Toxicity of Different Insecticides to Sitophilus oryzae, Tribolium castaeneum and Tribolium confusum Infesting Corn

Corn is an important cereal crop cultivated across various countries, including the United States. Corn serves multiple purposes such as human and livestock food and raw material for industrial processes. Despite being an important crop, corn production faces challenges due to factors such as weather fluctuations and the presence of insect pests and diseases, both in the field and during storage. Among these challenges, insect infestation poses a major threat, as the insect damage not only reduces the quantity and quality of the grain but also renders it unfit for human and animal consumption, leading to a decrease in market value. To address the challenges of insect infestation in stored grain, several control methods have been employed which include cleaning and drying grain to eliminate debris, the use of airtight containers or hermetic storage facilities, the application of botanicals with insecticidal properties, heat treatment, and the use of insecticide chemicals. Insecticides have been extensively used since 1950 to deal with various insect pests due to their effectiveness, ease of application, and long-lasting effects. This research examined the toxicity of four different insecticides namely, pirimiphos- methyl, deltamethrin, deltamethrin plus (S)-methoprene and malathion against Sitophilus oryzae, Tribolium castaneum, and Tribolium confusum infesting corn. The toxicity of each insecticide was determined in terms of LC50 by exposing target pests at different doses and the mortality was observed at 24 to 96 hours of exposure as well as until 10 days to determine delayed mortality. In the first study, the toxicity of the ementioned insecticides to S. oryzae was evaluated and the results revealed that pirimiphos-methyl insecticide was more toxic to S. oryzae, followed by malathion, deltamethrin, and deltamethrin plus (S)-methoprene. When the LC50 value of each insecticide formulation was compared with the label-recommended application rate, we found that only malathion was aligned with the label rate and did not need to be adjusted. Pirimiphos-methyl was over-applied, and the rate may be adjusted. In the second study, the toxicity of these insecticides was determined for T. castaneum and T. confusum. The results indicated that deltamethrin plus (S)-methoprene and deltamethrin displayed the highest toxicity against T. castaneum, followed by pirimiphos-methyl. Malathion displayed the lowest toxicity among all the insecticides tested. On the other hand, pirimiphos-methyl was highly toxic among all the insecticides, followed by deltamethrin plus (S)-methoprene, deltamethrin, and malathion against T. confusum. Although these two pests are closely related species, their susceptibility to the selected insecticides was different in such a way that T. confusum displayed higher susceptibility to pirimiphos-methyl compared to T. castaneum while T. castaneum exhibited greater susceptibility to deltamethrin, deltamethrin plus (S)-methoprene, and malathion compared to T. confusum. Compared the label recommended application rate with the LC50 found in the study, deltamethrin plus (S)-methoprene and malathion for T. confusum were appropriate and did not require any changes while the recommended label rate for pirimiphos-methyl exceeded the LC50 value for both species of flour beetles. These findings contribute to our understanding of insecticide toxicity to these stored-grain pests and provide valuable insights for their management

Toxicity of Different Insecticides to Sitophilus oryzae, Tribolium castaeneum and Tribolium confusum Infesting Corn

Corn is an important cereal crop cultivated across various countries, including the United States. Corn serves multiple purposes such as human and livestock food and raw material for industrial processes. Despite being an important crop, corn production faces challenges due to factors such as weather fluctuations and the presence of insect pests and diseases, both in the field and during storage. Among these challenges, insect infestation poses a major threat, as the insect damage not only reduces the quantity and quality of the grain but also renders it unfit for human and animal consumption, leading to a decrease in market value. To address the challenges of insect infestation in stored grain, several control methods have been employed which include cleaning and drying grain to eliminate debris, the use of airtight containers or hermetic storage facilities, the application of botanicals with insecticidal properties, heat treatment, and the use of insecticide chemicals. Insecticides have been extensively used since 1950 to deal with various insect pests due to their effectiveness, ease of application, and long-lasting effects. This research examined the toxicity of four different insecticides namely, pirimiphos- methyl, deltamethrin, deltamethrin plus (S)-methoprene and malathion against Sitophilus oryzae, Tribolium castaneum, and Tribolium confusum infesting corn. The toxicity of each insecticide was determined in terms of LC50 by exposing target pests at different doses and the mortality was observed at 24 to 96 hours of exposure as well as until 10 days to determine delayed mortality. In the first study, the toxicity of the ementioned insecticides to S. oryzae was evaluated and the results revealed that pirimiphos-methyl insecticide was more toxic to S. oryzae, followed by malathion, deltamethrin, and deltamethrin plus (S)-methoprene. When the LC50 value of each insecticide formulation was compared with the label-recommended application rate, we found that only malathion was aligned with the label rate and did not need to be adjusted. Pirimiphos-methyl was over-applied, and the rate may be adjusted. In the second study, the toxicity of these insecticides was determined for T. castaneum and T. confusum. The results indicated that deltamethrin plus (S)-methoprene and deltamethrin displayed the highest toxicity against T. castaneum, followed by pirimiphos-methyl. Malathion displayed the lowest toxicity among all the insecticides tested. On the other hand, pirimiphos-methyl was highly toxic among all the insecticides, followed by deltamethrin plus (S)-methoprene, deltamethrin, and malathion against T. confusum. Although these two pests are closely related species, their susceptibility to the selected insecticides was different in such a way that T. confusum displayed higher susceptibility to pirimiphos-methyl compared to T. castaneum while T. castaneum exhibited greater susceptibility to deltamethrin, deltamethrin plus (S)-methoprene, and malathion compared to T. confusum. Compared the label recommended application rate with the LC50 found in the study, deltamethrin plus (S)-methoprene and malathion for T. confusum were appropriate and did not require any changes while the recommended label rate for pirimiphos-methyl exceeded the LC50 value for both species of flour beetles. These findings contribute to our understanding of insecticide toxicity to these stored-grain pests and provide valuable insights for their management

A review of plant materials used for controlling insect pests of stored products (NRI Bulletin 65)

The use of plant materials for the protection of stored products against insect pests is reviewed. Following an outline of the different methods used for assessing their insecticidal activity, over 120 plants and plant products are listed alphabetically with a brief description of traditional uses (where appropriate), and a summary of the laboratory data currently available; where possible, information on constituents and toxicology is also included. Of these, only a few, all species in the genera Azadirachta, Acarus, Chenopodium, Eucalyptus, Mentha, Ocimum, Piper and Tetradenia, have been sufficiently tested in the laboratory to give an indication of their potential usefulness as stored product protectants. The neem tree, Azadirachta indica, is considered in a separate section as it has been more extensively studied than the other plants; the traditional uses of neem, its constituents, effects, pests and diseases, toxicity, field trials and commercial applications are outlined, and results of laboratory trials are summarized. It is concluded that more rigorous testing is required if the considerable potential for botanical protectants is to be realized . Standardized assessments of residual effectiveness against all stages of the insect's life cycle need to be undertaken, and mammalian toxicity requires more detailed evaluation. More attention may need to be given to the characterization and quantification of chemical constituents, and to variations which may occur in different karyotypes. It may also be necessary to determine the availability of appropriate extraction and application technology if the use of plant protectants is to be assimilated in developing countries

Effects of extreme temperatures on the survival of the quarantine stored-product pest, Trogoderma granarium (khapra beetle) and on its associated bacteria

Trogoderma granarium is a pest of stored-grain products in Asia and Africa, and a quarantine pest for much of the rest of the world. To evaluate extreme temperatures as a control strategy for this pest, I investigated the effect of high and low temperatures on the survival (Chapters 2 and 3) and on the microbiome (Chapter 4) of T. granarium. The most cold- and heat-tolerant life stages were diapausing-acclimated larvae and diapausing larvae, respectively. Trogoderma granarium can be controlled (Probit 9) with an exposure of 70 d to −15°C or 1.2 h to 60°C. High temperatures affected the microbiome; an effect of low temperatures was not detected. While the microbiome changed with life stage, it was dominated by Spiroplasma bacteria. Further research is necessary to understand the Spiroplasma-T. granarium relationship. Future research should also investigate combinations of extreme temperatures with other techniques to shorten the time required for mortality

Induction and termination of diapause in khapra beetle, Trogoderma granarium

Larvae of the stored product pest, khapra beetle, Trogoderma granarium, are unusual in that they can enter a diapause state that confers protection against pest control methods. In Chapter 1, I reviewed the literature on general aspects of insect diapause induction and termination, particularly in Dermestidae. In Chapter 2, I assessed the effects of age of culture and diet quality on diapause termination. The results indicated that diapausing larvae can accumulate the nutrients required to terminate diapause and complete development when provided with an opportunity to do so. In Chapter 3, I assessed whether desiccation enhances cold tolerance in diapausing and non-diapausing larvae. Results showed that cold tolerance of diapausing larvae increased with greater desiccation stress. This finding suggests that the physiological mechanisms that protect diapausing larvae from desiccation may also increase cold-tolerance. Overall results of this research provide new information with potential application for control of this global pest

Genetic Glass Ceilings

As the world’s population rises to an expected ten billion in the next few generations, the challenges of feeding humanity and maintaining an ecological balance will dramatically increase. Today we rely on just four crops for 80 percent of all consumed calories: wheat, rice, corn, and soybeans. Indeed, reliance on these four crops may also mean we are one global plant disease outbreak away from major famine. In this revolutionary and controversial book, Jonathan Gressel argues that alternative plant crops lack the genetic diversity necessary for wider domestication and that even the Big Four have reached a “genetic glass ceiling”: no matter how much they are bred, there is simply not enough genetic diversity available to significantly improve their agricultural value. Gressel points the way through the glass ceiling by advocating transgenics—a technique where genes from one species are transferred to another. He maintains that with simple safeguards the technique is a safe solution to the genetic glass ceiling conundrum. Analyzing alternative crops—including palm oil, papaya, buckwheat, tef, and sorghum—Gressel demonstrates how gene manipulation could enhance their potential for widespread domestication and reduce our dependency on the Big Four. He also describes a number of ecological benefits that could be derived with the aid of transgenics. A compelling synthesis of ideas from agronomy, medicine, breeding, physiology, population genetics, molecular biology, and biotechnology, Genetic Glass Ceilings presents transgenics as an inevitable and desperately necessary approach to securing and diversifying the world's food supply

Development of next-generation biopesticides as a control method for the small hive beetle Aethina tumida murray (coleoptera: nitidulidae), a serious pest of the European honey bee Apis mellifera

The small hive beetle (Aethina tumida) is a serious pest of the European honey bee (Apis mellifera), responsible for causing significant economic damage to the apiculture industry in North America and Australia. In 2014 A. tumida was detected in Italy, highlighting the potential for an outbreak within the UK. Current control measures rely on the use of organophosphate and permethrin, both are highly toxic to honeybees and continued use can give rise to resistance. Given these issues alternative control strategies are urgently required. The aims of this thesis were to explore potential for the development of next generation biopesticides, including RNA interference (RNAi) and fusion protein technology, as an alternative control method for A. tumida The sequence specificity of RNAi makes it an ideal strategy to combat this parasite of honey bees. Here we report that microinjection of low (2-10 ng) doses of V-ATPase subunit A and Laccase 2 dsRNAs resulted in 100 % mortality of A. tumida larvae. Quantitative PCR analysis confirmed that injections induced significant decreases in mRNA levels of the target genes with an enhancement of gene suppression over time providing evidence for systemic RNAi effects. Whilst oral delivery of V-ATPase subunit A dsRNA via “soaking” in dsRNA solutions resulted in 50 % mortality and malformed survivors, gene suppression could not be verified by qPCR analysis. Our results showed that dsRNAs are prone to degradation by extracellular nucleases following ingestion by feeding, but not wandering stage, larvae. We suggest that the lack of consistent RNAi effects in feeding experiments was a consequence of dsRNA degradation within the gut of A. tumida. Target specificity was confirmed by a lack of effect on survival or gene expression in honey bees injected with A. tumida dsRNAs. A. tumida show a robust response to injected dsRNA but further research is required to develop methods to induce RNAi effects via ingestion. The spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) is highly potent by injection to a range of insects, but not vertebrates making it an ideal candidate for the development of bioinsecticides. Oral delivery of the toxin is largely ineffective due to failure to access its site of action in the central nervous system (CNS). Fusion protein technology allows oral delivery of Hv1a to the CNS via fusion to a “carrier” protein, snowdrop lectin Galanthus nivalis agglutinin (GNA), directing transport of the toxin across the insect gut to the circulatory system. Constructs encoding Hv1a or modified Hv1a (K>Q modification to remove potential KEX2 cleavage site) linked to the N- or C-terminus of snowdrop lectin (GNA) were used to produce recombinant GNA/Hv1a, Hv1a/GNA fusion proteins and K>Q. All four fusion proteins were toxic by injection to A. tumida. The LD50’s for GNA/Hv1a and GNA/Hv1a(K>Q) were a similar 0.44 and 0.47 µg/µl, whilst Hv1a/GNA and Hv1a(K>Q)/GNA LD50’s were slightly lower, at a respective 0.33 and 0.25 µg/µl. In contrast no effects on honeybee survival were observed when 20 fold higher doses were injected. When fed to A. tumida larvae, GNA/Hv1a was 2x more effective than Hv1a/GNA, GNA/Hv1a(K>Q) and Hv1a(K>Q)/GNA (LC50s of 0.52, 1.14, 1.18 and 0.89 mg/ml, respectively). When fed to A. tumida adults no mortality was recorded for GNA/Hv1a(K>Q) or Hv1a(K>Q)/GNA treatments. However, both Hv1a/GNA and GNA/Hv1a were toxic to adults, with similar LC50s of 2.52 and 2.02 mg/ml, respectively. Reduced efficacy of Hv1a/GNA and K>Q variants against larvae was shown to be attributable to differences in the stability of the fusion proteins in the presence of extracellular gut proteases. In laboratory assays A. tumida larval survival was significantly reduced when brood, inoculated with eggs, was treated with GNA/Hv1a. The dominant digestive protease in A. tumida larvae was identified as trypsin. Consequently, a trypsin inhibitor (Soybean Kunitz trypsin inhibitor: SKTI) was incubated together with A. tumida gut extracts and GNA/Hv1a and Hv1/GNA, with both fusion protein remaining fully intact after 24 hr. This contrasted with previous analysis that showed no intact GNA/Hv1a or Hv1a/GNA after incubation with gut extracts in the absence of the trypsin inhibitor under comparable conditions. Consequently, SKTI was evaluated as an alternative carrier protein to GNA for the delivery of Hv1a to the circulatory system of A tumida. Preliminary studies indicated transport of SKTI into the haemolymph, suggesting SKTI could be used as an alternative carrier protein. An initial construct was designed based on GNA/Hv1a, however no biological activity was observed after injection into A. tumida larvae. It was speculated that the lack of insecticidal activity was attributed to the misfolding of the toxin during expression in the yeast cells. As such two additional fusion proteins were designed incorporating either a flexible (Gly-Gly-Gly-Gly-Ser motif) or rigid linker (Proline rich motif region) to improve protein folding and function. Only inclusion of a rigid linker showed limited biological activity after injection into A. tumida larvae, again suggesting misfolding of the toxin. Both RNAi and fusion protein technology hold enormous potential for the control of A. tumida in apiculture and to our knowledge this is the first study to demonstrate the use of a protein based biopesticide and RNAi as a possible control method for A. tumida