Synergistic combinations of native Syrian Lysinibacillus sphaericus and Bacillus thuringiensis isolates reduce resistance to Lysinibacillus sphaericus in Culex pipiens

Culex pipiens is a major vector of West Nile, Japanese encephalitis, and Rift Valley fever viruses in the Middle East and North Africa. Effective control of this vector is threatened by resistance to Lysinibacillus sphaericus (Ls). Native bacterial isolates may offer synergistic combinations for resistance management. However, not all isolates exhibit synergy, and even when synergy occurs, it is highly dependent on the mixing ratio. These critical aspects remain unexplored for native Syrian isolates against Ls-resistant Culex. To evaluate synergism, resistance to Ls2362 was developed by selective pressure over 25 generations reaching 27.1-fold (vs. parental) and 40.7-fold (vs. susceptible sister colony) resistance ratios. No cross-resistance to Bti was observed. Native Syrian Bacillus thuringiensis (Bt) and Ls isolates were evaluated individually and in binary combinations against field, susceptible, and resistant C. pipiens larvae. Sixteen 1:1 mixtures were screened, and three promising combinations were tested at different volumetric ratios. Synergism was quantified using fractional insecticidal concentration (FIC). Among individual isolates, Bt.Mb.C.D.2 and Ls.As.I.A.15 were the most potent. Screening of 1:1 mixtures revealed strong synergism only for Bt.As.C.B.18 with Ls.As.I.A.15 (FIC=0.11), Ls.Il.I.A.7 (0.32), or Ls2362 (0.43). The 1:3 mixture of Bt.As.C.B.18 + Ls.Il.I.A.7 as the most synergistic (FIC = 0.074), restoring Ls susceptibility by 50.3-fold. Ratios with excess Bt (3:2) were consistently antagonistic. The native Syrian isolate Bt.As.C.B.18 exhibits exceptional synergizing ability; low proportions in mixtures can substantially restore susceptibility to high-level Ls resistance. Such synergistic wild-type combinations offer a potential strategy for resistance management in Culex control programmes.