, larval and pupal stages of onion and cabbage maggots. These insects are not commercially available.

Soldier beetles (Chauliognathus marginatus and C. pennsylvanicus): These beetles are also called leatherwing beetle because of texture of their wings. Larvae of this insect mainly feed on grasshopper eggs, both adult and nymphal stages of aphids, soft bodied larvae of many insects (cutworms, gypsy moths) whereas adults mainly feed on adult aphids and other soft bodied insects. These predators also feed on snails and slugs. These insects are not pest any plant species but they can eat nector or pollen if insect hosts are not around.

Spined soldier bug (Podisus maculiventris): This is a "true bug" that also named as a stink bug because it emits a strong stinky odour when disturbed. Like Pirate bugs, this bug also uses its sharp beak to suck the body content of its prey. This predator feeds on immature stages of many insect pests including beet armyworm, cabbage loopers, cabbageworm, colorado potato beetle, corn earworm, diamond backmoth, Eropean corn borer, fall armyworms, flea beetles, Mexican bean beetle and velvetbean caterpillars. These insect predators are commercially available.

List of insects susceptible to various species of entomopathogenic nematodes

Insect Species: Entomopathogenic nematode species

Ø Apopka weevil (Diaprepes abbreviatus): S. carpocapsae All strain

Ø Armyworm (Heliothis armigera): S. carpocapsae All strain

Ø Billbugs (Sphenophorus purvulus): H. bacteriophora & S. carpocapsae All strain

Ø Black vine weevil (Otiorhynchus salcatus): S. carpocapsae All & UK strains, S. feltiae, S. glaseri & H. megidis UK 211 strain

Ø Blue grass weevil (Listronotus maculicollis): H. bacteriophora & S. carpocapsae

Ø Carpenter worms (Cossus cossus): S. carpocapsae

Ø Carrot weevil (Listronotus oregonensis): S. feltiae

Ø Cat fleas (Ctenocephalides felis): S. carpocapsae

Ø Citrus root weevil (Pachnaeus litus): S. carpocapsae All strain

Ø Clover root weevil (Sitona hispidulus): S. feltiae & H. bacteriophora

Ø Codling moth (Cydia pomonella): S. carpocapsae

Ø Crane flies (Tipula spp.): S. carpocapsae & H. megidis

Ø Cutworms (Agrotis ipsilon, A. segetum): S. carpocapsae All strain

Ø Dog fleas (Ctenocephalides cannis): S. carpocapsae

Ø Face fly (Musca autumnalis): S. carpocapsae, H. bacteriophora & S. feltiae

Ø Fall web worms (Hyphantria cunea): S. carpocapsae

Ø Flea beetles (Phyllotreta spp.): S. carpocapsae

Ø Fungus gnats (Bradysis spp.): H. bacteriophora, H. indica, H. zealandica, S. anomali, S. carpocapsae, S. feltiae SN strain & S. riobrave

Ø House flies (Musca domestica): S. carpocapsae, H. bacteriophora & S. feltiae

Ø Hunting billbug (Sphenophorus venatus venatus): S. carpocapsae All strain

Ø Japanese beetle (Popillia japonica): H. bacteriophora, H. indica, H. marelata, H. megidis, H. zealandica, S. anomali, S. carpocapsae, S. feltiae, S. glaseri, S. kushidai, S. riobrave, S. scapterisci & S. scarabae

Ø Leaf minors (Liriomyza trifolii): S. carpocapsae & S. feltiae

Ø Leopard moth (Zeuzera pyrina): S. carpocapsae

Ø Mole crickets (Gryllotapla gryllotapla): S. riobravis & S. scapterisci

Ø Peach borer moth (Synanthedon exitiosa): S. carpocapsae

Ø Pecan weevil (Curculio caryae): H. bacteriophora

Ø Pine weevil (Hylobius abietis): S. carpocapsae, S. feltiae & H. downesi

Ø Plum weevil (Conotrachelus nenuphar): S. riobrave 355 strain

Ø Shore flies (Scatella stagnalis): H. megidis, S. carpocapsae, S. feltiae & S. anomaly

Ø Sod webworm (Herpetogramma phaeopteralis): S. carpocapsae All strain

Ø Stable fly (Stomoxys calcitrans): S. carpocapsae, H. bacteriophora & S. feltiae

Ø Strawberry root borer (Nemocestes incomptus): S. carpocapsae

Ø Sugarcane borer (Diaprepes abbreviatus): S. carpocapsae All strain

Ø Sweet potato weevil (Cylasformicarius elegantulus): S. carpocapsae All strain & H. bacteriophora HP88 strain

Ø Western flower thrips (Frankliniella occidentalis): H. bacteriophora, H. indica, H. marelata, S. abassi, S. arenarium, S. bicornutum, S. carpocapsae, S. feltiae

Ø White grubs (Amphimallon solstitiale): S. glaseri

Ø White grubs (Anomala orientalis): H. bacteriophora, H. megidis, H. zealandica, S. carpocapsae, S. glaseri, S. longicaudum, S. scarabae

Ø White grubs (Ataenius spretulus): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Costelytra zealandica): H. bacteriophora & S. glaseri

Ø White grubs (Cotinus nitida): H. bacteriophora, S. carpocapsae, S. feltiae, S. glaseri & S. scarabae

Ø White grubs (Cyclocephala borealis): H. bacteriophora, H. indica, H. marelata, H. megidis, H. zealandica, S. glaseri & S. scarabae

Ø White grubs (Cyclocephala hirta): H. bacteriophora, H. megidis, S. carpocapsae, S. feltiae, S. glaseri, S. kushidai, S. riobrave & S. scarabae

Ø White grubs (Cyclocephala lurida): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Cyclocephala pasadenae): H. bacteriophora, S. glaseri, S. kushidai & S. scarabae

Ø White grubs (Hoplia philanthus): H. megidis, S. feltiae & S. glaseri

Ø White grubs (Maladera castanea): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Melolontha melolontha): H. bacteriophora, H. marelata, H. megidis, S. arenaria, S. feltiae, S. glaseri & S. riobrave

Ø White grubs (Phyllophaga congrua): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Phyllophaga crinita): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Phyllophaga georgiana): H. bacteriophora, S. glaseri & S. scarabae

Ø White grubs (Rhizotrogus majalis): H. bacteriophora, H. megidis, H. zealandica, S. carpocapsae, S. feltiae, S. glaseri & S. scarabae

For more information on insect pathogenic nematodes read following books:

Ø Nematodes As Biocontrol Agents by Grewal, P.S. Ehlers, R.-U., Shapiro-Ilan, D. (eds.). CAB publishing, CAB International, Oxon.

Ø Entomopathogenic Nematodes in Biological Control by Gaugler, R. and Kaya, H. K. (eds.), CRC Press, Boca Raton

Ø Entomopathogenic Nematology by Gaugler, R. (Ed.), CABI