Synergy Between Aedes aegypti Trypsin Modulating Oostatic Factor and δ-Endotoxins

Abstract

Starved first instar Aedes aegypti larvae were 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis (Bti) toxins than fed larvae. Feeding larvae Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with Escherichia coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) indicate that TMOF and Cry toxins are synergisitic. tmfA was cloned and expressed in the cyanobacterium Anabaena PCC 7120 and the hormone was purified by HPLC and identified by ELISA. The amount of TMOF synthesized by Anabaena was low (0.5 -1 μg in 10 8 cells). P. pastoris, which synthesizes high amounts of heterologous proteins in the presence of methanol and is readily consumed by mosquito larvae, was genetically engineered to produce more TMOF. Codon-optimized synthetic genes, cry11Aa-tmfA and gst-cry11Aa-tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae caused 87.5% mortality in 5 days. GST (glutathione-S-transferase) enhanced the activity of Cry11A-TMOF and protected it from heat denaturation. Cell free extracts of recombinant P. pastoris cells killed 40% of tested 4 th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa. This report shows for the first time that Cry toxins and TMOF are synergists to Ae. aegypti larvae when jointly fed or expressed in recombinant P. pastoris.

Residual effects of TMOF–Bti formulations against 1st instar Aedes aegypti Linnaeus larvae outside laboratory

“…aegypti larvae were 35-fold more sensitive to Bti toxins than fed larvae. TMOF enhances the effect of Bti toxins in organic rich environments that are abundant in nature [13] . From [12] .…”

“…So, the use of both toxins in early developmental stages of larvae is expected to enhance the effect of low toxin doses and reduce the amount of Bti required in sewage treatment ponds in which it is not effective alone [13] . The activation of the 毮-endotoxins in Bti may be accomplished by pre-existing endogenous larval gut trypsin before its level in the midgut is reduced by TMOF.…”

“…In addition, larval starvation, which occurs later on, will slow down repair of sub-lethal damaged gut epithelium by 毮-endotoxins enhancing gut absorption of TMOF into the hemolymph [18] . Starved larvae may also eat more recombinant yeast cells bearing toxins thus enhancing larval demise [13] . Toxicity of Cry1C against advanced larval stages (third and fifth instars) of Spodoptera littoralis is, indeed, reduced due to high levels of protease activity in the gut which rapidly degrades the ingested toxin [19] .…”

“…aegypti larvae outside laboratory. For all concentrations of (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mg), 100% larval mortality occurred at the end of second week exposure. At the end of the third week larval mortality indicated 95%-100% with 6% for the control.…”

“…aegypti 1 st instar larvae in the laboratory was found to be able to improve the activity of these product by giving quick mortality effect within 1 hour treatment and prolong their residual effect (no larvae survived) against all larval stages until four weeks of treatment [12] . TMOF-Bti are the combination of TMOF and B. thuringiensis israeliensis which showed increased efficacy in killing mosquitoe larvae [13] . The present study was aimed to determine the outdoor residual effect of TMOF-Bti in rice husk and wettable powder formulations at different weights in the area of UKM campus against 1 st instar Ae.…”