Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Efficacy of biorational insecticides against Bemisia tabaci (Genn.) and their selectivity for its parasitoid Encarsia formosa Gahan on Bt cotton.

The toxicity of seven biorational insecticides [five insect growth regulators (Buprofezin, Fenoxycarb, Pyriproxyfen, Methoxyfenozide, and Tebufenozide) and two oil-extracts of neem and bitter gourd seeds] against Bemisia tabaci and their selectivity for its parasitoid, Encarsia formosa were evaluated in laboratory and field conditions for 2 years (2018-2019) in Pakistan. Toxicity results demonstrate that Pyriproxyfen, Buprofezin, and Fenoxycarb proved to be effective (80-91% mortality and 66.3-84.2% population-reduction) against B. tabaci followed by Methoxyfenozide, Tebufenozide (50-75% mortality and 47.8-52.4% population-reduction), and then oil-extracts of neem and bitter gourd (25-50% mortality and 36.5-39.8% population-reduction) in the laboratory [72 h post-application exposure interval (PAEI)] and field trails (168 h PAEI), respectively. All tested biorationals, except Methoxyfenozide [(slightly-harmful/Class-II), i.e., causing mortality of parasitoids between a range of 25-50%] and Tebufenozide [(moderately-harmful/Class-III), i.e., causing mortality of parasitoids between the ranges of 51-75%], proved harmless/Class-I biorationals at PAEI of 7-days in the field (parasitism-reduction < 25%) and 3-days in the lab (effect < 30%). In laboratory bioassays, exposure of parasitized-pseudopupae and adult-parasitoids to neem and bitter gourd oils demonstrated that these compounds proved harmless/Class-I biorationals (< 30% mortality). Alternatively, Pyriproxyfen, Buprofezin, Fenoxycarb, Methoxyfenozide, and Tebufenozide were slightly-harmful biorationals (30-79% mortality) against the respective stages of E. formosa. We conclude that most of the tested biorationals proved harmless or slightly harmful to E. formosa, except tebufenozide after PAEI of 7-days (168 h) in the field and, therefore, may be used strategically in Integrated Pest Management (IPM) of B. tabaci.

RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae).

BACKGROUND: Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time. RESULTS: From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci. Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes (Dicer-2/Aubergine/Staufen) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci. CONCLUSION: The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci, which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest.

Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein Loss: Evaluation of Insecticidal Activity and Biosecurity.

Bacillus thuringiensis (Bt) can produce Cry proteins during the sporulation phase, and Cry protein is effective against lepidopteran, coleopteran, and dipteran insects and nematodes. However, Cry protein tends to be discharged into soil and nontarget plants through rainwater runoff, leading to reduced effective period toward target insects. In the present study, nano-Mg(OH)2 (magnesium hydroxide nanoparticles, MHNPs) were synthesized to control the loss of Cry1Ac protein and deliver protein to Helicoverpa armigera (Lepidoptera: Noctuidae). The results showed that Cry1Ac protein could be loaded onto MHNPs through electrostatic adsorption, and both MHNPs and Cry protein were stable during the adsorption process. Meanwhile, the Cry1Ac-loaded MHNPs could remain on the surface of cotton leaves, resulting in enhanced adhesion of Cry1Ac protein by 59.50% and increased pest mortality by 75.00%. Additionally, MHNPs could be slowly decomposed by acid medium and MHNPs showed no obvious influence on cotton, Bt, Escherichia coli, and H. armigera. Therefore, MHNPs could serve as an efficient nanocarrier for delivery of Cry1Ac protein and be used as a potential adjuvant for biopesticide in agricultural applications.

Comparative host selection responses of specialist (Helicoverpa assulta) and generalist (Helicoverpa armigera) moths in complex plant environments.

We tested the behavioral responses of ovipositing females and natal larvae of two sibling species, a generalist Helicoverpa armigera (Hübner) and a specialist Helicoverpa assulta (Guenée), to odor sources emitted from different combinations of six plant species (tobacco, Nicotiana tabacum; hot pepper, Capsicum annuum; tomato, Solanum esculentum; cotton, Gossypium hirsutum; peanut, Arachis hypogaea; maize, Zea mays). Under the conditions of plant materials versus corresponding controls, both stages of both species could find their corresponding host plants. However, H. assulta females and larvae exhibited a supersensitive and an insensitive response, respectively. Under the conditions of tobacco paired with each plant species, H. assulta females exhibited more specialized ovipositional response to tobacco than its sibling. When each plant species were combined with tobacco and tested against tobacco reference, peanut played an opposite role in the two species in their ovipositional responses to tobacco, and cotton can enhance the approaching response of H. armigera larvae when combined with tobacco. It seems that two attractive host plants also can act antagonistically with respect to host selection of the generalist via volatile exchange. Tomato should better be excluded from host list of H. assulta.

Pollen-mediated gene flow from transgenic cotton under greenhouse conditions is dependent on different pollinators.

With the large-scale release of genetically modified (GM) crops, there are ecological concerns on transgene movement from GM crops to non-GM counterparts and wild relatives. In this research, we conducted greenhouse experiments to measure pollen-mediated gene flow (PGF) in the absence and presence of pollinators (Bombus ignitus, Apis mellifera and Pieris rapae) in one GM cotton (resistant to the insect Helicoverpa armigera and the herbicide glyphosate) and two non-GM lines (Shiyuan321 and Hai7124) during 2012 and 2013. Our results revealed that: (1) PGF varied depending on the pollinator species, and was highest with B. ignitus (10.83%) and lowest with P. rapae (2.71%); (2) PGF with B. ignitus depended on the distance between GM and non-GM cottons; (3) total PGF to Shiyuan321 (8.61%) was higher than to Hai7124 (4.10%). To confirm gene flow, we tested hybrids carrying transgenes for their resistance to glyphosate and H. armigera, and most hybrids showed strong resistance to the herbicide and insect. Our research confirmed that PGF depended on pollinator species, distance between plants and the receptor plant.

Cloning of a putative extracellular Cu/Zn superoxide dismutase and functional differences of superoxide dismutases in invasive and indigenous whiteflies.

Superoxide dismutases (SODs) are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase (ecCuZnSOD) complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of BtecCuZnSOD was more than 10-fold higher in the invasive Middle East Asia Minor 1 (MEAM1) than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature (4 °C), the expression of Bt-ecCuZnSOD gene was significantly up-regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level of B. tabaci intracellular CuZnSOD (Bt-icCuZnSOD), Bt-ecCuZnSOD and mitochondrial MnSOD (Bt-mMnSOD) was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable (cotton) to unfavorable host plants (tobacco). On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt-ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt-mMnSOD was expressed equally in both species on cotton, Bt-mMnSOD messenger RNA was up-regulated in MEAM1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion of MEAM1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.

Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.

The efficacy of a petroleum spray oil against Aphis gossypii Glover on cotton. Part 1: mortality rates and sources of variation.

Petroleum spray oils (PSOs) kill insect pests on contact, and the composition of modern PSOs is substantially different from the ones introduced earlier. The effects of direct application of a new nC24 PSO on the cotton aphid, Aphis gossypii Glover, were therefore determined. This covered not only aphid mortality rates but also the way in which the oils affected aphid behaviour at the time of contact with the oil. Direct application of the nC24 oil proved to be highly effective in controlling A. gossypii at a range of concentrations between 1 and 10% v/v. The oil killed cotton aphids quickly, with most of the mortality occurring within the first 10 min of spraying. The fast killing action of the oils prevented any behavioural responses by the aphids. Aphids killed by the oils became flaccid and their legs and antennae extended horizontally relative to the body axis. With time, their cuticle became very shiny and began to darken. The quick death of the aphids suggested a contact mode of action of the oils, an interpretation supported by the lack of any negative effect on aphids not initially reached by the oils. However, those aphids not hit by the oils, but that subsequently encountered oil-treated areas when they moved elsewhere, also died, indicating that the oil deposits are also toxic to the aphids. The mode of action of the oil thus seems to be versatile, and the means by which it kills the aphids may be more complex than anoxia, which is the widely claimed mechanism attributed to PSOs. This oil now needs to be tested for any possible indirect effects on the cotton aphid (e.g. through its host-plant acceptance behaviour). The implications of the present findings for cotton aphid control and assessment of PSO efficacy in the field are discussed.

The efficacy of a petroleum spray oil against Aphis gossypii Glover on cotton. Part 2: indirect effects of oil deposits.

The primary mode of action of petroleum spray oils (PSOs) on pest insects is through direct contact. Indirect effects are, however, also possible, and deposits of the oils may influence pest populations by killing insects and/or by influencing their behaviour. The indirect effects of deposits of a new nC24 oil against the cotton aphid, Aphis gossypii Glover, were therefore determined. The effects of oil deposits on the acceptance of cotton as host plant by the aphids were assessed, as well as aphid mortality rates and their success in the establishment of colonies. The efficacy of deposits of a heavier oil (nC27) was also evaluated. Deposits of PSO were toxic to A. gossypii and remained effective until 8 days after spraying. Mortality decreased with time, so that, the older the deposit, the lower was the mortality. Significantly higher aphid mortalities were achieved on younger leaves than on mature ones. Thus, leaf age proved a significant factor in the efficacy of the deposits. Consecutive prophylactic applications (at 9 day intervals) did not have a cumulative effect, and their killing power proved to be independent of one another. Thus, applying the oil prior to aphid infestations would confer only minimal protection. The mortality inflicted by the deposits was not improved by increasing the molecular mass of the oil used (nC27 oil), but the toxic life of the oil deposit was increased. Oil deposits did not deter alates from landing on oil-sprayed plants. Oil deposits did, however, affect subsequent alate and nymphal survival, and thus the establishment of aphid colonies. The impact that the oils could have on the longer-term development of aphid populations in the field was thus demonstrated. First- and second-instar nymphs were the most susceptible life stages, with > 50% mortality compared with < 10% for the other stages. These nymphs did not show the typical signs of oil-induced mortality observed in aphids killed by direct oil applications, which suggests an alternative mode of action to that of the directly applied oil. Anoxia does not seem to be involved in either process, and alternative modes of action of the oil deposits are discussed. The implications of these findings for cotton aphid control are also considered, primarily in relation to the timing and frequency of oil application.

Antifeedant and toxic activity of some plant extracts against larvae of cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae).

The biological activity of crude petroleum ether extracts of Oshar (Calotropis procera); Harmal (Rhazya stricta) and Hargal (Solenostemma argel) were assessed using the 4th larval instar of cotton leafworm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). All extracts exhibited a significant antifeedant activity at the LC50 levels. Harmal extract deterred feeding potential of insect larvae by 52.96% but decreased to 26.76 and 18.00% for Hargal and Oshar, respectively. In nutritional assays, all plant extracts affected Growth Rate (GR mg) where Harmal caused the highest rate of efficiency and followed by Oshar against 4th larval instar fed for two days on castor leaves treated with botanical extracts (LC50) and three days on un-treated leaves after that. Hargal didn't show a significant effect on growth rate compared with un-treated larvae. The differences between Consumption Index (CI mg) of larvae treated with Hramal and Hargal after five days of feeding in comparison with un-treated larvae were significant. Efficiency of Conversion of Ingested food (ECI%) to biomass reached 31.81 +/- 1.49, 26.06 +/- 1.89 and 48.67 +/- 1.54 after five days of treatment by Oshar, Harmal and Hargal, respectively. These values were 49.61 +/- 1.72, 39.12 +/- 0.54 and 53.20 +/- 0.96 for digested food utilization (ECD%) in larvae treated with the aforementioned extracts after five days, respectively. There was a remarkable inhibitory activity of plant extracts on the digestive carbohydrate enzymes, amylase and invertase in vitro. It was noticed that Harmal had a remarkable inhibitory action causing an average of 42.58% inhibition rate on amylase and 16.27% on invertase followed by Hargel and Oshar with inhibition rates of 33.27 and 19.58% against amylase, while these values averaged 11.19 and 5.97% of inhibition in case of invertase, respectively.

Effect of Zohar LQ-215, a biorational surfactant, on the sweetpotato whitefly Bemisia tabaci (biotype b).

Zohar LQ-215, a surfactant based on plant oils, able to control nymphs of the whitefly, Bemisia tabaci (Gennadius), under laboratory and field conditions. To evaluate the effects of the surfactant on the nymph stages of whitefly under laboratory conditions, potted cotton seedlings infested with 1st- or 3rd-instars were treated with the compound aqueous solutions. LC90 values of Zohar LQ-215 on 1st and 3rd-nymphs, based on mortality curves, were 0.78% and 1.14%, respectively. Adult mortality of 12% and 19% was obtained at concentrations of 0.5% and 1%, respectively. Under both laboratory and, in some cases, under field conditions, greater mortality was achieved when combining Zohar LQ-215 with the insect growth regulator buprofezin than when either insecticide was applied separately. Our results indicate that Zohar LQ-215 could serve as a potential compound for controlling whiteflies under light to moderate infestation and could be used in combination with other rational insecticides such as buprofezin for controlling whiteflies in integrated pest management programs.

Feeding deterrent and growth inhibitory properties of limonoids from Khaya senegalensis against the cotton leafworm, Spodoptera littoralis.

Three rearranged phragmalin-type limonoids, khayanolide A, khayanolide B and 1-O-acetylkhayanolide B, and a mexicanolide-type limonoid, khayalactol, have been isolated from the stem bark of Khaya senegalensis (Desr) A Juss (Meliaceae). The antifeedant and growth-inhibitory activities of the isolated compounds were evaluated on Spodoptera littoralis (Boisduval). When added to an artificial diet, khayanolide A, khayanolide B and 1-O-acetylkhayanolide B showed antifeedant activity in a concentration-dependent manner. Khayalactol exhibited strong antifeedant activity without significant differences at all of the tested concentrations (7.5-100 mg kg(-1)). Khayanolide B was the most potent antifeedant with an ECso of 2.19 mg kg(-1). The results also revealed that the isolated compounds caused marked larval growth inhibition on S littoralis after 7 days of feeding on treated diet; this effect was concentration-dependent. Khayanolide B was the most active growth inhibitor among the isolated compounds, with an EC50 of 6.96 mg kg(-1).

Field accumulation of aflatoxin in cottonseed as influenced by irrigation termination dates and pink bollworm infestation.

Aflatoxin accumulation in Deltapine 16 cottonseed, grown in Yuma, Ariz., in a 3-year study, was significantly influenced by the timing of irrigation terminations and by level of pink bollworm infestations. In 1971 and 1972, termination of irrigations by early August resulted in significantly less aflatoxin than in plots where two additional irrigations were applied. Significantly less aflatoxin also was found in the 1971 and 1973 plots where low levels of pink bollworm infestations were maintained.