Transcriptomic analysis of Spodoptera frugiperda Sf9 cells resistant to Bacillus thuringiensis Cry1Ca toxin reveals that extracellular Ca2+, Mg2+ and production of cAMP are involved in toxicity.

Bacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model - referred to as the pore forming model - is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+-dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca-resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical 'loss of toxin binding'. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover.

Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut.

Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. Here, mimicking environmental conditions, we show that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, we show that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes.

Microbial Pest Control Agents: Are they a Specific And Safe Tool for Insect Pest Management?

Microorganisms (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA). They are used as alternative strategies to chemical insecticides to counteract the development of resistances and to reduce adverse effects on both environment and human health. These natural entomopathogenic agents, which have specific modes of action, are generally considered safer as compared to conventional chemical insecticides. Baculoviruses are the only viruses being used as the safest biological control agents. They infect insects and have narrow host ranges. Bacillus thuringiensis (Bt) is the most widely and successfully used bioinsecticide in the integrated pest management programs in the world. Bt mainly produces crystal delta-endotoxins and secreted toxins. However, the Bt toxins are not stable for a very long time and are highly sensitive to solar UV. So genetically modified plants that express toxins have been developed and represent a large part of the phytosanitary biological products. Finally, entomopathogenic fungi and particularly, Beauveria bassiana and Metarhizium anisopliae, are also used for their insecticidal properties. Most studies on various aspects of the safety of MPCA to human, non-target organisms and environment have only reported acute but not chronic toxicity. This paper reviews the modes of action of MPCA, their toxicological risks to human health and ecotoxicological profiles together with their environmental persistence. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined.

In the agricultural environment, honey bees may be exposed to combinations of pesticides. Until now, the effects of these combinations on honey bee health have been poorly investigated. In this study, we assessed the impacts of biological and chemical insecticides, combining low dietary concentrations of Bacillus thuringiensis (Bt) spores (100 and 1000µg/L) with the chemical insecticide fipronil (1µg/L). In order to assess the possible effects of Cry toxins, the Bt kurstaki strain (Btk) was compared with a Bt strain devoid of toxin-encoding plasmids (Bt Cry(-)). The oral exposure to fipronil and Bt spores from both strains for 10 days did not elicit significant effects on the feeding behavior and survival after 25 days. Local and systemic physiological effects were investigated by measuring the activities of enzymes involved in the intermediary and detoxication metabolisms at two sampling dates (day 10 and day 20). Attention was focused on head and midgut glutathione-S-transferase (GST), midgut alkaline phosphatase (ALP), abdomen glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD). We found that Bt Cry(-) and Btk spores induced physiological modifications by differentially modulating enzyme activities. Fipronil influenced the enzyme activities differently at days 10 and 20 and, when combined with Bt spores, elicited modulations of some spore-induced physiological responses. These results show that an apparent absence of toxicity may hide physiological disruptions that could be potentially damaging for the bees, especially in the case of combined exposures to other environmental stressors.

Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication.

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera.

The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae.

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae).

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.

Effects of a mosquitocidal toxin on a mammalian epithelial cell line expressing its target receptor.

The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an alpha-glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays alpha-glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.

Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test.

Populations of the codling moth, Cydia pomonella L (Lepidoptera, Tortricidae) have developed resistance to several classes of insecticide such as benzoylureas, juvenile hormone analogues, ecdysone agonists and pyrethroids, but the corresponding resistance mechanisms have not been extensively studied. Knockdown resistance (kdr) to pyrethroid insecticides has been associated with point mutations in the para sodium channel gene in a great variety of insect pest species. We have studied two susceptible strains (S and Sv) and two resistant strains (Rt and Rv) of C pomonella that exhibited 4- and 80-fold resistance ratios to deltamethrin, respectively. The region of the voltage-dependent sodium channel gene which includes the position where kdr and super-kdr mutations have been found in Musca domestica L was amplified. The kdr mutation, a leucine-to-phenylalanine replacement at position 1014, was found only in the Rv strain. In contrast, the super-kdr mutation, a methionine-to-threonine replacement at position 918, was not detected in any C pomonella strain. These data allowed us to develop a PCR-based diagnostic test (PASA) to monitor the frequency of the kdr mutation in natural populations of C pomonella in order to define appropriate insecticide treatments in orchards.

Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance.

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an alpha-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1(GEO) cDNA differs from the sequence we previously reported for a susceptible strain (cpm1(IP)) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1(IP) was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1(GEO) resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1(GEO) and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.