Highly Effective Broad Spectrum Chimeric Larvicide That Targets Vector Mosquitoes Using a Lipophilic Protein

Two mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis (Bti) and Lysinibacillus sphaericus (Ls) are the active ingredients of commercial larvicides used widely to control vector mosquitoes. Bti’s efficacy is due to synergistic interactions among four proteins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, whereas Ls’s activity is caused by Bin, a heterodimer consisting of BinA, the toxin, and BinB, a midgut-binding protein. Cyt1Aa is lipophilic and synergizes Bti Cry proteins by increasing midgut binding. We fused Bti’s Cyt1Aa to Ls’s BinA yielding a broad-spectrum chimeric protein highly mosquitocidal to important vector species including Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti, the latter an important Zika and Dengue virus vector insensitive to Ls Bin. Aside from its vector control potential, our bioassay data, in contrast to numerous other reports, provide strong evidence that BinA does not require conformational interactions with BinB or microvillar membrane lipids to bind to its intracellular target and kill mosquitoes.

major toxins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa), was the most potent (LC 50s 3.6-7.1 ng/ml, LC 95s from 18.5-88 ng/ml). Ls 2362 was active against Cx. quinquefasciatus, and An. gambiae and An. stephensi, but not against Ae. aegypti and Cx. quinquefasciatus BS-R, a strain selected for high levels of resistance to Bin; the LC 50 s and LC 95 s of Ls 2362 were >1,000,000 ng/ml. The Cyt1Aa-BinA chimeric strain, however, was highly toxic to larvae of species belonging to all three major genera of disease vectors, Culex, Aedes and Anopheles, with LC 50 s ranging from 9.2 to 61.9 ng/ml, and LC 95 s from 30 to 271 ng/ml ( Table 1). Toxicity of the chimera was high by 24 hours post-treatment (Table 1), which typically only occurs by 48 hours when Ls is tested against larvae (Table 2).
Interestingly, with regard to both LC 50 s and LC 95 s, the relative toxicities of the Cyt1Aa-BinA chimera or Bti 4Q5 (with the wild type parasporal body) against all larvae assayed, with the exception of Ae. aegypti, were not significantly different, as they ranged from 0.4-2.1, even against the BinA/BinB-resistant Cx. quinquefasciatus BS-R strain (Table 1). Against the anopheline species, although fiducial limits of LC 50 s of the Cyt1Aa-BinA protein (23.0 ng/ml) and Bti 4Q5 (26.5 ng/ml) against An. gambiae overlapped, those of Cyt1Aa-BinA (28.9 ng/ml) and Bti 4Q5 (14.8 ng/ml) against An. stephensi did not. However, their LC 95 s completely overlapped against both species indicating that the Cyt1Aa-BinA fusion protein alone was as effective as the wild-type Bti 4Q5.
Perhaps most interesting are the LC 50 s and LC 95 s toxicities observed for Cyt1Aa-BinA against Ae. aegypti, respectively, 61.9 ng/ml and 271.1 ng/ml, when compared to Ls (>1,000,000 ng/ml), i.e., the chimera was >16,155 and >3689 more toxic than Ls.
Preliminary histological studies of treated versus control larvae showed that the midgut epithelium was completely destroyed in moribund and dead larvae by eight hours post-treatment at the LC 95 level (Fig. 2D). Most midgut cells had sloughed from the basement membrane and had lysed. Those that still had a recognizable cellular structure lacked microvilli and had one or two large vacuoles in the cytoplasm, the chacteristic cytopathology resulting from Ls Bin intoxication.
In the present study we fused the protoxins, not the activated toxins, so that the protoxin chimera contained proteolytic cleavage sites of each partner. Once activated in the midgut lumen each partner should then act independently, Cyt1Aa causing midgut microvillar membrane lesions through which BinA would enter the cytoplasm to reach its internal target site, killing the cell within 24 hr rather than the 48 hours required by the BinAB complex 5, 22, 23 . Our trypsin activation and Western blot results (Fig. 2C) indicate the two partners separated Control midgut epithelium, (i) and (ii), respectively, 100x and 400x magnification. Midgut epithelium of a treated larva (iii) and (iv), respectively 100x and 600x magnification. Note the vacuoles in cells designated by arrows in D (iv) that have sloughed from the midgut basement membrane (C, 100x; D, 600x). The central circular area in A is the food column surrounded by the peritrophic membrane. MW, protein molecular mass standards; kDa, kilodaltons. and acted independently, achieving toxicity within 24 hr for all mosquito species and strains tested (Table 1) as opposed to 48 hr with wild type BinAB. Our purpose did not include determining the type of Cyt1Aa lesion formed. However, with a diameter of about 3 nm, BinA is too large 6 to be a cation ion channel (1-2 nm) 9, 24 , and more likely forms an irregular lipid fault 25 as opposed to a larger semicircular pore. In fact, in a previous study 7 we showed that activated the BinAB complex, about 6 nm in diameter 6 , can enter Cx. quinquefasciatus midgut cells resistant to Bin in vivo through Cyt1Aa lesions without binding to microvilli.

Discussion
High toxicity to Ae. aegypti (Table 1) was unexpected because this species does not have a Bin receptor and Cyt1Aa's effect is negligible. However, we previously showed combination of Ls technical powder with purified Cyt1Aa crystals at a 10:1 ratio increased toxicity slightly to Ae. aegypti, with LC 50 and LC 95 values of 3,800 ng/ ml and 31,500 ng/ml, and synergism factors of 2.1 and 8.6, respectively 26 . Compared with these results, LC 50 and LC 95 values for the chimera (a 1:1 ratio of Cyt1Aa:BinA) increased toxicity to this species by, respectively, 55-fold and 116.2-fold. The marked differences in LC 50 and LC 95 values between these two studies cannot be compared directly due to variations in toxin constructs, but our chimera's high toxicity to four mosquito species indicates that the intracellular target for BinA is present in Ae. aegypti, and thus probably in all mosquito species. Moreover, against An. gambiae, it is the most toxic of any strain we tested (Table 1). Based on our SDS-PAGE results we estimate BinA is only about 10% (dry weight) of spore/parasporal body complex tested, indicating its activated peptide is one of the most potent mosquitocidal toxins known, if not the most toxic. Although not as toxic to Ae. aegypti as Bti 4Q5 with the wild type parasporal body (LC 50 = 3.6 ng/ml, LC 95 = 18.5 ng/ml), these results demonstrate that the Cyt1Aa-BinA chimera strain extended the target spectrum of Ls BinA (Table 1). Thus, rather than using a mixture of Bti and Ls, as is currently done is some current commercial products, the Cyt1Aa-BinA chimera combines the properties of high toxicity against a broad vector target spectrum with the known resistance management properties of Cyt1A 11 .
Aside from potential vector control applications, the Cyt1Aa-BinA chimera could prove useful for clarifying how BinA kills midgut cells causing mosquito death. The literature on these topics is full of disparate and often contradictory results. Whereas Bin's intoxication has been well described cytologically 5, 16-18 , its mode of action at the molecular level remains unknown. In many studies over the past decade it appears to be assumed that BinA and BinB cystallize separately in Ls, dissolve after ingestion in the midgut, are activated, and then associate to form an activated dimer or tetramer 20 independent dissociation or tetramer formation is lacking in any of these studies. In earlier studies 13,14 it was shown expression of the bin operon, i.e., binA and binB, yielded only a single crystal, demonstrating BinA and BinB formed a heterodimer, not separate crystals, which was confirmed recently by the the solution of Bin's crystal structure 6 . Another problem with a report that BinA and BinB prepared separately and then mixed together formed a tetramer 28 is that in a subsequent study it was shown the Ls protein complex studied 29 by the former group was a spore coat protein complex, not the Bin toxin. In other studies it has been suggested reassociation of BinA and BinB may be required for important conformational changes essential to both molecules so that activated Bin can bind receptors, interact with membrane lipids for additional structural alterations, and induce its internalization 18,20,27 . We do not question these results under the conditions tested, but our in vivo results reported here provide strong evidence that BinA once activated is highly toxic without requiring BinB for conformational changes, nor does it appear to require interactions with microvillar membrane lipids for toxicity. This suggests that BinA's hydrophobic domains may target this toxin to an intracellular organelle, such as the endoplasmic reticulum, rather than act by forming pores in the microvillar membrane.
Bacterial strains and purification of parasporal bodies. Ls 2362 was grown in MBS broth 25 , and Bti strains 4Q5, 4Q7/pWF45, 4Q7/pBU-cyt1Aa-binA, and 4Q7/p45S1 were grown in 50 ml of NBG 13, 14 appropriately supplemented with 25 μg/ml erythromycin and 3 μg/ml tetracycline, at 28 °C for 4 days by which time >95% of the cells had sporulated and lysed. Spores and crystals were collected by centrifugation at 6,500 g for 15 min, washed 2x in double-distilled (dd) H 2 O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash, and lyophilized (FreezeZone 4.5, Labconco) for storage. To isolate parasporal bodies, spore/parasporal body mixtures collected from 50 ml cultures were resuspended in 15 ml ddH 2 O and sonicated twice at 50% duty cycle for 15 s using the Ultrasonic Homogenizer 4710 (Cole-Parmer Instrument Co.). Five-milliliter samples were loaded onto a sucrose gradient cushion (30-65% w/v), which was then centrifuged at 20,000 g for 45 min at 20 °C in a Beckman L7-55 ultracentrifuge using the