Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor

Abstract

BackgroundMosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni.MethodsIn this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin.ResultsCalcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa.ConclusionsOur results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

Production and characterization of Trichoderma asperellum chitinases and their use in synergy with Bacillus thuringiensis for lepidopteran control

“…Interestingly, in S. littoralis larvae a significantly increased toxicity was observed when the chitinase preparation was administered with sublethal concentrations of the commercial Bt ‐based product Xentari™. This synergistic effect was likely due to the action of chitinases altering the structural integrity of the peritrophic matrix, thus facilitating the action of Bt toxins, as previously observed with other enhancing agents 6–8,76 . This result is particularly promising from an applicative perspective to enhance the impact and the long‐term efficacy of Bt spray formulations.…”

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

“…Although Cry toxins are very efficient molecules and only very minute quantities are required for toxicity, the obsession to improve their efficiency has led to the development of diverse strategies [28]. The developed strategies include (i) the combination of several Cry toxins to increase efficiency toward a specific target [29]; (ii) co-expression with other B. thuringiensis proteins such as the P20 protein to provide protection in the larval gut environment [29,30], or chitinases for peritrophic membrane degradation [31]; (iii) combination with chemical compounds such as calcofluor for peritrophic membrane digestion [32], or coating with Mg(OH) 2 to increase their resistance to UV light [33]; (iv) combination with other insecticidal toxins such as Cyt toxins [34][35][36][37], VIP toxins [38], Bin toxins [39], Metalloproteinase Bmp1 [40], or insect-specific scorpion toxins [41] that synergize their effect; (v) combination with insect chaperones such as Hsp90 chaperone [42]; or (vi) expression of Cry toxins in other backgrounds such as baculovirus [43]. Other strategies developed to increase potency include the fusion of 3D-Cry to other toxins such as neurotoxins (Huwentoxin-I [44], ω-ACTX-Hv1a [45], or huwentoxin XI [46]) present in spiders venom, VIP proteins [47], the N-terminal region of PirB toxin from Photorhabdus luminescens [48], or the fusion to other proteins that provide interesting domains such as garlic lectin [49,50], cellulase-binding peptides [51], or Escherichia coli maltose binding protein (MBP) [52], thus rendering chimeras with improved activity.…”

Co-Toxicity Factor Analysis Reveals Numerous Plant Essential Oils Are Synergists of Natural Pyrethrins against Aedes aegypti Mosquitoes

“…Moreover, if these oils increase insecticide penetration, it would be valuable to better understand the physicochemical factors underlying these activities. Increased penetration may be facilitated via improved passive diffusion across the cuticle (as in the case of calcofluor [ 22 , 23 ]) or through the inhibition of drug efflux pumps, similar to the mechanism of verapamil, an ATP-binding cassette (ABC) transporter inhibitor [ 24 ].…”