A Strain of Bacillus sphaericus Causes Slower Development of Resistance in Culex quinquefasciatus

Abstract

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.

Mosquitoes transmit some of the world's most serious vector-borne diseases, such as malaria, encephalitis, filariasis, yellow fever, and dengue. Vector control is recognized as an effective tool for controlling some tropical diseases (23). Due to the increasing resistance of mosquitoes to chemical pesticides, as well as their risks to humankind and the environment, mosquito control using the entomopathogenic bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis has continuously increased in different regions of the world. Compared to B. thuringiensis subsp. israelensis, B. sphaericus offers a distinct advantage, having an increased duration of larvicidal activity against certain mosquito species, especially in organically enriched larval habitats. There is also evidence of spore recycling in dead mosquito larvae in certain environments. B. sphaericus, therefore, has been considered a promising agent for mosquito control, especially for Culex spp. in urban environments (18, 26).

For many years, it was assumed that the use of microbial larvicides based on B. sphaericus would not lead to resistance in mosquitoes. However, recent reports have shown that B. sphaericus toxins are not free from this problem (5). Under continuous selection pressure, mosquito populations develop resistance to B. sphaericus binary toxin (Bin), both in the laboratory and in the field. It has been demonstrated that Culex quinquefasciatus Say can develop from 35- to 150,000- and from 10- to 10,000-fold resistance to B. sphaericus in the laboratory (9, 15, 21) and in the field (1, 20, 25, 31; G. Sinègre, M. Babinot, J. M. Quermei, and B. Gaven, Abstr. 8th Eur. Meet. Soc. Vector Ecol., P17, 1994), respectively. Laboratory studies have shown that the resistance developed to certain strains of B. sphaericus confers more or less cross-resistance to other strains of the same species of toxin-producing organisms (16, 22, 27). Therefore, the resistance of mosquito populations to B. sphaericus Bin toxin will seriously threaten the sustainability of current mosquito control programs using this microbial insecticide.

B. sphaericus C3-41, a highly active strain isolated from a mosquito breeding site in China in 1987, has different levels of toxicity against Culex spp., Anopheles spp., and Aedes spp. (13, 32, 33). This strain belongs to the flagellum serotype H5a5b, like strains 2362 and 1593. DNA sequence analysis revealed that the sequence of Bin2 toxin from C3-41 was identical to that of Bin2 toxin from strain 2362 (30), and it has only several amino acid differences in comparison to Bin1 from strain IAB59, Bin3 from strain 2297, and Bin4 from strain LP1-G (19, 30, 29). A C3-41 liquid formulation has been used throughout the last decade for controlling mosquito larvae in more than 40 cities and towns in China (Z. Yuan, Y. Zhang, and E. Liu, unpublished data). In certain southern cities, such as Shenzhen and Dongguan, Guangdong Province, this formulation has been intensively used as the sole larvicide to treat mosquito breeding sites for more than 8 years. Under this high selection pressure, C. quinquefasciatus evolved a 22,600-fold resistance to this agent. Interestingly, after a 6-month treatment interval with a B. thuringiensis subsp. israelensis formulation in the formerly B. sphaericus-treated area, the resistant mosquito population became susceptible to B. sphaericus C3-41; The resistance ratio of the field-collected larvae dropped from 22,600- to 5.76-fold (31). Recent studies (16, 27) have shown that B. sphaericus strain IAB59, which produces Bin1 during its sporulation and belongs to serotype H6, had not only a very high toxicity to susceptible C. quinquefasciatus colonies but also a moderate toxicity to a B. sphaericus-resistant Culex colony.

Our studies were done to investigate the evolution of resistance to B. sphaericus strains C3-41, 2362, and IAB59 in field-collected populations of C. quinquefasciatus from China and Brazil under laboratory conditions. Particular attention was paid to strain IAB59 for its toxicity against B. sphaericus-resistant mosquito larvae, with the aim of investigating whether this strain could be an alternative to the already commercialized B. sphaericus strains. The stability of resistance in the selected mosquito colonies and their cross-resistance to B. sphaericus strains C3-41, 2362, and IAB59 and B. thuringiensis subsp. israelensis were also investigated. The information will be of value for understanding the inheritance of resistance and for developing approaches for resistance detection and monitoring, as well as for management strategies for resistant mosquito colonies.

MATERIALS AND METHODS

Bacterial strains and preparation.

B. sphaericus C3-41, 2362, and IAB59 and B. thuringiensis subsp. israelensis (IPS82) lyophilized powders were provided by the Unité de Bactéries Entomopathogènes, Institut Pasteur, Paris, France. The strains were grown in a 20-liter fermentor in MBS medium (11), and the final sporulated cultures were washed and lyophilized separately.

A recombinant B. thuringiensis strain expressing the binary toxin of IAB59 was constructed to test the toxicity of this Bin1 toxin. A 3.5-kb HindIII chromosomal fragment containing the bin1 operon from strain IAB59 (kindly provided by Colin Berry, Cardiff School of Biosciences, Cardiff, United Kingdom) was inserted into the Escherichia coli-B. thuringiensis shuttle vector pBU4, and the resulting recombinant plasmid was used for expression of the subcloned operon in a crystal-minus B. thuringiensis subsp. israelensis strain, 4Q2-81, following the procedures of Bourgouin et al. (3). This recombinant strain, called BinIAB59 and expressing Bin1, was grown in a 20-liter fermentor in UG liquid medium containing 25 μg of tetracycline per ml for 72 h, and the final sporulated and lysed whole cultures were lyophilized to powders.

To assure good suspensions for selection and bioassay procedures, stock suspensions (1%) of the primary powders were prepared in distilled water by vigorously shaking 0.5 g of the powder in 50 ml of water in screw-cap glass vials. Serial dilutions were prepared in distilled water, and aliquots of appropriate dilutions were used in bioassays and selection according to the related procedures. All stocks and dilutions were kept refrigerated for no more than 4 months.

Protein analysis.

Powders of all four strains, C3-41, 2362, IAB59, and BinIAB59, were used to estimate the content of protein toxin per milligram of powder. Fifty milligrams of powder was suspended in 900 μl of water and 100 μl of the protease inhibitor phenylmethylsulfonyl fluoride (10 mM). The powder suspensions were solubilized for 1 h in 50 mM NaOH (final concentration) at 30°C with agitation and then centrifuged at 8,000 × g for 5 min. The supernatants were analyzed by the Bradford protein assay (4) using bovine serum albumin as a standard and the Bio-Rad microdosage dye. Proteins in supernatants were separated and analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) (12). Approximately 5 μg of protein was loaded into each well and stained with Coomassie brilliant blue after electrophoresis.

Mosquito colonies.

The susceptible laboratory C. quinquefasciatus (SLCq) colony, which was established from a laboratory-reared colony maintained at Hubei Academy of Medical Sciences for more than 10 years, was reared without exposure to B. sphaericus and did not show any marked change in its susceptibility to B. sphaericus C3-41 powder.

Two parental C. quinquefasciatus mosquito colonies have been used for resistance selection in the laboratory. One field-collected colony (called RFCq2) was established from egg rafts collected from the mosquito breeding sites in Dongguan, China, where all mosquito breeding sites had been routinely treated with B. sphaericus C3-41 for 8 years, followed by treatment at 6-month intervals with B. thuringiensis subsp. israelensis. The resistance level of this colony against B. sphaericus was 8.8-fold in comparison with the susceptible SLCq control colony (31). Another susceptible colony of C. quinquefasciatus (called CqSF) was established from egg rafts collected from mosquito breeding sites in the Coque district of Recife, Pernambuco State, Brazil. The above-mentioned two colonies were used as parental populations in selection with B. sphaericus strains C3-41, 2362, and IAB59.

Four resistant laboratory-selected colonies also obtained for this work. The two colonies, RLCq1/C3-41 and RLCq2/IAB59, originated from a field-collected colony, RFCq2, treated with B. sphaericus C3-41 and IAB59 powders, respectively, in China. Another two colonies, CqRL1/2362 and CqRL2/IAB59, were obtained from a field-collected susceptible CqSF colony continuously exposed to B. sphaericus 2362 and IAB59 powders, respectively, in Brazil.

Larvae of all colonies were reared in enamel pans filled with dechlorinated tap water and fed with a mixture of yeast powder and wheat mill or cat chow. The pupae were removed from the pans every day and placed in screen cages for emergence. The adults were allowed to feed on 10% sucrose solution, and the females were fed with blood from mice or chickens. All larvae and adults were held at 26 to 28°C and a photoperiod of 12:12 (light-dark) h.

Selection procedure.

Two C. quinquefasciatus colonies were selected with B. sphaericus C3-41 and IAB59 in China and with strains 2362 and IAB59 in Brazil. Selection pressures were applied to third and fourth instars of every generation at the dosages providing 70 to 90% mortality within 48 h (China) and 70 to 90% cumulative preadult mortality (Brazil). The selected colonies were reared generation after generation from the larvae that survived exposure to the B. sphaericus powders. Newly estimated treatment dosages were used as selection pressure in the next generation. For each generation, about 6,000 to 21,000 third- and fourth-instar larvae were treated with C3-41, 2362, or IAB59 powder. Each treatment contained 2 liters of dechlorinated tap water with a surface area of 760 cm² and about 2,000 to 5,000 larvae. The surviving larvae were retrieved 48 h after treatment, placed in dechlorinated tap water in enamel pans, and reared to adulthood to form the next generation.

Bioassays.

Bioassays were done according to the standard method recommended by the World Health Organization (28). The bioassays were undertaken by placing groups of 20 early fourth-instar larvae in 100 ml of distilled water in 125-ml plastic cups with the desired concentration of B. sphaericus powders. At least five concentrations giving mortalities between 2 and 98% were tested, and mortality was recorded after 24 h for B. thuringiensis subsp. israelensis and 48 h for B. sphaericus. One drop of larval food was added to each cup. The tests were replicated on at least three different days. The 50% lethal concentration (LC₅₀) and LC₉₀ were determined using probit analysis (7) with a program indicating means and standard error. The LC₅₀ and LC₉₀ were expressed in milligrams per liter. Resistance ratios (RR) for each resistant colony were calculated by comparing the LC₅₀ of the resistant colony to that of the susceptible one.

Stability of resistance.

After the selections were finished, the two resistant colonies (RLCq1/C3-41 and RLCq2/IAB59) were reared without B. sphaericus in separate places. After every five generations, bioassays were done to determine the susceptibilities of the colonies to B. sphaericus C3-41 and IAB59 and to investigate the stability of resistance to the selection agents.

RESULTS

Analysis of bacterial strains.

Powders of the three natural B. sphaericus strains C3-41, 2362, and IAB59 were used for selections of resistance and for evaluating the level of cross-resistances. The LC₅₀s of these three powders against the larvae of the SLCq colony were 0.00692, 0.00513, and 0.0640 mg/liter, respectively. The recombinant strain BinIAB59 was found to express the Bin1 toxin in high concentrations as two major bands (Fig. 1, lane 3). The toxicity of crystals of Bin1 isolated from this strain was in the same range as that of crystals of Bin2 (strain 1593) when tested on susceptible larvae: 0.008 and 0.033 mg/liter at the LC₅₀ and LC₉₀, respectively (C. Nielsen-LeRoux, unpublished data).

FIG. 1.

Protein profile of alkali-solubilized whole-culture powders of B. sphaericus strains 2362 (lane 1), IAB59 (lane 2), BinIAB59 (lane 3), and C3-41 (lane 4) after separation by SDS-12% PAGE and Coomassie blue staining.

The protein concentrations of alkali-solubilized powders were determined. The protein contents relative to milligrams of powder were 26 mg/liter for strain 2362, 52 mg/liter for C3-41, 37 mg/liter for IAB59, and 72 mg/liter for the recombinant BinIAB59. There was some variation in the amount of protein (active compound) among strains, which may influence the quantity of the toxic compound ingested and possibly the selection pressure. The SDS-12% PAGE protein profiles showed the presence of the binary toxin (41.9 and 51.4 kDa) in all strains (Fig. 1). The protein profile of strain IAB59 (lane 2) exhibits two extra bands different from those of strains 2362 and C3-41: one is just below the 51-kDa marker protein (with a molecular mass of about 49 kDa), and the other is close to the 27-kDa marker protein.

Evolution of resistance to B. sphaericus in C. quinquefasciatus.

Laboratory selections of Culex colonies for resistance to B. sphaericus strains C3-41, 2362, and IAB59 were done in China and Brazil. Under a high selection pressure, both the susceptible mosquito colony (CqSF) and the low-level-resistant colony (RFCq2) decreased their susceptibilities, particularly to B. sphaericus strains C3-41and 2362 but to a much lesser extent to IAB59. After the RFCq2 larvae were successively treated with C3-41 powder, the probit lines of that colony remained near to that of the parental population up to F5 (Fig. 2A). However, a significant change in susceptibility occurred in F7, with an LC₅₀ of 6.322 mg/liter compared with an LC₅₀ of 0.0607 mg/liter for the parental colony (Table 1). The probit lines for the next generations shifted further to the right from those of the previous generations (Fig. 2A), indicating the increase of resistance. With further selection, the RR reached >16,474 and >144,000 at F13 in comparison with the RFCq2 colony and the susceptible colony (SLCq), respectively (Table 2).

FIG. 2.

Probit lines for resistances of two C. quinquefasciatus colonies (RFCq2 and CqSF) to B. sphaericus C3-41, 2362, and IAB59 lyophilized powders throughout the laboratory selection carried on with those strains.

TABLE 1.

Larvioidal activities of B. sphaerious strains (C3-41, 2362, and IAB59) and B. thuringiensis subsp. israelensis to susceptible and resistant colonies of C. quinquefasciatus

Colonya	Selected	Generation	LC50 (range) (mg/liter)
			C3-41	2362	IAB59	B. thurigiensis serovar isvaeleusis
SLCq	None		0.0069 (0.0053-0.0087)	0.0051 (0.0035-0.0072)	0.0640 (0.00511-0.0812)	0.0064 (0.0041-0.0092)
RFCq2	None	F0	0.0607 (0.0542-0.0683)	0.0598 (0.0522-0.0664)	0.0667 (0.037-0.083)	0.0055 (0.0035-0.0078)
RLCq1/C3-41	C3-41	F6	0.4401 (0.0380-0.7520)		0.660 (0.035-0.091)	0.0087 (0.0056-0.0141)
		F7	6.322 (4.961-8.762)		0.912 (0.726-1.132)	0.0074 (0.0046-0.0093)
		F13	>1,000	672.8 (428.2-1211)	0.584 (1.10-2.59)	0.0079 (0.0042-0.0092)
RLCq27IAB59	IAB59	F6	0.0420 (0.0310-0.582)		0.308 (0.182-0.434)	0.0076 (0.0043-0.0102)
		F14	3.706 (2.465-7.720)		0.334 (1.314-1.367)	0.0068 (0.0045-0,0083)
		F18	>1,000	552.7 (421.1-1021)	3.090 (3.010-3.244)	0.0084 (0.0055-0.0097)
CqSF	None	F0	0.0141 (0.0121-0.0162)	0.0372 (0.0325-0.0413)	0.0411 (0.0365-0.0446)	0.0150 (0.0132-0.0166)
CqRL1/2362	2362	F18		0.6710 (0.4621-0.8923)		0.0128 (0.093-0.01777)
		F35	1.429 (0.399-2.930)	92.45 (83.64-100.2)	0.3234 (0.2242-0.4940)	0.01192 (0.0083-0.0163)
		F46	448.0 (361.8-688.9)	>4,400	1.011 (0.8921-1.742)	0.0120 (0.0098-0.1672)
CqRL2/IAB59	IAB59	F6			0.0388 (0.0105-0.1147)
		F8			0.0721 (0.0483-0.9624)
		F12	0.8210 (0.5123-1.021)	0.1510 (0.1221-0.2012)	0.1642 (0.1326-0.2115)

^aSLCq, susceptible laboratory colony; RFCq2, field-collected low-level-resistant colony; RLCq1/C3-41 and RLCq2/IAB59, resistant laboratory colonies selected from RFCq2 with C3-41 and IAB59 in China; CqSF, field-collected susceptible colony; CqRL1/2362 and CqRL2/IAB59, resistant laboratory colonies selected from CqSF with 2362 and IAB59 in Brazil.

TABLE 2.

Levels of resistance and cross-resistance of C. quinquefasciatus colonies selected with B. sphaericus 2362, IAB59, and C3-41 to B. sphaericus and B. thuringiensis subsp. israelensis

Colony	Selection	Generationa	RR tob:
			C3-41	IAB59	BinIAB59	2362	B. thuringiensis subsp. israelensis
RLCq1/C3-41	C3-41	F13	>144,000	23.7		108,000	1.4
RLCq2/IAB59	IAB59	F18	>144,000	46.3		132,000	1.5
CqRL1/2362	2362	F46	32,000	26	>162,000	>162,000	1
CqRL2/IAB59	IAB59	F12	57.1	4.1		5.5

^aLast generation exposed to selection pressure

^bRR for each resistant colony was calculated by comparing the LC₅₀ of the resistant colony to those of the susceptible ones (SL Cq in China and CqSF in Brazil).

The selection pressure of B. sphaericus 2362 on colony CqSF was lower than that for C3-41. CqRL1/2362, the 2362-selected colony, did not show any marked change in susceptibility to 2362 up to F15. At F18, only a low degree of resistance was recorded, with an LC₅₀ of 0.6710 mg/liter for strain 2362 and an RR of 18.1 compared to the parental population (CqSF). Resistance developed slowly in the CqRL1/2362 colony throughout the first 30 generations but then rose to 137-fold at F32. At F35, a much higher level of resistance was observed, with an LC₅₀ of 93.24 mg/liter and an RR of 2,500-fold. The resistance level at F38 further increased, and this change can be clearly seen from the shift of the probit line further to the right (Fig 2B). With continued selection, a very high level of resistance was obtained at F46, with an RR of >162,000 (Table 2).

In the selection of resistance to IAB59, after selection for five or six generations, larvae of the RFCq2 (from China) and CqSF (from Brazil) colonies were less susceptible to IAB59 than the parental colonies, but their probit lines shifted only slightly to the right (Fig. 2). Then the two selected colonies developed low-level resistance to this agent. After 18 and 12 generations of selection in China and Brazil, respectively, the selected colonies lost their susceptibilities to IAB59 powder (Fig. 2 and Table 1), the selected RFCq2/IABb59 colony developed low-level resistance reaching an RR of 46.3 at the last generation (F18) exposed, and the selected CqRL2/IAB59 colony attained an RR of 4.1 at F12 (Table 2).

Cross-resistance to B. sphaericus and B. thuringiensis subsp. israelensis.

The susceptibility of the resistant RLCq1/C3-41 colony to strain IAB59 and resistant RLCq2/IAB59 to strain C3-41 were evaluated at each generation. Under selection by C3-41, the selected colonies developed higher resistance to C3-41 more quickly but only developed lower resistance, and more slowly, to IAB59. Although a very high resistance to C3-41 has been detected in RLCq1/C3-41 (F13), it is remarkable that this colony showed only a low-level cross-resistance to IAB59 (RR = 23.7) (Fig. 3; Table 2). The same response was observed in the resistant CqRL1/2362 colony, which had high-level resistance to strain 2362 (RR > 162,000) and low-level cross-resistance to strain IAB59 (RR = 26) (Table 2).

FIG. 3.

Evolution of resistance and cross-resistance of Bacillus sphaericus-selected C. quinquefasciatus colonies to B. sphaericus strains C3-41 and IAB59.

In contrast, under selection by IAB59, the SLCq colony developed a more slowly evolving and lower resistance to IAB59 and a very high level of cross-resistance to C3-41 (Fig. 3). In the last generation (F18), the RLCq2/IAB59 colony had a cross-resistance with an RR of >144,000-fold to C3-41, even though only a 46.3-fold resistance could be found towards IAB59 (Table 2). On the other hand, both the RLCq1/C3-41 and RLCq2/IAB59 colonies developed high levels of cross-resistance to strain 2362 (RR = 108,000 and 132,000, respectively). Likewise, a high level of cross-resistance was noted to strain C3-41 (RR = 32,000) and to strain BinIAB59 (RR > 162,000) in the CqRL1/2362 colony (Table 2).

The susceptibilities to B. thuringiensis subsp. israelensis of the two parental colonies (RFCq2 and CqSF) and the one susceptible colony (SLCq) were also studied during the B. sphaericus treatment period. B. thuringiensis subsp. israelensis had similar LC₅₀s for the three Culex colonies (Table 1). The susceptibilities of the B. sphaericus-resistant colonies to B. thuringiensis subsp. israelensis were measured as well, and they were similar to those of the susceptible colonies. Thus, no cross-resistance was detected (Tables 1 and 2).

Resistance stability.

The susceptibilities of the resistant RLCq1/C3-41 and RLCq2/IAB59 colonies, which were submitted to selection for 13 and 18 generations, respectively, were evaluated at different intervals after the selection pressure was interrupted. The resistances of RLCq1/C3-41 and RLCq2/IAB59 to strain C3-41 were stable throughout 17 generations in the absence of selection pressure. At the end of that time, the RR values were still very high: 123,410 and 112,717, respectively.

The IAB59 powder had LC₅₀s of 1.584 and 3.090 mg/liter for the RLCq1/C3-41 and RLCq2/IAB59 colonies, corresponding to 23.7- and 46.3-fold increases in resistance levels. Little or no decrease in the LC₅₀ of IAB59 powder for RLCq1/C3-41 larvae was observed, ranging from 1.326 to 2.254 mg/liter throughout the period in the absence of any pressure. However in the RLCq2/IAB59 colony, a slow increase in susceptibility to IAB59 was observed. The last assessment showed that the LC₅₀s of IAB59 for the RLCq2/IAB59 larvae at F35 decreased to 0.941 mg/liter, corresponding to 14.1-fold resistance after selection pressure was interrupted.

DISCUSSION

These studies have confirmed that selection of resistance in two distinct C. quinquefasciatus populations to commercial B. sphaericus strains 2362 and C3-41 is possible under laboratory conditions. However, B. sphaericus strain IAB59 appeared to induce a different evolution of resistance, causing much more slowly evolving and lower resistances in both the field-collected susceptible colony and the low-level-resistant colony after approximately the same number of generations were subjected to selection.

Data clearly showed that the resistances of strains C3-41 and 2362 in the two colonies progressed at different rates; the resistance in the RFCq2 colony to strain C3-41 progressed faster than that of the CqSF colony to strain 2362. The speed of resistance evolution, as also shown previously from laboratory studies (21, 27), may depend on both selection pressure and the genetic pool of the population. In the selection of RFCq2 with C3-41 in China, the selection pressure imposed caused 70 to 90% larval mortality in 48 h, while the selection pressure applied to CqSF with 2362 in Brazil induced 70 to 90% accumulated preadult mortality. It is evident that a higher selection pressure was imposed on RFCq2 in China than on CqSF in Brazil. The different responses of mosquitoes to selection agents suggest that the higher the selection pressure is, the more quickly C. quinquefasciatus larvae will develop resistance. In addition, CqSF is a field-collected susceptible colony while RFCq2 is a low-level-resistant colony. Previous exposure to strain C3-41 in the field possibly increased the frequency of the resistant alleles, favoring resistance to strain C3-41 in the laboratory. Information about exposure history is rarely reported in selection studies, but it may be crucial for interpreting the results of selection for resistance.

The field-collected susceptible and low-level-resistant mosquito colonies developed high levels of resistance to B. sphaericus C3-41 and 2362, strains that both carry the Bin2 toxin (2). On the other hand, these colonies expressed only low-level resistance when they were subjected to B. sphaericus IAB59. The contrasting responses in the evolution of resistance in Culex mosquitoes to various B. sphaericus strains suggests that these differences may be related to toxin characteristics of the strains. Based on our results, it is evident that the C3-41- and 2362-selected C. quinquefasciatus RLCq1/C3-41 and CqRF1/2362 colonies that had very high levels of resistance or cross-resistance to strains 2362 and C3-41 and to Bin1 from IAB59 exhibited only low-level cross-resistance to strain IAB59. Additionally, the IAB59-selected colonies evolved only low-level resistance to IAB59 and high-level cross-resistance to B. sphaericus C3-41 and 2362 and to the Bin1 of IAB59. This means that strain IAB59 may produce a new active factor besides the binary toxin, and its mode of action is probably different from that of binary toxin. This is in agreement with the fact that the Bin1 toxin from IAB59 had activity only against susceptible larvae but not against those resistant to strains C3-41 and 2362 (Bin2). Protein analysis demonstrated that B. sphaericus strains C3-41 and 2362 produced the binary toxin and that strain IAB59 produces, in addition to the binary toxin, another major protein with a molecular mass of about 49 kDa. It is supposed that the existence of this new active factor (or a combination of factors) probably explains the low-level cross-resistance to the wild-type strain in the binary toxin-selected mosquitoes RLCq1/C3-41 and CqRF1/2362 and the high-level cross-resistance in the IAB59-selected mosquitoes RLCq2/IAB59 and CqRL2/IAB59 to the Bin2 toxins of 2362 and C3-41. Furthermore, the much lower resistance and its slower evolution may be explained by the combined actions of different active compounds. The possibility that the additional toxic factor of strain IAB59 is the same as one suspected in B. sphaericus strain LP1-G is under study. The response of mosquitoes to laboratory selection provides a mechanism to study resistance development. However, many factors may influence the development of resistance in the field. Immigration seems particularly important, since susceptible individuals can move from untreated areas to the treated one. In addition, under laboratory conditions, selection pressure is stronger, while in the field a certain number of breeding sites do not always receive treatment, since complete coverage and application of high doses to all breeding sites is difficult in practice. Therefore, the magnitude and rate of resistance development generally are considered to be lower in the field than those obtained in the laboratory. For example, after a 32-month exposure to B. sphaericus 2362 in the laboratory, the CqRL1/2362 colony developed a >100,000-fold resistance, while field-collected larvae from a population exposed to a 26-month trial using strain 2362 were found to be only 10-fold less susceptible than populations from untreated areas (25). Another important factor leading to the slow process of stable field resistance is the fact that resistance in the field seems to decline quickly when treatments are suspended (24). However, other reports of field applications indicate that resistance can appear both more quickly and to a higher level (20; G. Sinègre, M. Babinot, J. M. Quermei, and B. Gaven, Abstr. 8th Eur. Meet. Soc. Vector Ecol., P17, 1994). These variations may be explained by different factors, such as selection pressure, genetic background, and migration. Additionally, a recent report indicates even more complex situations (6). Meanwhile, the knowledge of the evolution of B. sphaericus resistance in C. quinquefasciatus has provided a useful basis for developing solutions to prevent or delay the appearance of resistance in mosquitoes. It may also improve the efficacy of control strategies using B. sphaericus in integrated mosquito control programs.

Our results confirm the previously recorded data showing that Culex populations resistant to B. sphaericus are susceptible to B. thuringiensis subsp. israelensis (17, 25, 31). B. sphaericus binary toxin binds to a single specific class of receptors on the surfaces of midgut brush border membranes, and no B. thuringiensis subsp. israelensis toxin shares this binding site. Nielsen-LeRoux and Charles (14) and Georghiou et al. (10) showed that the natural variation in the susceptibility of C. quinquefasciatus to B. sphaericus was much higher than the variation in susceptibility to B. thuringiensis subsp. israelensis. Thus, selection of resistance to B. sphaericus is higher than that for B. thuringiensis subsp. israelensis. Additionally, selection of resistance to B. thuringiensis subsp. israelensis seems to be difficult due to its multitoxin complex (8). Therefore, B. thuringiensis subsp. israelensis can be considered a good candidate for use in rotation with B. sphaericus against C. quinquefasciatus, because it reduces selection pressure from B. sphaericus (31).

Whether strain IAB59 will be a good alternative in the management of resistance to B. sphaericus will depend on both its potency and its field performance compared to the commercialized strains (2362, 1593, and C3-41). Additional studies are needed with respect to these factors.