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Efficacy of Lysinibacillus sphaericus against mixed-cultures of field-collected and laboratory larvae of Aedes aegypti and Culex quinquefasciatus

Published online by Cambridge University Press:  22 May 2018

J.C. Santana-Martinez ,
J.J. Silva  and
J. Dussan
J.C. Santana-Martinez
Affiliation:
Departamento de Ciencias Biológicas, Centro de Investigaciones Microbiológicas (CIMIC), Universidad de los Andes, Bogotá, Colombia
J.J. Silva
Affiliation:
Departamento de Ciencias Biológicas, Centro de Investigaciones Microbiológicas (CIMIC), Universidad de los Andes, Bogotá, Colombia Department of Entomology, University of Cornell, Ithaca, New York, USA
J. Dussan*
Affiliation:
Departamento de Ciencias Biológicas, Centro de Investigaciones Microbiológicas (CIMIC), Universidad de los Andes, Bogotá, Colombia
*
*Author for correspondence Phone: +57-1-3394949 (ext. 3644) E-mail: jdussan@uniandes.edu.co
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Abstract

Lysinibacillus sphaericus (Bacillales: Planococcaceae) is a spore-forming bacillus used for the biological control of mosquitoes (Diptera: Culicidae) due to its larvicidal activity determined by various toxins and S-layer protein produced either during sporulation or by the vegetative cell. Aedes aegypti and Culex quinquefasciatus are the vectors of arboviruses that cause tropical diseases representing a current public health problem. Both species may coexist in the same larval development sites and are susceptible to the larvicidal activity of L. sphaericus. In this study, we compared the larvicidal effects of L. sphaericus 2362 (WHO Reference strain) and native strains III(3)7 and OT4b.25 against Cx. quinquefasciatus and Ae. aegypti in single-species and mixed-culture bioassays. Findings showed that L. sphaericus spores, vegetative cells and a combination thereof possessed high larvicidal activity against Cx. quinquefasciatus larvae, whereas only the formulation of L. sphaericus vegetative cells was effective against Ae. aegypti larvae. Similar results were obtained for field-collected larvae. We propose that a formulation of vegetative cells of L. sphaericus 2362 or III(3)7 could be a good alternative to chemical insecticides for the in situ control of mixed populations of Ae. aegypti and Cx. quinquefasciatus.

Keywords

Biological controlfield-collected strainvegetative cellstemephos-resistant
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 109 , Issue 1 , February 2019 , pp. 111 - 118
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Copyright
Copyright © Cambridge University Press 2018 

Introduction

Biological insecticides have proven to be effective for the control of mosquito populations since they can be easily recycled, and unlike artificial or chemical insecticides, are less harmful to the environment and to human health (Ali et al., Reference Ali, Ravikumar and Beula2013). More specifically, biological control is an efficient solution to insect pests given these benefits, and in addition, it uses natural pest antagonists or predators to regulate other populations (Van Driesche et al., Reference Van Driesche, Hoddle and Center2009).

Lysinibacillus sphaericus Ahmed (Bacillales: Planococcaceae) is a spore-forming bacterium with entomopathogenic activity. During the final stages of sporulation, L. sphaericus produces a binary toxin (BinAB), which is toxic against Culex spp. and Anopheles spp. (Davidson, Reference Davidson1988; Baumann et al., Reference Baumann, Clark, Baumann and Broadwell1991). After ingestion of BinAB, alkaline pH in the larvae activate the mechanism of each polypeptide: BinB binds to an α-glucosidase receptor identified as Cpm1 in epithelial midgut cells, allowing the entrance of BinA and causing cellular lysis (Davidson, Reference Davidson1988, Reference Davidson1989; Oei et al., Reference Oei, Hindley and Berry1992; Charles et al., Reference Charles, Nielson-LeRoux and Delécluse1996; Silva-Filha et al., Reference Silva-Filha, Nielsen-LeRoux and Charles1999; Darboux et al., Reference Darboux, Nielsen-LeRoux, Charles and Pauron2001).

Additionally, a proteinaceous structure found on the surface of several archaea and bacteria identified as the S-layer is expressed in L. sphaericus vegetative cells (Peña et al., Reference Peña, Miranda-Rios, de la Riva, Pardo-López, Soberón and Bravo2006). This structure contributes to larvicidal activity against Culex (Linnaeus) quinquefasciatus Say (Diptera: Culicidae) (Lozano et al., Reference Lozano, Ayala and Dussán2011) and have a synergistic effect with the spore (Lozano & Dussán, Reference Lozano and Dussán2017). Furthermore, L. sphaericus toxic strains express three mosquitocidal toxins in vegetative cells: Mtx1, Mtx2, and Mtx 3 (Thanabalu et al., Reference Thanabalu, Hindley, Jackson-Yap and Berry1991; Liu et al., Reference Liu, Porter, Wee and Thanabalu1996; Thanabalu & Porter, Reference Thanabalu and Porter1996). All three exhibit larvicidal activity but are sensitive to proteases from the sporulation phase (Thanabalu & Porter, Reference Thanabalu and Porter1995) and are not used in commercial products against vectors. Mtx1 is a 100 kDa protein that is very similar to ADPribosylation-type toxins, whereas Mtx2 and Mtx3 contain a domain characteristic of pore form toxins (Thanabalu et al., Reference Thanabalu, Hindley, Jackson-Yap and Berry1991, Reference Thanabalu, Berry and Hindley1993; Liu et al., Reference Liu, Porter, Wee and Thanabalu1996; Thanabalu & Porter, Reference Thanabalu and Porter1996; Marchler-Bauer et al., Reference Marchler-Bauer, Lu, Anderson, Chitsaz, Derbyshire, De Weese-Scott, Fong, Geer, Geer, Gonzales, Gwadz, Hurwitz, Jackson, Ke, Lanczycki, Lu, Marchler, Mullokandov, Omelchenko, Robertson, Song, Thanki, Yamashita, Zhang, Zhang, Zheng and Bryant2011).

Aedes (Stegomyia) aegypti Linnaeus (Diptera: Culicidae) and Cx. quinquefasciatus are the vectors of arboviruses that cause tropical diseases representing a current public health problem. Among these diseases, Ae. aegypti transmits dengue, Chikungunya, Zika, and yellow fever. Dengue is the most prevalent disease with approximately 390 million infections per year (Bhatt et al., Reference Bhatt, Gething, Brady, Messina, Farlow, Moyes, Drake, Brownstein, Hoen, Sankoh, Myers, George, Jaenisch, Wint, Simmons, Scott, Farrar and Hay2013). Chikungunya virus has caused over 2.5 million infections in the last decade and has more recently been spreading in the Americas (Staples & Fischer, Reference Staples and Fischer2014) and emerging in Europe (Schaffner et al., Reference Schaffner, Medlock and Bortel2013). Yellow fever has caused approximately 200,000 severe cases per year in Africa (Garske et al., Reference Garske, Van Kerkhove, Yactayo, Ronveaux, Lewis, Staples, Perea and Ferguson2014) and Zika virus approximately 4 million infections in the Americas (Boeuf et al., Reference Boeuf, Drummer, Richards, Scoullar and Beeson2016).

Cx. quinquefasciatus can among other virus transmit West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), and St. Louis Encephalitis Virus (SLEV), pathogenic protozoa and nematode (Bhattacharya & Basu, Reference Bhattacharya and Basu2016). WNV is geographically widespread in Africa, Western Asia, the Middle East and recently, East Europe and North America (Petersen & Roehrig, Reference Petersen and Roehrig2001), causing continuously outbreaks during the last decades (Chancey et al., Reference Chancey, Grinev, Volkova and Rios2015). JEV is the main cause of viral encephalitis in South-East Asia and the Western Pacific, with an estimated 68,000 clinical cases annually (Campbell et al., Reference Diaz, Quaglia, Konigheim, Boris, Aguilar, Komar and Contigiani2011). SLEV is a reemerging arbovirus in the southern cone of South America (Rocco et al., Reference Rocco, Santos, Bisordi, Petrella, Pereira, Souza, Marti, Barbosa, Cerroni, Katz and Suzuki2005; Diaz et al., Reference Diaz, Quaglia, Konigheim, Boris, Aguilar, Komar and Contigiani2016), and a serious public health threat in North America (Reisen, Reference Reisen2003). Another concern is that these diseases are common in the tropics where the climate conditions allow the development of vectors (Rueda et al., Reference Rueda, Patel, Axtell and Stinner1990) and where the predominant expansion of cities and towns surrounding bodies of water leads to an increase in incidence (Ali et al., Reference Ali, Ravikumar and Beula2013). As the mosquitoes seek stagnant water in which they can lay eggs, the chances that they find artificial water containers are high due to increasing human settlement in or near mosquito habitats (Rozendaal, Reference Rozendaal1997). Therefore, control efforts of Ae. aegypti and Cx. quinquefasciatus populations have been conducted to reduce the risk of diseases transmission. These control programs, are mostly based on the application of chemical insecticides such as DDT, malation and temephos, and the overuse of these compounds has led to increased resistance in Ae. aegypti (Lima et al., Reference Lima, Da-Cunha, Júnior, Galardo, Soares, Braga, Pimentel and Valle2003; Bisset et al., Reference Bisset, Magdalena, Fernandez and Perez2004; Fonseca-González, Reference Fonseca-González, Quiñones, Lenhart and Brogdon2011). Ae. Aegypti and Cx. quinquefasciatus tend to colonize urban and suburban areas in the tropics and can be found in the same larval development sites (Rios et al., Reference Rios, Machado-Allison, Rabinovich and Rodriguez1978; Obándo et al., Reference Obándo, Gamboa, Perafán, Suárez and Lerma2007; Burke et al., Reference Burke, Barrera, Lewis, Kluchinsky and Claborn2010; Leisnham et al., Reference Leisnham, LaDeau and Juliano2014), despite the differences in ecological requirements for each species. Cx. quinquefasciatus has a predilection for polluted waters rich in organic matter, whereas Ae. aegypti prefers less polluted waters (Rozendaal, Reference Rozendaal1997) with few exceptions (Barrera et al., Reference Barrera, Amador, Diaz, Smith, Muñoz-Jordan and Rosario2008; Burke et al., Reference Burke, Barrera, Lewis, Kluchinsky and Claborn2010).

Given that Ae. aegypti and Cx. quinquefasciatus are two species of epidemiological importance sharing larval development sites in the tropics, the objective was to assess the larvicidal activity of L. sphaericus spores, vegetative cells and a combination thereof against mixed-cultures of field-collected, temephos-resistant Ae. aegypti and field-collected Cx. quinquefasciatus. This is in order to evaluate a new alternative to chemical insecticides commonly used.

Materials and methods

Bacterial and mosquito culture conditions

Three L. sphaericus strains previously reported as highly toxigenic against laboratory Cx. quinquefasciatus (Lozano & Dussán, Reference Lozano and Dussán2013) were evaluated in the study. L. sphaericus III(3)7 originally isolated from soil samples in an oak forest near Bogotá D.C., Colombia and L. sphaericus OT4b.25 originally isolated from coleopteran larvae in an oak forest near Bogota D.C., Colombia; both belonging to the CIMIC culture collection; and the WHO reference strain L. sphaericus 2362, originally isolated from adult Simulium damnosum (Diptera: Simuliidae) samples in Nigeria and obtained from the Pasteur Institute (Charles et al., Reference Charles, Nielson-LeRoux and Delécluse1996).

Following the protocol of Lozano & Dussán (Reference Lozano and Dussán2013), the three L. sphaericus strains: 2362, OT4b.25 and III(3)7 were subjected to a synchronization procedure. An initial inoculum of each strain was cultivated in a liquid culture of Nutrient Broth (Oxoid) for 16 h. This was followed by five cycles of cultivation in acetate broth (composed of sodium acetate 5.00 g l−1, yeast extract 3 g l−1, MgCl2 1 × 10−3 M, CaCl2 7 × 10−4 M and MnCl2 5 × 10−5 M), with incubation at 30 °C and subjection to thermal shock at 90 °C for 20 min until 90% of cells have sporulated. Laboratory strains of Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña were obtained from the Entomology Laboratory at National Institute of Health of Colombia (INS). Ae. aegypti Rockefeller was first collected by the CDC of San Juan, Puerto Rico and Cx. quinquefasciatus was collected by the INS from Muña wetlands, Cundinamarca, Colombia. In order to assess the efficiency of L. sphaericus against field-collected larvae, Ae. aegypti and Cx. quinquefasciatus larvae were collected at La Mesa, Cundinamarca, Colombia (4°38′02.9″ N and 72°27′43.42″ W) and Cordoba wetlands in Bogotá, Colombia (4°42′10.1″ N and 74°04′07.2″ W), respectively. The field-collected population of Ae. aegypti was previously reported resistant to the commonly used larvicide temephos (Santacoloma et al., Reference Santacoloma, Chaves and Brochero2012).

Field-collected larvae were identified based on the identification key compiled by Gualdron (Reference Gualdron2007). Laboratory- and field-collected larvae were maintained at 30 °C with 70% of relative humidity and a 12-h light/dark photoperiod.

Dose-dependent bioassays

Preliminary bioassays of testing vegetative cells against Ae. aegypti showed larvae mortality (data not shown). Therefore, we determined the dose-dependent response of Ae. aegypti Rockefeller to bacterial vegetative cells (colony-forming units (CFU) per ml) and estimated the LC50. Four concentrations of 2362 (2.10 × 104 CFU ml−1, 2.10 × 105 CFU ml−1, 2.10 × 106 CFU ml−1, and 2.10 × 107 CFU ml−1) and III(3)7 (7.29 × 104 CFU ml−1, 7.29 × 105 CFU ml−1, 7.29 × 106 CFU ml−1, and 7.29 × 107 CFU ml−1) vegetative cells were tested and mortality at 48 h was recorded.

Larvicidal bioassays

The larvicidal activity of the L. sphaericus strains was assayed against early fourth instar larvae of Cx. quinquefasciatus and Ae. aegypti. In order to determine the toxicity of L. sphaericus vegetative cells, cells from synchronized cultures were grown in Nutrient Agar at 30 °C and after 12 h, the cells were resuspended in 1 ml of sterile distilled water obtaining the inocula for the subsequent bioassays. To determine the toxicity of sporulated cultures, the inocula were prepared by separating 1 ml aliquots of the synchronized strains grown in acetate broth.

Single-species bioassays were performed by adding an inoculum of vegetative cells or sporulated cultures to 99 ml of chlorine-free tap water with 10 larvae of Ae. aegypti or Cx. quinquefasciatus, to have a final exposure of 107 CFU ml−1. Mixed-cultures bioassays consisting of a mixture of larvae from each species were performed by adding an inoculum of vegetative cells, sporulated cultures or the mixture of both to 99 ml of chlorine-free tap water with 20 larvae of Ae. aegypti and 20 larvae of Cx. quinquefasciatus, to have a final exposure of 107 CFU ml−1. The bioassays were incubated at 30 °C for 48 h after inoculation and larval mortality was recorded at 24 and 48 h. Negative controls consisted of 100 ml chlorine-free tap water with larvae without bacteria. Each treatment was replicated three times and the procedure was repeated twice for both single-species and mixed-cultures bioassays.

Statistical analyses

The R v3.1.1 software was used for statistical analyses (R Core Team, 2012). Shapiro–Wilcoxon test (Korkmaz et al., Reference Korkmaz, Goksuluk and Zararsiz2014) was used to test the data for normality. Single-species bioassays and mixed-cultures bioassays results were analyzed by analysis of variance (ANOVA) followed by a Tukey–Kramer test to separate averages among the different strains evaluated and control with no bacteria. If the data were not normally distributed, a Kruskal–Wallis test, followed by a Mann–Whitney U test to establish significant differences between different strains and control with no bacteria were used. The Toxicity Relationship Analysis Program (TRAP version 1.30a) was used to determine the LC50 of bacterial concentration against Ae. aegypti by probit analysis (US EPA, 2015).

Results

Ae. aegypti Rockefeller dose-dependent bioassays

Results shown on table 1 suggest that III(3)7 and 2362 strains have similar dose effect on Ae. aegypti 4th instar larvae. Subsequent bioassays were therefore performed with doses of 107CFU ml−1 as described in material and methods.

Table 1. Larvicidal activity of L. sphaericus highly toxigenic strains against Ae. aegypti fourth larvae.

Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña single-species bioassays

Single-species bioassays indicated that there was no difference in the percentage mortality of Ae. aegypti Rockefeller larvae exposed to spores of L. sphaericus III(3)7, OT4b.25, and 2362 and the control with no bacteria (fig. 1; Kruskal–Wallis : H = 0.2188, P = 0.9745). On the other hand, the Cx. quinquefasciatus Muña larvae exposed to spores, showed high mortality with all the bacterial strains when compared with the control with no bacteria (fig. 1; Kruskal–Wallis: H = 15.22, P = 0.0016).

Fig. 1. Mortality of Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña fourth-instar larvae in presence of L. sphaericus spores (107 CFU ml−1) in single-species bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Regarding vegetative cells after 48 h of exposure, mortality of Ae. aegypti Rockefeller larvae showed significant differences between treatments and control with no bacteria (Kruskal–Wallis: H = 15.25 P = 0.0016, fig. 2). Likewise, Cx. quinquefasciatus Muña larvae exposed to vegetative cells after 48 h showed significant differences between treatments and control with no bacteria (Kruskal–Wallis: H = 16.15, P = 0.0010, fig. 2).

Fig. 2. Mortality of Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña fourth-instar larvae in presence of L. sphaericus vegetative cells (107 CFU ml−1) in single-species bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Field-collected larvae of Ae. aegypti and Cx. quinquefasciatus mixed-culture bioassays

Larvae exposed to L. sphaericus spores for 48 h showed significant differences in mortality of Ae. aegypti field-collected larvae between treatments and control with no bacteria (ANOVA: F = 9.611, P = 0.0050; fig. 3a) and Cx. quinquefasciatus field-collected larvae between treatments and control with no bacteria (Kruskal–Wallis: F = 462.2 P < 0.0001; fig. 3). Similar results were obtained for Ae. aegypti Rockefeller (ANOVA: F = 29.79, P < 0.0001; fig. 3) and Cx. quinquefasciatus Muña (Kruskal–Wallis: H = 8.314, P = 0.0399; fig. 3), indicating that spores have similar larvicidal activity against field-collected and laboratory-reared larvae.

Fig. 3. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus spores (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Likewise, larvae exposed to L. sphaericus vegetative cells for 48 h showed significant differences in mortality of Ae. aegypti field-collected larvae between treatments and control with no bacteria (ANOVA: F = 59.67, P < 0.0001; fig. 4) and Cx. quinquefasciatus field-collected larvae between treatments and control with no bacteria (Kruskal–Wallis: H = 9.358 P = 0.0248; fig. 4); and mortality of Ae. aegypti Rockefeller larvae between treatments and control with no bacteria (ANOVA: F = 35.93, P < 0.0001; fig. 4) and Cx. quinquefasciatus Muña larvae between treatments and control with no bacteria (Kruskal–Wallis: H = 8.521, P = 0.0228; fig. 4). In this context, vegetative cells have similar larvicidal activity against field-collected and laboratory-reared larvae, but in contrast to the spores, L. sphaericus vegetative cells showed high mortality against Ae. aegypti.

Fig. 4. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus vegetative cells (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Bioassays of larvae exposed to vegetative cells and spores also showed significant differences in mortality of Ae. aegypti field-collected larvae between treatments and control with no bacteria (ANOVA: F = 17.17, P = 0.0007; fig. 5) and Cx. quinquefasciatus field-collected larvae between treatments and control with no bacteria (ANOVA: F = 484, P < 0.0001; fig. 5); and mortality of Ae. aegypti Rockefeller larvae between treatments and control with no bacteria (ANOVA: F = 4.187, P = 0.0468; fig. 5) and Cx. quinquefasciatus Muña larvae between treatments and control with no bacteria (Kruskal–Wallis: H = 8.895, P = 0.0307; fig. 5). As for the exposure of the larvae solely to spores, both toxicity of sporulated cultures and vegetative cells did not show high mortality against Ae. aegypti.

Fig. 5. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus spores and vegetative cells (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

All treatments showed that laboratory reared and field-collected populations of Cx. quinquefasciatus were highly sensitive to all three formulations of L. sphaericus, in contrast to Ae. aegypti populations, which were only highly sensitive to vegetative cells. Furthermore, mixed-cultures with no bacteria showed a mortality rate of 18.06 ± 4.89% for Cx. quinquefasciatus, which was interestingly higher than the mortality rate of Ae. aegypti (3.89 ± 4.39%).

Discussion

This study showed that L. sphaericus spores, vegetative cells and the combination of both exerted high larvicidal activity against Cx. quinquefasciatus larvae under coexisting and non-coexisting conditions (figs 1–5). These findings are consistent with previous studies showing that L. sphaericus III(3)7, 2362 and OT4b.25 are highly toxigenic against Cx. quinquefasciatus (Lozano & Dussán, Reference Lozano and Dussán2013). Therefore, we conclude that sensitivity of Cx. quinquefasciatus was not affected by the presence of Ae. aegypti in any of the three formulations.

With respect to the formulations of L. sphaericus against Ae. aegypti, only vegetative cells were toxigenic against both coexisting and non-coexisting Ae. aegypti larvae (figs 2 and 4). These results correspond with previous studies (Nielsen-Leroux & Charles, Reference Nielsen-Leroux and Charles1992; Lekakarn et al., Reference Lekakarn, Promdonkoy and Boonserm2015), which reported that binary toxin present in the spores of L. sphaericus has no toxic effect against Ae. aegypti. The Aam1 midgut receptor present in Ae. aegypti has been identified as homologous to the Cx. quinquefasciatus α-glucosidase midgut receptor, but the former is found at a very low concentration. Furthermore, the binding capacity of any such homolog of the toxin BinA and BinB is very low (Nielsen-Leroux & Charles, Reference Nielsen-Leroux and Charles1992; Lekakarn et al., Reference Lekakarn, Promdonkoy and Boonserm2015). However, we found a statistically significant low mortality rate in the mixed-culture bioassay treated with spores (fig. 3), presumably explained by L. sphaericus spore germination in mosquito larval cadavers. Correa & Yousten (Reference Correa and Yousten1995) showed that naturally occurring mosquito toxic strains of L. sphaericus are able to recycle in mosquito larval cadavers. Therefore, we suggest that spores might germinate inside the dead Cx. quinquefasciatus larvae, which promotes direct contact of either S-layer or Mtx toxins from the vegetative cell stage with Ae. Aegypti and thus, causing mortality.

As expected, all Cx. quinquefasciatus treatments presented high mortality to L. sphaericus vegetative cells due to the capacity to produce vegetative mosquitocidal toxins (Mtx) and S-layer (Thanabalu et al., Reference Thanabalu, Berry and Hindley1993; Liu et al., Reference Liu, Porter, Wee and Thanabalu1996; Thanabalu & Porter, Reference Thanabalu and Porter1996; Promdonkoy et al., Reference Promdonkoy, Promdonkoy, Tanapongpipat, Luxananil, Chewawiwat, Audtho and Panyim2004; Lozano et al., Reference Lozano, Ayala and Dussán2011). In the case of Ae. aegpyti, this finding is interesting because this mosquito is not typically reported as a biocontrol target for L. sphaericus by the binary toxin. Instead, Ae. aegpyti mortality could be explained by the presence of the other toxins previously mentioned only expressed at vegetative cell stage that presumably exhibit high synergistic activity when co-expressed (Rungrod et al., Reference Rungrod, Tjahaja, Soonsanga, Audtho and Promdonkoy2009).

Berry (Reference Berry2012) suggested that preparations with spores and a mixture of vegetative mosquitocidal toxins would act synergistically, enhancing the effectiveness of L. sphaericus. Our study showed that contrary to expectations, there was no synergistic effect in the larvicidal effect of L. sphaericus using the two stages of the bacteria. Presumably, proteases produced during the sporulation phase of the bacteria could be degrading the Mtx1, Mtx2, and Mtx3 toxins present in vegetative cells (Thanabalu & Porter, Reference Thanabalu and Porter1995), hence reducing the effectiveness of the formulation. Further investigations are needed to elucidate synergism between BinAB and vegetative mosquitocidal toxins. For this purpose, protease-negative L. sphaericus strains expressing Mtx might be used.

Mixed-cultures with no bacteria showed a higher mortality of Cx. quinquefasciatus, contrary to Ae. aegypti mortality. Santana-Martínez et al. (Reference Santana-Martínez, Molina and Dussán2017), showed that Ae. aegypti is a superior resource competitor and appears to be capable of competitively affecting Cx. quinquefasciatus under larval coexistence. In this sense, we suggest that a synergistic effect between the larvicide and natural dynamics of populations to control might occurs. However, we conclude that L. sphaericus has a biological control potential as a formula intended for mixed populations, due to L. sphaericus vegetative cells are highly toxigenic against both Ae. aegypti and Cx. quinquefasciatus individuals whether they are coexisting or not (figs 2 and 4).

This study shows that L. sphaericus 2362, III(3)7 and OT4b.25 are good candidates to control Ae. aegpyti and Cx. quinquefasciatus coexisting populations in vitro and, ultimately, in situ while they are vegetative cells but not as spores. The explanation for this phenomenon may be that L. sphaericus cells produce several mosquitocidal toxins, other than a binary toxin, that when co-expressed may increase its toxicity. Since Ae. aegypti is poorly sensitive to the binary toxin and several studies found the resistance of Cx. quinquefasciatus against L. sphaericus binary toxin (Nielsen-Leroux et al., Reference Nielsen-Leroux, Charles, Thiery and Georghiou1995; Chalegre et al., Reference Chalegre, Romão, Tavares, Santos, Ferreira, Oliveira, de-Melo-Neto and Silva-Filha2012; Guo et al., Reference Guo, Cai, Yan, Hu, Zheng and Yuan2013), we recommend further studies on vegetative cells toxins to elucidate mechanisms of action and effectiveness surrounding synergism. Given the low LC50 values of L. sphaericus 2362 and III(3)7, these strains could be a suitable alternative to control Ae. aegypti and Cx. quinquefasciatus mixed populations and deal with insecticide resistance, through a formulation of vegetative cells.

Acknowledgements

The authors are grateful to the Faculty of Sciences at Universidad de Los Andes in Colombia for funding this research. Special thanks to Patricia Fuya, Ligia Lugo, and the members of the Entomology Laboratory at Instituto Nacional de Salud of Colombia (INS) for supplying mosquito reference strains, to Néstor Pinto and Marlon Salgado at Secretaria de Mayor de Cundinamarca in Colombia and to Angélica María Aguirre and Daniel David Rodríguez for collecting field mosquito larvae; and special thanks to A. Delecluse from the Pasteur Institute for kindly donating the L. sphaericus WHO reference strain. Also, special thanks to Nestor Pinto for identifying the field-collected larvae and special thanks to Paula Andrea Rojas for the assistance.

References

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Figure 0

Table 1. Larvicidal activity of L. sphaericus highly toxigenic strains against Ae. aegypti fourth larvae.

Figure 1

Fig. 1. Mortality of Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña fourth-instar larvae in presence of L. sphaericus spores (107 CFU ml−1) in single-species bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Figure 2

Fig. 2. Mortality of Ae. aegypti Rockefeller and Cx. quinquefasciatus Muña fourth-instar larvae in presence of L. sphaericus vegetative cells (107 CFU ml−1) in single-species bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Figure 3

Fig. 3. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus spores (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Figure 4

Fig. 4. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus vegetative cells (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.

Figure 5

Fig. 5. Mortality of field-collected Ae. aegypti and Cx. quinquefasciatus and laboratory Ae. aegypti and Cx. quinquefasciatus fourth-instar larvae in presence of L. sphaericus spores and vegetative cells (107 CFU ml−1) in mixed-culture bioassays after 48 h of exposure. Upper or lower-case letters refer to statistical comparisons within the same species. Boxes with the same letter are not significantly different according to Tukey–Kramer test or Mann–Whitney U test in case of no normality. Horizontal bars, capped bars, and circles indicate median values, maximum and minimum values and outliers, respectively.