Interactions between Bacillus thuringiensis and entomophagous insects

The interactions between insect pathogens, parasitoids, and predators have been proposed as “intraguild predation (IGP).” It occurs when two species share a host or prey (and therefore may compete) or when they engage in a trophic interaction with each other (parasitism or predation). Laboratory studies revealed that many predacious species attack and consume Bacillus thuringiensis (Bt)-infected prey with no detrimental effects on the biological parameters of the predators. However, some studies indicated adverse impact of Bt on development and biology of the predators. As for parasitoids, some studies showed no detrimental effects on the biology of the parasitoids when reared on Bt-infected hosts and others revealed negative effects on their biological aspects. Such negative effects were found to be related to Bt concentration and timing of treatment (parasitism or infection). The biological parameters of the offspring of the adult parasitoids that had emerged from infected parasitized larvae were not affected. Also, ingestion of Bt by adult parasitoids did not affect the longevity or fecundity of such parasitoids. Concerning Bt crops, a recent published report in 2019 stated that over the past 20+ years, extensive experience and insight have been gained through laboratory and field studies of non-target effects of crops producing Cry proteins. Overall, the vast majority of the studies demonstrated that the insecticidal proteins developed today do not cause adverse effects on natural enemies (parasitoids and predators) of target pests.


Background
The interactions between insect pathogens, parasitoids, and predators have been proposed as "intraguild predation (IGP)" (Rosenheim et al. 1995). It occurs when two species share a host or prey (and therefore may compete) or when they engage in a trophic interaction with each other (parasitism or predation). This interaction may impact the population dynamics of biological control agents and target pests. Common forms of IGP include pathogens that infect both herbivores and their parasitoids, facultative hyperparasitoids which can parasitize either the herbivore or a primary parasitoid of the herbivore, and predators that feed upon parasitized herbivores and cannibal predators. Thus, IGP can be intense resulting in high levels of mortality for one or both of natural enemies, while the total mortality imposed on the target pest populations is minimal (Rosenheim et al. 1995). In this respect, Labauda and Griffin (2018) reported that despite such negative interactions occur in the laboratory, it is less often documented in the field. Although laboratory-reared insects are more susceptible to pathogens, the ecological components and behavioral responses of the natural enemies should be taken into consideration to explain the low or scarce incidence of entomopathogens infecting natural enemies (predators and parasitoids) under field conditions (Scorsetti et al. 2017). Beline (2018) reported that entomopathogens and other biological control agents can be synergistic, additive, or antagonistic depending on the specific biological control agents as well as their rate, timing of application, and the host species. As demonstrated by Ferguson and Stiling (1996), synergistic interactions result in a higher mortality than the combined individual mortalities of the pest population. Additive interactions occur if the natural enemies do not interact, and thus, the total level of mortality is equivalent to the combined individual mortalities caused by each agent. The antagonistic interactions occur if the total mortality is less than when either natural enemy acts alone. Roy and Pell (2000) reported that synergistic interactions between pathogens and insect predators or parasitoids can enhance control efficacy, whereas antagonistic interactions reduce total control efficacy. Roy et al. (1998) stated that predators may consume pathogen-infected prey and thus may remove the pathogen from the environment. However, Caceres (2009) mentioned that such predators may defecate viable amount of a pathogen which increases the numbers of infected hosts and, consequently, the distribution of the pathogen in the environment. In addition, Fazal (2004) stated that integration of biological control agents may substantially contribute to sustainable management of damage caused by insects in both greenhouses and fields.

Bacillus thuringiensis
B. thuringiensis (Bt) is a Gram-positive, soil-dwelling bacterium commonly used as a biological control insecticide. It was first discovered in 1901 by the Japanese biologist Shigetane Ishiwatari as a cause of sotto disease that was killing silkworms, and named Bacillus sotto. In 1911, Ernst Berliner isolated this bacterium from dead Mediterranean flour moth in Thuringia, Germany, and named it B. thuringiensis. In 1915, Berliner reported the existence of a parasporal body or crystalline inclusion (called crystal) close to the endospore within Bt spore but the activity of the crystal was not then discovered. In 1956, it was found that the main insecticidal activity against insects was due to the parasporal crystal (Abbas 2018).
During sporulation process, Bt produces crystalline proteinaceous inclusion (parasporal inclusion body) adjacent to the endospore, which have has been found to be toxic for invertebrates, mainly insects from the orders Lepidoptera, Coleoptera, and Diptera) (Andrews et al. 1987). These parasporal inclusions (or α-endotoxins) are formed by different insecticidal crystal proteins and are encoded by Cry) from crystal) and Cyt (from cytoletic) genes (Hofte and Whitely 1989). Bt subspecies can synthesize more than one inclusion which may contain different Cry toxins (Hofte and Whitely 1989).

Mode of action of Bacillus thuringiensis
Bt spores have to be ingested by the susceptible insect to cause infection. The parasporal body (or crystal, or crystalline inclusion, known as Cry toxin) in Bt spore becomes active by proteoletic enzymes in the alkaline gut juice of the host (pH 8-10). Most Cry toxins are actually pro-toxins of about 130 to 140 kDa, and after activation, they become 60-70 kDa (Bravo et al. 2007). The activated toxin passes through the peritrophic membrane and binds to specific receptors on apical microvillar brush border membrane of the epithelial cells of the midgut making pores through which the toxin penetrates to such cells. The cells then become swollen, and the swelling continues until the cells lyse, separate from the basement membrane, and fall in the lumen of the midgut. The alkaline gut juices then leak into the hemocoel causing increase of hemolymph pH which leads to paralysis and death of the insect (Soberon et al. 2010). However, Broderick et al. (2006) mentioned that the naturally occurring bacteria in the hostgut (Escherichia coli and Enterobacter) penetrate to the hemocoel through the disrupted epithelium, caused by Bt toxins, and multiply causing sepsis of the hemolymph and death of the insect. In moderately susceptible insects to Bt, such as Spodoptera spp., the endospore has a considerable role in killing the insect by producing toxins during its vegetative growth in the hemolymph (Crickmore et al. 2014). However, Gill et al. (1992) reported that the insects or any living organisms that do not have the receptors in gut epithelial cells are not killed by Bt.

Bacillus thuringiensis crops (Bt crops)
Bt crops are plants genetically engineered (modified) to contain the toxins (Cry toxins) from the crystalline parasporal body to be resistant to certain insect pests. "Plant Genetic Systems," in Belgium, was the first company to produce a Bt crop (tobacco) in laboratory in 1985, but the crop was not commercially successful. However, in 1995, the Environmental Protection Agency (EPA) in the USA approved the commercial production and distribution of the Bt crops (corn, cotton, potato, and tobacco). Recently, Bt crops are currently planted in almost 100 million ha worldwide. Adoption of Bt crops has greatly reduced the abundance of targeted pests in such crops and, in addition, has enabled the application of IPM programs and the increase of natural enemy populations (Abbas 2018). Bt toxins can be found in the whole plant including roots, stems, leaves, pollen, and fruits. However, concentrations can vary considerably in different plant tissues across different developmental stages of the plant and among different Bt toxins (Eisenring et al. 2017).

Interactions between B. thuringiensis and predators
Laboratory studies Mahmoud (1992) found that both the 4th larval instar and adults of the coccinellid predator, Coccinella undecimpunctata (L.) (Coleoptera), attacked and consumed Bt-infected larvae of Spodoptera littoralis Boisd. (Lep.: Noctuidae). No statistical differences in prey consumption were found between healthy and infected prey. In addition, no detrimental effects on the predator larvae were observed when fed on infected prey and they completed their development to adults which were capable of mating and ovipositing. The larvae and adults that fed on infected prey were found to excrete viable spores in their feces. Carvalho et al. (2012) studied the effect of Bt-infected larvae of Plutella xylostella (L.) as prey, on the nymphs and adults of the pentatomid predator, Podisus nigrispinus (Pallas) (Hemiptera), and found that Bt did not affect their biological parameters compared to the control. Similarly, Megalhaes et al. (2015) investigated the influence of Bt var. kurstaki and the commercial product "Agree" (a combination of Bt var. kurstaki and Bt var. aizawai) on the biological aspects of P. nigrispinus when fed on infected larvae of P. xylostella. They found that the infected larvae were consumed more than the healthy ones throughout the nymphal development with no effect on nymphal survival or the population growth parameters. However, de Carvalho et al. (2018) reported that rearing P. nigrispinus nymphs on Bt-treated larvae of Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) affected the oviposition periods and the total egg numbers deposited by the predator's female.
Field studies Dutton et al. (2003) assessed the effects of spraying Dipel (a Bt commercial product) against Spodoptera littoralis larvae infesting maize, on Chrysoperla carnea Steph. larvae in a greenhouse. The results showed negative impact on the predator's larvae including a significant increase in mortality, a prolonged developmental period, and a slight decrease in adult`s weight.

Interactions between B. thuringiensis and parasitoids
Laboratory studies Survival of parasitoid progeny in Bt-infected hosts Mahmoud (1992) reported that Bt-infected Spodoptera littoralis larvae had no significant effect on percentages of parasitism by the braconid larval parasitoid, Microplitis rufiventris Kok. When the Bt-treated larvae were exposed to the parasitoid 1, 2, 3, 4, and 5 days post-infection, percentages of successful parasitism averaged 65, 35, 45, 60, and 60%, respectively, which did not differ significantly from those in healthy larvae. Blumberg et al. (1997) stated that development of the immatures of Microplitis croceipes (Cresson) in Helicoverpa armigera (H.) host larvae Bt-infected 3 days before exposure to the parasitoid was detrimental to the parasitoid because of premature host mortality. However, feeding parasitoid adults on Bt preparation mixed with honey was not harmful to the wasps. Atwood et al. (1997) evaluated the impact of Bt var. kurstaki on Cotesia marginiventris (Cresson) survival in parasitized Heliothis virescens (Fab.) larvae. The infected larvae were exposed to the parasitoid females immediately, 24 or 48 h after parasitism. The results showed that combination of Bt and Cotesia increased mortality of host larvae than that of either alone. Emergence of C. marginiventris larvae from parasitized host larvae was inversely related to Bt concentration and the timing of Bt treatment after parasitism. In all instances, greater adult parasitoid emergence was noted when host exposure to Bt was delayed for 48 h post-parasitism. Similar results were obtained by Atwood et al. (1999) with Bt, the braconid Microplitis croceipes, and the host H. virescens and at the same concentrations of Bt. Ebrahimi et al. (2012) evaluated the effects of Bt var. kurstaki on immature stages of the ichneumonid parasitoid, Diadegma insulare (Cresson), within larvae of its host Plutella xylostella and on the adult stage as a Bt-honey solution. They found that percent formation of the parasitoid pupae from host larvae treated with LC 10 , LC 30 , and LC 50 of Bt and then exposed to the parasitoid females after 48 h were 5, 3, and 2%, respectively, which were much lower than that of the parasitoid alone (41%). They also found that the survival of adult parasitoid fed on Bt-honey solution at the field rate of Bt product (1000 ppm) did not differ significantly (95%) from that of the control (97%).

Discrimination between Bt-infected and healthy larvae
Pre-feeding of H. armigera on an artificial diet treated with Dipel at lethal concentrations of 0.08 and 0.16 mg/g of the diet did not prevent Microplitis croceipes from oviposition in the infected host larvae (Blumberg et al. 1997). Mohan et al. (2008) found that the female parasitoid, Campoletis chlorideae Uchida, did not discriminate between healthy and Bt-infected larvae of Helicoverpa armigera for oviposition. Schoenmaker et al. (2011) found that the females of the ichneumonid parasitoid, Tranosema rostrale rostrale (Brishke), were able to detect and avoid Bt-infected host larvae (the spruce budworm, Choristoneura fumiferana (Clemens) (Lep.: Tortricidae)) that exhibited a lethal response to the pathogen and, to a lesser extent, larvae that could survive Bt infection. Wallner et al. (1983) noticed an indirect effect on the braconid parasitoid, Rogas lymantriae Watanabe, parasitizing the gypsy moth, Lymantria dispar (L.), larvae that fed on artificial diet mixed with Bt. The sex ratio among the parasitoid offspring was found to lean towards males as the female parasitoid deposited more fertilized eggs in larger untreated host larvae. Dequech et al. (2005) investigated the effect of Bt var. aizawai on larvae of Spodoptera frugiperda (Smith) and its parasitoid, Campoletis flavicincta (Ashmead). They found that the average mortality percentage of both (parasitized and infected) larvae was higher (96.0%) than the only parasitized (78.4%) and the only infected ones (44.3%). The biological parameters of the offspring of the parasitoids' adults that had emerged from infected parasitized larvae were not affected. Mohan et al. (2008) reported that the growth and survival of the parasitoid, Campoletis chlorideae, in its host larvae, Helicoverpa armigera, were normal when the host larvae were fed with sub-lethal doses or subjected to short time exposure to lethal doses of Btk. However, the parasitoid offspring developed slowly, and the pupal and adult durations as well as adult emergence rate were significantly reduced if the parasitoid was developing inside a severely Bt-infected host larvae. Oluwafemi et al. (2009) found that Bt or Habrobracon hebetor Say alone caused 41.7 and 35.4% mortality, respectively, in Pludia interpunctella (H.) larvae whereas the combined treatment with Bt and the parasitoid increased host mortality to 86%. Famil et al. (2012) investigated the longevity, host preference, lengths of life stages, and numbers of offspring of the ecto-larval parasitoid, H. hebetor, on its host, Ephestia kuehniella Zeller, larvae treated with lethal and sub-lethal doses of Bt. They found that the average daily number of eggs deposited/parasitoid female decreased from 3.9 in the control to 1.4 and 2.7 in lethal and sublethal doses, respectively. The respective average of longevity of adult parasitoid decreased from 15.6 to 12.3 and 13.7 days. At the lethal dose of Bt treatment, duration of the egg and larval stages of the parasitoid showed significant prolongation compared to the control but the pupal stage duration decreased.

Effect of Bt on biological parameters of the parasitoids
Similarly, Sedaratian et al. (2014) investigated the effects of Bt on biological parameters of H. hebetor parasitizing H. armigera. The neonate larvae were treated with sublethal doses of Bt (LC 25 ), and the surviving larvae that reached the 4th instar were then exposed to the parasitoid for 24 h. The results showed that the successful development of immature stages (from egg to adult) was 89% in the control compared to 61% in Bt-treated larvae. The total developmental period of the parasitoid (from egg to adult) averaged 14.1 and 12.3 days, respectively. The respective total deposited eggs/female were 140.8 and 41.2. Salles et al. (2015) reported that Btk infection of Spodoptera exigua (H.) larvae previously parasitized with Campoletis flavicincta did not affect, significantly, the developmental periods of the parasitoid or its longevity. de Carvalho et al. (2018) investigated the possible impact of Bt on the pupal parasitoid Palmistichus elaeisis Delvare and LaSalle (Hym.: Eulophidae) of Tenebrio molitor when the larvae were fed on wheat bran treated with 6 different concentrations of Bt ("Agree"; commercial product) (0-4 gm/kg). When the formed pupae were exposed to Palmistichus females. The biological characters of emerged adult parasitoids were not affected.
Direct effect of Bt on adult parasitoids Mohan et al. (2008) reported that there were no differences in longevity of the parasitoids of, Campoletis chlorideae adults fed on bee honey solution containing different concentrations of Bt compared to those fed only on honey solution. Marchetti et al. (2012) studied the effect of feeding adults of Exorista larvarum (L.) (Dipt.: Tachinidae), a parasitoid of Galleria mellonella (L.) larvae, on sugar soaked with the commercial Bt var. kurstaki (Foray 48B) at 3 times the dose which is highly virulent to lepidopteran insects. The study showed that the longevity of males and females of the parasitoid, the number of eggs laid by the female, and the percentage of larvae that developed to pupae were not significantly affected compared to feeding on distilled water.

Field studies
Nealis and van Frankenhuyzen (1990) investigated the interactions between Bt and the braconid parasitoid, Apanteles fumiferanae (Vireck), of the spruce budworm, Choristoneura fumiferana, in a semi-field trial. Treatment was carried out by placing parasitized late 3rd instar larvae on foliage before spraying Bt at the recommended application rate. The results showed that Bt reduced the parasitoid populations by 50-60 % by killing the host before parasitoid emergence. This negative impact of Bt was decreased when spraying the parasitized larvae with Bt after reaching late 4th instar. Schoenmaker et al. (2011), in a semi-field experiment, stated that when Bt-treated and untreated larvae of the spruce budworm, C. fumiferana, were exposed (in caged pots) for 1 week to a complex of indigenous parasitoids, there were no differences between treatments in the rates of parasitism by either the echneumonid, Tranosema rostrale rostrale, or the tachinid, Actia interrupta Curran, parasitoids. Percentages of parasitism averaged 91 and 92% for untreated and treated larvae, respectively. Also, Singh and Mathew (2015), investigating the effects of Bt sprays on natural enemies of the spruce budworm, C. fumiferana, found a range of effects from synergism, repellency, and toxicity to no effect.

Effect of Bt crops on predators
Laboratory studies By feeding on Bt toxins Hilbeck et al. (1998) fed the larvae of Chrysoperla carnea on artificial diet mixed with Cry1Ab toxin and found that the total mortality in larvae was significantly higher (57%) than in the untreated control (30%). Also, significantly more larvae died (29%) when received Cry1Ab later during their development compared to the control ones (17%). However, no differences in developmental period were observed between treated and untreated larvae. In another study, Hilbeck et al. (1999) obtained almost similar results when C. carnea larvae were fed on Spodoptera littoralis larvae fed on diet mixed with Cry1Ab and Cry2A toxins at different concentrations.

By feeding on pollen
Mendelsohn et al. (2003) found that pollen from Bt corn, containing Cry toxins, which was at relatively very high doses, was not toxic to coccinellids or Chrysoperla spp. Adults of the predator, Chrysoperla carnea, were found to feed exclusively on pollen and nectar (Li et al. 2010), and its larvae can supplement their diet with pollen (Meissle et al. 2014). Liu et al. (2016) reared the larvae of the coccinellid, Propylea japonica, on the pollen of Bt or non-Bt corn from the first instar larvae until pupation. They found that over 70% of larvae of both groups developed to adults and pupation rates (83 and 84%, respectively) and eclusion rates (75 and 74%, respectively) did not differ significantly. Romeis et al. (2019) reported that insect predators such as chrysopids (Neoroptera), coccinellids (Coleoptera), and anthocorids (Hemiptera) were found to feed on the plant pollen especially in the absence of their prey.
By feeding in on prey fed on Bt plants Romeis et al. (2019) reported that the predators, Geocoris spp. (Hem.:Geocoridae) and Nabis spp.(Hem.:Nabidae), were noticed to feed directly on the tissues of plant leaves by sucking the sap, whereas the soil predators such as earwigs (Dermaptera: Labiduridae) and Calosoma spp. (Col: Carabidae) might feed on plant roots in the soil. Such habits will make these predators come in contact with Cry toxins in Bt crops. However, it does not seem that Bt toxins occur in phloem tissue sap in the plants or they may be less occurring than in the green tissues. Consequently, the aphids which suck plant sap as well as their predators will not be harmed in Bt crops. In addition, the honeydew of aphids, which is an important source of energy to parasitoids and predators, did not contain Bt toxins. Ponsard et al. (2002) investigated the effect of Bt cotton on adult longevity of 4 hemipteran predators of cotton pests. The adults were fed on Spodoptera exigua larvae reared on Bt cotton leaves. They found that the longevity of Orius tristicolor (White) (Fam. Anthocoridae) and Geocoris punctipes (Say) (Fam. Geocoridae) decreased by 28 and 27%, respectively, compared to control adults. No effects were found for Nabis sp. (Fam. Nabidae) and Zelus sp. (Fam. Reduviidae). However, Head et al. (2005) found a non-significant negative impact of Bt cotton on Geocoris spp., Orius spp., ladybeetles, and spiders in comparison with non-Bt cotton. Also, Torres and Ruberson (2006) found no detrimental effects on development and reproduction of G. punctipes fed on prey reared on leaves of Bt cotton.
Similarly, Bai et al. (2006) investigated the effects of Bt rice, containing Cry1Ab toxin, on the coccinellid, Propylea japonica (Thunberg), by feeding its larvae on the nymphs of the plant hopper, Nilaparvata lugens (Stal) (Hem.: Delphacidae), reared on leaves of Bt rice. The results showed that the developmental period, pupation rate, adult eclusion, and weight of pupae and adults of the predator did not differ significantly than from those of the control. Zhao et al. (2013) evaluated the effect of Bt toxins on P. japonica when fed on Aphis gossypii Glover infesting Bt cotton expressing Cry1Ac toxin. The results indicated that there were no distinct differences in the larval development, emerged female weight, or fecundity between predators reared on aphids infesting Bt cotton or conventional cotton. Romeis et al. (2012) reported that the red spider mite, Tetranichus urtica Koch, a natural prey for the larvae of the coccinellid, Adalia bipunctata (L.), was fed on Bt corn before exposure to the predator larvae. The mortality rate among the treated Adalia larvae did not differ significantly from that of the control group which was fed on mites reared on conventional corn.

Field studies
In Bt crops in general Men et al. (2003) and Pilcher et al. (2005) reported decreased populations of predators and parasitoids in transgenic crops compared to conventional ones. Mendelsohn et al. (2003) stated that beneficial arthropods were noticed to be substantially more abundant in Bt crops than in crops treated with chemical pesticides. Naranjo (2009) reported that until 2008, over 63 field studies had been conducted to assess the potential impacts of Bt crops on non-target arthropods (in 6 classes, 21 orders, and 185 species) with vast majority of these being natural enemies. All these studies beside tens of studies, as reported (Dang et al. 2017 andPelligrino et al. 2018), concluded that Bt crops have minimal or negligible effects on non-target arthropods, mainly predators and parasitoids.
Similarly, Romeis et al. (2019) reported that over the past 20+ years, extensive experience and insight have been gained through laboratory and field studies of nontarget effects of crops producing Cry proteins. Overall, the vast majority of the studies demonstrated that the insecticidal proteins developed today do not cause adverse effects on natural enemies of target pests. Furthermore, when Bt crops replace synthetic chemical insecticides for pest control, this creates an environmental support of the conservation of natural enemies.

In Bt cotton
A 3-year field study carried out by Moar et al. (2002) revealed that there were no adverse effects on non-target arthropods (parasitoids and predators) in Bt cotton fields compared to conventionally grown cotton. In a 6-year field study, Naranjo (2005) assessed the long-term impact of Bt cotton producing Cry1Ac toxin on 22 species and strains of foliar-dwelling natural enemies in Arizona.

Abbas Egyptian Journal of Biological Pest Control
(2020) 30:51 Page 5 of 9 The study revealed no chronic or long-term effects of Bt cotton on such natural enemies. Almost similar results were obtained by Sharma et al. (2007) who recorded higher populations of chrysopids, coccinellids, and spiders in Bt cotton than in conventional cotton. Also, Mellet and Scheoman (2007) found that Bt cotton had no adverse impact on the abundance of coccinellids. Yao et al. (2012) studied the potential effects of Bt cotton on the seasonal abundance of 5 groups of predators during 3 years (2009)(2010)(2011) in China. The groups were as follows: (1) the coccinellids, C. undecimpunctata (L.), Adonia variegata (Goeze), and Stethorus sp.; (2) the chrysopids, Chrysopa formosa Brauer and Chrysoperla sinica (Tjeder); (3) the thripid, Acolothrips fasciatus (L.); (4) the anthocorid, Orius similis Zhang; and (5) the spider, Misumenopos tricuspidata (Fabricius) (Acari: Thumicidae). They found that species composition and seasonal abundance of such predators did not differ significantly between those in Bt and non-Bt cotton every year. Lu et al. (2012) reported a remarkable decline in aphid populations in Bt cotton fields in 36 locations in 6 districts north of China. They related this decline to the increase of populations of the coccinellids, chrysopids, and spiders. In addition, these increased populations of the predators on Bt cotton were found to have a considerable role for insect biological control on cotton, corn, and peanut crops adjacent to Bt cotton. Also, Dahi (2013) reported that Bt cotton producing Cry1Ac and Cry2Ab did not affect the populations or abundance of common predators-species prevailing in cotton fields in Egypt. These results agree with those reported by Dhillon and Sharma (2013). Arshad et al. (2015) estimated the populations of insect predators from the 1st week of July to the 2nd week of November in Bt cotton and conventional cotton fields. They found that the population densities and abundance of the predators, C. carnea, Coccinella septempunctata, Geocoris spp., and Minochilus sexmaculatus (Fab.) (Coccinellidae), did not differ significantly in both fields.

In Bt corn
Al- Deeb and Wilde (2003) found that the populations of prevailing predators in a Bt corn field did not differ significantly from those on a conventional corn field. These predators were Hyppodamia convergens Guer, Orius insidiosus (Say), and Scymnuss spp. Similarly, Wolfenbarger et al. (2008) detected no significant negative effects for the predators, Orius spp., Coleomegilla spp., Hypodamia spp., or Chrysoperla spp., in Bt maize.

Effect of Bt crops on parasitoids
Laboratory studies Effect on biological parameters of the parasitoids Baur and Boethel (2003) stated that the parasitoid, Cotesia marginiventris, developed significantly faster in Pseudaletia includens (Walker) larvae fed on the conventional cotton than on Bt cotton. However, the parasitoid which developed in host larvae feeding on Bt cotton suffered reduced longevity and lower fecundity. Also, Bt cotton affected the development of P. includens parasitized with the egg-larval parasitoid, Copidosoma floridanum (Ashmead), as well as the biological parameters of the parasitoid. Fewer C. floridanum adults were found to emerge from hosts fed on Bt cotton, but the longevity of such adults was not affected. Prutz and Dettner (2004) assessed the potential effect of Bt corn leaves on Chilo partellus (Swinhoe) (Lep.: Crambidae) and its braconid larval parasitoid, Cotesia flavipes (Cameron). The percentage of parasitized host larvae which gave rise to adult parasitoids was reduced, and the fresh weight of parasitoid pupae and the dry weight of adults were lower than those from the control. Similarly, Vojtech et al. (2005) stated that Cotesia marginiventris (Cresson), the larval parasitoid of Spodoptera littoralis, was affected when its host larvae were reared on leaves and stems of Bt corn. The survival of parasitoid larvae and their development period inside the host larvae as well as the cocoon weight were significantly negatively affected. Cui et al. (2005) reported that the survival and developmental rates of Microplitis sp. and Campoletis chlorideae, the parasitoids of H. armigera, decreased when their host was reared on leaves of Bt cotton. Sanders et al. (2007) investigated the impact of Bt maize expressing Cry1Ab toxin on the development and behavior of the parasitoid Campoletis sonorensis (Cameron). The results showed that adult parasitoids which emerged from Spodoptera frugiperda larvae fed on Bt maize were significantly smaller (by 15-30%) than those emerged from larvae fed on conventional maize. However, the total developmental period of the parasitoid was not affected by Bt maize treatment. In choice tests, female parasitoids displayed no preference for host larvae fed on Bt maize or conventional maize. No Cry1Ab was detected within adult parasitoids. Salles et al. (2015) investigated the interactions between Bt crops, S. frugiperda, and its larval parasitoid, Campoletis flavicincta. They found that when parasitized and unparasitized larvae were fed on Bt maize (expressing Cry1Ab) for 10 days, their mortality did not differ significantly (90 and 87.7%, respectively). Solmaz et al. (2014) studied the effect of Bt cotton on some biological parameters of Encarsia formosa Gahan, a parasitoid of Bemisia tabaci (Genn.). The results indicated that the total developmental period and the pre-oviposition period of parasitoid females which emerged from the host reared in the Bt cotton were significantly longer than those from non-Bt cotton. The total numbers of eggs deposited/female were 23.6 and 43.8/female in the Bt and non-Bt cotton, respectively.
Discrimination between Bt-infected and uninfected host Sanders et al. (2007), in choice tests, found that females of Campoletis sonorensis (Cameron) displayed no preference for its host larva (S. frugiperda) fed on Bt maize or conventional maize.
Direct effect of Bt toxins on adult parasitoids Marchetti et al. (2012) studied the effect of feeding adults of Exorista larvarum (Dipt.: Tachinidae), a parasitoid of Galleria mellonella (L.) larvae, on sugar soaked with the Cry9Aa from Bt var. galleriae at 3 times the dose which is highly virulent to lepidopteran insects. The study showed that the longevity of males and females of the parasitoid, the numbers of eggs laid by the female, and the percentage of larvae that gave rise to pupae were not significantly affected compared to feeding on distilled water. Yang et al. (2005) reported that population densities of the parasitoids, Trichogramma confusum, Campoletis chlorideae, and Meteorus pulchroconis, were significantly lower in Bt cotton fields than in conventional cotton ones. The authors claimed that such lower populations of parasitoids in Bt crops could be due to the reduced density of the host insect, Helicoverpa armigera. Also, Wu and Guo (2005) recorded lower populations of Trichogramma spp., Micropletis Microplitis sp., and Campoletis sp. parasitoids in Bt cotton as compared to non-Bt cotton.

Field studies
In contrast, Romeis et al. (2006) reported that field studies confirmed that the abundance and activity of parasitoids were found to be similar in Bt and non-Bt crops. Also, Fernandes et al. (2007) found no adverse effects of Bt maize on the populations of Trichogramma parasitoids. Arshad et al. (2015) estimated the populations of insect parasitoids from the 1st week of July to the 2nd week of November in Bt cotton and conventional cotton fields. They found that the abundance of the parasitoids, Trichogramma spp. and Apanteles spp., did not differ significantly in both fields.

Conclusion
Some laboratory studies revealed that B. thuringiensis did not cause detrimental effects on the biological parameters of parasitoids and predators whereas others showed significant negative impact on such natural enemies. The negative impact of Bt-infected larvae on the development of parasitoids of such larvae is related to Bt concentration and timing of infection. Overall, the vast majority of the studies demonstrated that the insecticidal proteins developed today do not cause adverse effects on natural enemies (parasitoids and predators) of target pests.