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Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi
Accepted Manuscript Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi Il-Hwan Kim, Jerald Ensign, Do-Young Kim, Hoe-Yune Jung, Na-Ri Kim, BoHwa Choi, Sun-Min Park, Que Lan, Walter G. Goodman PII: DOI: Reference:
S0022-2011(17)30096-4 http://dx.doi.org/10.1016/j.jip.2017.07.002 YJIPA 6970
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
25 March 2017 1 July 2017 10 July 2017
Please cite this article as: Kim, I-H., Ensign, J., Kim, D-Y., Jung, H-Y., Kim, N-R., Choi, B-H., Park, S-M., Lan, Q., Goodman, W.G., Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.07.002
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Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi
Il-Hwan Kim1,5, Jerald Ensign2, Do-Young Kim3, Hoe-Yune Jung3,4, Na-Ri Kim3, Bo-Hwa Choi3, Sun-Min Park3, Que Lan5,6 and Walter G. Goodman5
1
Vector Biology Section, Laboratory of Malaria and Vector Research; National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA 2
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA
3
Advanced Bio Convergence Center, Pohang Technopark, Jigok-dong, Pohang, Republic of
Korea 4
R&D Center, NovMetaPharma Co., Ltd., Jigok-dong, Pohang, Republic of Korea
5
Department of Entomology, University of Wisconsin-Madison, Madison, WI, 53706, USA
6
Deceased
Correspondence: e-mail: [email protected]; phone: (301)-219-0569
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Abstract Reduction of mosquito-borne diseases relies, in part, on the use of synthetic pesticides to control pest mosquitoes. This reliance has led to genetic resistance, environmental contamination and the nondiscriminatory elimination of both pest and non-pest species. To expand our options for control, we screened entomopathogenic bacteria for potential larvicidal activity. A lipopeptide from the bacterium, Xenorhabdus innexi, was discovered that displayed potent larvicidal activity. The LC50s of the lipopeptide towards Aedes aegypti, Culex pipiens and Anopheles gambiae larvae were 1.81, 1.25 and 1.86 parts-per-million, respectively. No mortality was observed in other insect species tested. The putative mode of action of the lipopeptide suggested that after orally ingestion, it bound to the apical membrane of anterior midgut cells and created pores in the cellular membranes. The rapid neutralization of midgut pH suggested the pores disabled the H+-V-ATPase on the basal membrane and led to epithelial cell death. Specificity and toxicity towards mosquito larvae and the unique mode of action makes this lipopeptide a potentially attractive bacterial insecticide for control of mosquitoes. Keywords: Aedes aegypti; Xenorhabdus innexi; bacterial secondary metabolites; biolarvicide
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1. Introduction Mosquitoes transmit life-threatening diseases such as malaria, dengue, Chikungunya (CHIK), West Nile (WNF) and yellow fever (Gratz, 1999). Recent outbreaks and emergence of Zika virus underscores the public health threat posed by certain mosquitoes (Malone et al., 2016). There are several strategies to reduce mosquito-borne disease; vaccinations, therapeutic drugs and mosquito population control (Scholte et al., 2005 and Hemingway et al., 2006). Where medical intervention is impractical, synthetic insecticides are often used to control mosquito populations. Chemical control measures, while effective, have significant drawbacks including genetic resistance, non-target effects, and environmental pollution (Hemingway & Ranson 2000, Smith & Stratton 1986, Casida & Quistad, 1998 and Hao et al., 2016a). Several non-Bacillus entomopathogenic bacterial species, Xenorhabdus and Photorhabdus, have been studied for their insecticidal activities against agricultural pest insects (Bowen & Ensign 1998 and Sergeant et al., 2003). Toxin complex proteins (Tc), produced by Photorhabdus luminescens, have modes of action that are different from Bacillus thuringiensis (Bt) toxins, and may be considered potential alternatives to Bt toxins in agricultural pest management systems (Meusch et al., 2014 and Dowling & Waterfield 2007). Isolates from the bacterium Xenorhabdus innexi display potent insecticidal activities against medically important mosquito species including Aedes, Culex, and Anopheles larvae (Ensign et al., 2014). Preliminary structural analysis of the purified compound (Xlt) indicated that the larvicidal toxin is a low-molecular weight lipopeptide and its molecular weight ranges between 1195 to 1478 (Ensign et al., 2014). The peptide component of Xlt
includes amino acids such as histidine, glycine,
asparagine/aspartate, diaminobutyric acid and serine (Ensign et al., 2014). Variation in its molecular weight is due to the differences in its fatty acid component and its lipid moiety
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includes at least one oxo-fatty acid of C8 to C20 (Ensign et al., 2014). In this study, Xlt target specificity and mode of action is investigated.
2. Materials and methods 2.1 Bacterial strain X. innexi strain PTA-6826 was grown in modified minimal media broth (50 mM Na2HPO4, 50 mM KH2PO4, 20 mM (NH4)2SO4, 1 mM MgSO4, 0.25% (w/v) yeast extract and 100 mM glucose) at 30 °C for 72 h. The mosquitocidal lipopeptide toxin, Xlt, was extracted and purified as described previously (Ensign et al., 2014). Purified Xlt, which has a molecular weight of 1350, was quantified using Nanodrop ND-1000 spectrophotometer (Thermo scientific, Waltham, MA).
2.2 Mosquitoes and other test insects Laboratory colonies of Aedes aegypti, Anopheles gambiae and Culex pipiens larvae were reared at 26 °C under a 14L: 10D photoperiod. Larvae were fed fish food (Spectrum Brands, Middleton, WI). Late 3rd and early 4th instars were used in bioassays. Manduca sexta (Madison strain) were reared at 25 °C under a 16L: 8D photoperiod. Larvae were fed a wheat-germ based artificial diet (MP Biomedicals, Aurora, OH). Second instars were used in bioassays. Apis mellifera adults were obtained from Dr. Jon Roll (Department of Bacteriology, University of WisconsinMadison). Leptinotarsa decemlineata adults were collected at Hancock Agricultural Experiment Station (Hancock, WI).
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2.3 Insect and human cell culture Aedes aegypti cell line-2 (Aag-2) was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS). Aedes albopictus cells (C6/36) were propagated in minimal essential medium (MEM) supplemented with 10% FBS. Both cell lines were maintained at 28 °C with 5% CO2 and passed every 7 days with a 1:10 dilution. Drosophila cells (S2) were grown in SF-900 serum-free medium at 28 °C and M. sexta cells (GV1) were cultured in modified Grace’s medium supplemented with 10% FBS at 28 °C. Human fibroblasts cells (Hs68) were grown in DMEM supplemented with 1% antibioticantimycotic (Gibco) and 10% FBS and maintained at 37 °C and 5% CO2. Cells between the 20th and 25th passages were sub-cultured following trypsinization and used for experiments. Human mast cells (HMC-1) were cultured in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 2 mM L-glutamine, 25 mM HEPES, 10% FBS and 1% penicillin and streptomycin, and incubated in a 95% humidity-controlled incubator with 5% CO2 at 37 °C.
2.3 Bioassay of Xlt on mosquito larvae, pupae and adults Larval bioassays were conducted following standard protocols outlined by the World Health Organization (2005). Late 3rd instars of Aedes aegypti, An. gambiae, and C. pipiens were placed in 12-well plastic plates (Becton Dickinson Labware, Franklin Lakes, NJ) containing 5 ml of double distilled water. Ten mosquito larvae were placed in a single well and each bioassay series tested at seven different concentrations of Xlt. Larval mortality was determined 24 h after treatment. Approximately 240 larvae were tested in three independent replications. The lethal concentration (LC50) for each species was calculated by probit analysis (SAS Institute, 2009;
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Huseth & Groves 2013). Newly emerged Ae. aegypti pupae were tested for susceptibility to Xlt following the protocol described for the larval bioassay. Ae. aegypti adult mosquitoes were maintained at 26 °C under a 14L: 10D photoperiod at 70-80% humidity. One day after emergence, ten female mosquitoes were transferred to a small cage and provided with a cotton ball soaked in a 10% (w/v) sucrose solution. After an equilibration period of 24 h, adults were provided with cotton balls soaked in 10% sucrose and varying concentrations of Xlt. Mortality was determined after 24 h and the LC50 calculated by probit analysis.
2.4 Bioassay of Xlt on A. mellifera, L. decemlineata, and M. sexta Between 20 and 30 adult A. mellifera were transferred to a cage and maintained at 26 °C under a 14L: 10D photoperiod at 70-80% humidity. A. mellifera fed on 2 M sucrose solution from 50 ml conical tube that contained small feeding holes. Different concentrations of Xlt (0, 50, 75 and 100 ppm) were dissolved separately in sucrose solutions and placed in the feeder. Mortality was checked at 24 h and 48 h. The bioassay was repeated three times and the LC50 was calculated by probit analysis. Field-collected L. decemlineata adults were maintained at 26 °C in 70-80% humidity under a 14L: 10D photoperiod. Adults were provided with fresh potato leaves. To conduct the feeding bioassay, three adults were placed in a petri dish (8.5 cm diameter) and provided with 1 cm2 of potato leaf treated with either Xlt (1, 5, 10, 50 or 100 ppm) or distilled water. A freshly Xlttreated or untreated potato leaf was provided every three hours and mortality assessed at 24 h and
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48 h. Nine beetles were tested per treatment in each bioassay series and each dosage was repeated three times. The LC50 was calculated by probit analysis. Second instar M. sexta were selected for feeding bioassays using methods described by Schesser et al. (1976). Individual M. sexta larvae were placed into a plastic cup and provided with an Xlttreated (0, 1, 5, 10, 25, 50, 75 or 100 ppm) cube (1 cm3) of artificial diet. Diets were changed every 12 h; body weights of larvae were measured after 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. The average body weight increase at 48 h was calculated and one-way ANOVA and Tukey’s multiple comparisons test was conducted to evaluate whether Xlt affected the weight increase.
2.5 SYTOX® Green nucleic acid stain of Xlt treated insect cell lines Aag-2, S2, or GV1 cells were cultured in 12-well plates (1 x 106 cells/well), and then treated with 2 ppm of Xlt or distilled water and 8 µM of SYTOX® Green nucleic acid dye (Invitrogen). Cells were incubated at 28 °C with 5% CO2 for 6 h. Cell images were captured using a brightfield and fluorescence microscope equipped with 488 nm argon-ion laser (excitation/emission: 504/523 nm). The experiment was repeated four times with three replications in each series.
2.6 Cell viability assay Cell viability was determined using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay with modification (Jung et al., 2014). Briefly, Aag-2, C6/36, Hs68 or HMC-1 cells cultured in 6-well plates (4 X 106 cells/well) were treated with five
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concentrations (1, 5, 10, 50, and 100 ppm) of Xlt and then incubated for 24 h in serum-free medium. Cells were then incubated with 1 mg/ml MTT for 2 h at 37 °C. MTT/formazan was extracted by overnight incubation at 37 °C with 1 ml extraction buffer (10% triton X-100, 89% isopropanol and 11% 1N HCl) and optical densities measured at 570 nm. The percent cell viability was calculated as the number of cells in the Xlt-treated samples divided by the number of cells in the control treatment based on the optical densities measured at 570 nm. Each experiment was repeated four times for Aag-2 and C6/36, and three times for Hs68 and DLD-1. Tukey’s multiple comparisons test was conducted to examine whether the Xlt-treated cell populations were significantly different from the untreated cells.
2.7 pH detection in Ae. aegypti larval midgut Larval bioassays were prepared following the protocol described above. Ten early 4th instar Ae. aegypti were placed in 5 ml of water in a well of 12-well plates, and treated with Xlt (2 or 6 ppm) or distilled water. m-cresol purple (0.04% ,w/v; Sigma-Aldrich, St. Louis, MO) was added to each well to detect pH changes in larval midguts. After 2 h or 6 h, treated larvae were dissected and the midgut imaged under bright-field microscope. The experiment was repeated three times.
2.8 SYTOX® Green stain of Bti endotoxins and Xlt fed mosquitoes SYTOX® Green stain was used to detect the location of cells undergoing apoptosis in Xlt-fed larval mosquito midguts. Mosquito larval bioassays were conducted as described above. Late 3rd instar An. gambiae and Ae. aegypti were treated with 2 ppm of Bti endotoxins (Summit Chemical, Baltimore, MD) or Xlt, followed by addition of SYTOX® Green stain to bring it to 8 µM in the
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well. After 2 h, images of larvae were taken with a compound microscope with 488 nm argonion laser (excitation/emission: 504/523 nm). The experiment was replicated five times and fifty larvae were examined in each treatment.
2.9 Confocal microscopy of Xlt treated Ae. aegypti larval midgut The late 3rd instar Ae. aegypti larvae were treated with 2 ppm of Xlt as described above. After 2 h, both Xlt treated and healthy larval midguts were dissected and fixed in 4% paraformaldehyde for 1 h. Fixed midguts were washed 3 times in 0.1M PBS-0.05% Tween 20 (PBS-T) for 10 minutes, and then blocked in 0.1% bovine serum albumin in PBS-T for 1 hour. Washed midguts were treated with a 1:100 dilution of Xlt-specific polyclonal antibody and incubated overnight on a shaking platform at 4 °C. After washing 3 times in PBS-T for 10 minutes, midguts were treated with a 1:200 dilution of Alexa-555® (Thermo Fisher Scientific, Waltham, MA) for 1 h. Midguts were then washed 3 times in PBS-T and once in 0.1M PBS for 10 minutes. Midguts were placed into depression microslides filled with mounting media. Antibody-treated midguts were observed under bright field and red fluorophore spectrum (561 nm laser line) and examined for immunofluorescence. The experiment was conducted five times and a total of fifty Ae. aegypti larvae were dissected and examined.
3. Results 3.1 Xlt insecticidal activity against larvae of various mosquito species
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Preliminary observations demonstrated that X. innexi culture supernatants have insecticidal activities against the larvae of the Yellow fever mosquito, Ae. aegypti. Xlt, was purified from X. innexi cultured in minimal medium as described previously (Ensign et al., 2014). Ae. aegypti, C. pipiens, and An. gambiae larvae were treated with varying doses of purified Xlt (1, 5, 10, 25, 50, 75, and 100 ppm) for 24 h to assess the median lethal concentrations (LC50). The LC50 for late 3rd instar Ae. aegypti was 1.81 ppm. Table 1 indicates that 3rd instar C. pipiens and An. gambiae were also as sensitive to Xlt as Ae. aegypti. The LC50 for C. pipiens and An. gambiae was 1.25 and 1.86 ppm, respectively. Bioassays were conducted on Ae. aegypti pupae and adult females to examine Xlt lethality in other developmental stages. Xlt did not induce acute toxicity in pupae or adults during a 24 h treatment. The LC50 for pupae and adults could not be determined since the highest concentration tested, 100 ppm, did not cause mortality (Table 1). The bioassay results suggested that Xlt is an effective mosquito larvicide at low concentrations and its toxicity is exclusively directed towards the larval stage.
Xlt lacks lethal or insect growth inhibitor To investigate the potential non-target effects of Xlt on other insects, we conducted bioassays on representative holometabolan species including L. decemlineata adults, A. mellifera adults and M. sexta larvae. These experiments were designed to measure short-term (acute) toxicity and growth inhibition. At any concentration tested, Xlt had no lethal effects on these species (Table 1). The LC50 for L. decemlineata, A. mellifera and M. sexta were not calculated since the highest concentrations, 100 ppm, did not cause mortality.
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We also examined the possibility that Xlt functions as an insect growth inhibitor. Body weight of Xlt-treated and untreated M. sexta were measured every 12 h for 2 days. Weight increases between untreated and Xlt treated larvae were not significantly different (p=0.3776 as determined by one-way ANOVA and p>0.05 by Tukey’s multiple comparisons test; Fig. 1). Although body weights of L. decemlineata larvae was not measured, we observed that the larvae rapidly consumed Xlt-treated potato leaves. Moreover, A. mellifera adults that repeatedly fed on 2 M sucrose solutions treated with Xlt displayed no short-term effects. Thus, Xlt is not lethal to L. decemlineata, A. mellifera nor does it affect M. sexta growth.
3.2 Xlt causes acute toxicity specific in mosquito cell lines To examine Xlt effects at the cellular level, different insect cell lines including Ae. aegypti (Aag2), D. melanogaster (S2) and M. sexta (GV1) cells were treated with 2 ppm of Xlt, which represents the LC50 concentration for mosquito larvae. Cells were first treated with Xlt and then stained with SYTOX® Green, a nucleic acid stain that does not penetrate live cells but fluoresces green when it interacts with nucleic acids of dying cells (Gaforio et al., 2002). Light microscope images showed that when S2 and GV1 cell were treated for 6 h with Xlt, cell morphology remained unchanged (Fig. 2I and 2K). However, Aag-2 cells underwent aggregation and apoptosis following a 6 h treatment (Fig. 2G). Green fluorescence was observed in Xlt-treated Aag-2 cells, which further confirmed that Xlt induced apoptosis (Fig. 2H). Neither S2 nor GV1 cells displayed green fluorescence, indicating a lack of acute cytotoxicity in response to Xlt treatment (Fig. 2J and 2L). This demonstrates that Xlt at low concentrations is not toxic to nontarget cells but is effective against mosquito cells.
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The toxicity of Xlt on human cell lines was tested using human skin fibroblast cells (Hs68) and human mast cells (HMC-1), and compared with two mosquito cell lines, Ae. aegypti (Aag-2) and Ae. albopictus (C6/36). At 24 h post-treatment, fewer than 20% of Aag-2 and C6/36 cells survived treatment with 1 ppm, consistent with their susceptibility to Xlt toxicity (Fig. 3A and 3B). Unlike mosquito cell lines, Hs68 cells were not affected until treated with 50 ppm of Xlt. Tukey’s multiple comparisons test indicated that a significant population decrease occurred in Hs68 cells treated with 50 ppm and 100 ppm of Xlt (Fig. 3C); however, the percent viability at these concentrations were significantly higher than that of Aag-2 and C6/36 cells. The population of HMC-1 cells increased significantly at 10 and 50 ppm of Xlt treatment. At 100 ppm, the HMC-1 cell viability decreased significantly with fewer than 20% of cells surviving (Fig. 3D). Thus, Xlt is acutely toxic to mosquito cells but much less so in human cells. 3.3 Xlt induces pH changes within mosquito larval midgut Xlt is orally toxic to mosquito larvae raising the possibility that Xlt disrupts mosquito larval midgut in a fashion similar to the Bti endotoxins (Bravo et al., 2007). To test this idea, early 4th instar Ae. aegypti were treated with 2 and 6 ppm of Xlt, and then stained with 0.04% m-cresol purple to observe potential pH changes in the midgut. Disruption of midgut integrity would be expected to reduce the midgut pH (12-13) of control mosquito larvae (Boudko et al., 2001). As expected, untreated larvae displayed a midgut pH of 12 or higher (Fig. 4A and 4B). When treated with 2 ppm of Xlt, the anterior and central midgut became more acidic within 2 h (Fig. 4C and 4D). Larvae subjected to 6 ppm displayed lower pH levels in the anterior and middle midguts (Fig. 4E). After 6 h of treatment with 6 ppm, Ae. aegypti midguts were yellow, indicating that the entire midgut pH fell below pH 7 (Fig. 4F).
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3.4 Xlt induced cell death at the anterior midgut To identify the initial sites of Xlt-induced toxicity in midgut epithelial cells, we stained Xlttreated mosquito larvae midguts with SYTOX® Green stain. As a comparison, we included larvae fed Bti endotoxins, which targets the posterior midgut (Chen et al., 2009a,b). Untreated An. gambiae midguts showed no green fluorescence indicating a lack of cytotoxicity (Fig. 5B). Larvae treated with 2 ppm of Bti endotoxin showed green fluorescence throughout the midgut 2 h after treatment (Fig. 5D), consistent with published observations (Chen et al., 2009a,b). By contrast, larvae treated with 2 ppm of Xlt displayed green fluorescence only in the anterior midgut (Fig. 5F). This suggests, unlike Bti endotoxins, Xlt induces cell death in the anterior midgut. Similar observations were made in Ae. aegypti larvae (Fig. 5H and 5J). These data indicate that while both Bti endotoxins and Xlt cause midgut epithelial cell apoptosis, their initial cellular targets are spatially distinct, possibly indicating different binding sites.
3.5 Xlt binds specifically in the anterior midgut region We used immunocytochemistry to locate Xlt within mosquito larval midguts. Late 3rd instar Ae. aegypti were treated with Xlt (2 ppm) for 2 h, and then their midguts were dissected and incubated with Xlt-specific antibody followed by Alexa-555® dye (red fluorescence signal) to probe the potential binding sites for Xlt. Confocal microscopy of untreated Ae. aegypti larval midguts indicated a lack of red fluorescence (Fig. 6A, 6B and 6C). By contrast, Xlt-treated midguts displayed areas of strong red fluorescence in the anterior midgut (Fig. 6D). No immunofluorescence was observed in the central mid- and posterior midgut of Xlt treated larvae
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(Fig. 6E and 6F). This agrees with our observation that Xlt-induced apoptosis occurs in the anterior regions of the larval midgut.
4. Discussion 4.1 Specificity of Xlt in whole organisms and in cell lines The LC50s for Xlt treated Ae. aegypti, An. gambiae, and C. pipiens indicated that Xlt is highly toxic to larvae of the three mosquito species tested. Xlt proved inactive against several other insect species from different orders including L. decemlineata, A. mellifera, and M. sexta. We also considered that Xlt may function as an insect growth regulator by examining the growth and development of Xlt-fed M. sexta larvae. The results strongly suggested that Xlt does not function as an insect growth inhibitor. Thus, Xlt appears to be specific and it does not affect other insect orders at the concentrations that kills mosquito larvae. Cells are sensitive to toxic stress and in vitro cytotoxicity assay using DNA-binding dye such as SYTOX® Green or propidium iodide is a frequently used method to measure the acute toxicity of a compound (Eisenbrand et al., 2002, Roth et al., 1997 and Hao et al., 2016b). Our study demonstrated that mosquito cells are highly susceptible to Xlt showing rapid cellular degradation. However, Xlt did not elicit cytotoxicity in D. melanogaster or M. sexta cells at comparable concentrations that killed mosquito cells. Since human toxicity is of critical importance to the deployment of this novel compound as a larvicide, we examined the toxicity of Xlt on several human cell lines. Human epithelial cells are often tested to evaluate drugs, chemicals, personal care products, and potential toxicants (Elmore et al., 1999). Although results indicated that human skin cells were affected by high
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concentrations of Xlt, the concentrations required to reduce viability were significantly higher than that in mosquito cell lines. Human mast cells were also tested for the potential cytotoxicity of Xlt. Mast cells serve an important role in immune surveillance and contribute to the human defense system. Various host and foreign stimuli are known to activate mast cells at different locations (Krishnaswamy et al., 2001). Unlike human skin cells, mast cell populations increased significantly when Xlt concentrations of 10 and 50 ppm were introduced. This may stem from the natural response of human mast cells to foreign stimuli since certain lipoproteins and peptides activate mast cells (Krishnaswamy et al., 2001 and Niyonsaba et al., 2002). However, at an elevated concentration of 100 ppm, fewer than 20% survived, suggesting that Xlt could be cytotoxic towards human mast cells if used at high levels.
4.2 Xlt modifies gut function After ingestion, Xlt appears to alter the midgut pH, changing it from a relatively alkaline condition to a neutral/acidic condition. In larval mosquito midguts, H+ V-ATPases contribute to luminal alkalization (Zhuang et al., 1999, Boudko et al., 2001, Patrick et al., 2006, Okech et al., 2007, and Onken et al., 2008). In the anterior midgut, H+ V-ATPases are located in the basal membranes of gut cells (Zhuang et al., 1999). High proton concentrations are found near the basal membrane and establish a gradient that promotes proton movement into the hemolymph (Boudko et al., 2001). By contrast, a neutral pH is observed in the posterior midgut where fewer H+ V-ATPases are located and leads to a lower concentration of protons moving into the lumen which maintains the pH level within the midgut (Zhuang et al., 1999, Boudko et al., 2001).
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SYTOX® Green stain of Xlt-treated midguts indicates that Xlt induces a region of localized cell death in the anterior midgut in both Ae. aegypti and An. gambiae larvae. Images from in vitro cytotoxicity assays showed that Xlt caused Aag-2 cells to undergo both a morphological change and a cellular membrane degradation which leads to their cell death. Thus, the localized cell death in the anterior midgut is likely due to the membrane disintegration of the apical membrane of midgut epithelial cells. As a result, the H+ V-ATPases on the basal membrane is disabled, thereby disrupting the proton gradient and leading to the neutralization of pH in the midgut lumen. This hypothesis is supported by our studies using the pH sensitive indicator, m-cresol purple. Cellular differences in the anatomy of the anterior and posterior midgut may also play an important role in localization of Xlt. Midgut epithelial cells of mosquito larvae display considerable divergence in cellular structure (Zhuang et al., 1999). Anterior midgut apical microvilli are short and broad whereas the posterior midgut apical microvilli are, long thin and numerous (Zhuang et al., 1999). The structure of the apical membranes in anterior midgut cells perhaps provides enhanced surface features that are compatible for interactions with Xlt. Using immunofluorescence, Xlt appeared to be localized in the anterior midgut whereas none was detected in the posterior midgut. We propose that Xlt, following oral ingestion, interacts with the apical membrane of anterior midgut cells and creates pores on the cellular membranes. This leads to epithelial cell death in the anterior midgut which disables the H+ V-ATPase on the basal membrane. The midgut epithelium, now breached, cannot maintain the alkaline pH. Cell deaths and loss of cellular functions in larval midgut leads to the mortality of mosquito larvae. It is yet unclear whether the nature of the Xlt binding is specific or non-specific.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϳ
A comparison of another family of bacterial-derived pesticides, the Bti delta endotoxins, may provide insight into the molecular mode of action of Xlt. Although not well understood, studies suggest that the Bti endotoxins interact with H+ V-ATPases in the posterior midgut of larval mosquitoes where H+ V-ATPases are located on the apical membrane (Bayyareddy et al., 2009, Likitvivatanavong et al., 2011 and Tetreau et al., 2012). Other membrane receptors of Bti endotoxins including alkaline phosphatase, aminopeptidase-N, and cadherin are also exclusively located in the posterior midgut of mosquito larvae (Fernandez et al., 2006 and Chen et al., 2009a,b). Since our observations indicate that Xlt is localized in the anterior midgut, it is evident that Xlt does not interact with known binding receptors of the Bti delta-endotoxins. Xlt may not require specific membrane receptors; the epithelial cell surface may facilitate the non-specific binding of Xlt. This mode of action may be similar to that observed for the Bti cytolytic deltaendotoxin, Cyt1A, which directly interacts with membrane lipids to create pores (Knowles et al., 1989 and Butko 2003).
5. Conclusion Xlt displays characteristics that are favorable for its deployment as a larvicide. It is stable to heat, UV irradiation and proteases (Ensign et al., 2014), characteristics not observed in other bacterial derived insect toxins. The relative stability observed in the lab suggests that the molecule will be more environmentally stable. Xlt appears to display a different mode of action from other biological insecticides such as the Bti delta-endotoxins. The synergistic effect of applying multiple insecticides with varying modes of action, such as Bti delta endotoxins and Xlt, could substantially enhance mosquito larvicidal activities and delay development of resistance in the
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϴ
field (Gill et al., 1992). Indeed, higher efficacy is observed when using mixtures of insecticides with varying modes of actions rather than a single compound (Poopathi 2012).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϵ
Acknowledgement We thank Dr. Jon Roll and Dr. Justin Clements for generously providing A. mellifera and L. decemlineata used in this study. We also thank Dr. Yeon Soo Han and Dr. Yong Hun Jo for their technical assistance in confocal microscopy. This study was supported by the College of Agricultural and Life Sciences, the Graduate School, University of Wisconsin-Madison and the National Science Foundation - East Asia and Pacific Summer Institutes for U.S. Graduate Students Fellowship.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϬ
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<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϮ
Meusch, D., Gatsogiannis, C., Efremov, R.G., Lang, A.E., Hofnagel, O., Vetter, I.R., Aktories, K. & Raunser, S. (2014). Mechanism of Tc toxin action revealed in molecular detail. Nature, 508(7494), 61-65. Niyonsaba, F., Iwabuchi, K., Someya, A., Hirata, M., Matsuda, H., Ogawa, H., & Nagaoka, I. (2002). A cathelicidin family of human antibacterial peptide LLǦ37 induces mast cell chemotaxis. Immunology, 106(1), 20-26. Okech, B. A., Boudko, D. Y., Linser, P. J., & Harvey, W. R. (2008). Cationic pathway of pH regulation in larvae of Anopheles gambiae. Journal of Experimental Biology, 211(6), 957-968. Onken, H., Moffett, S. B., & Moffett, D. F. (2008). Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti. Journal of Insect Science, 8(1), 46. Patrick, M. L., Aimanova, K., Sanders, H. R., & Gill, S. S. (2006). P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti. Journal of Experimental Biology, 209(23), 4638-4651. Poopathi, S. (2012). Current trends in the control of mosquito vectors by means of biological larvicides. Journal of Biofertilizers and Biopesticides, 3:125. Roth, B.L., Poot, M., Yue, S.T. and Millard, P.J., (1997). Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Applied and environmental microbiology, 63(6), 2421-2431. SAS Institute (2009). SAS 9.3.1 Help and documentation. SAS Institute, Cary, NC. Schesser, J. H., Kramer, K. J., & Bulla, L. A. (1977). Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta. Applied and Environmental Microbiology, 33(4), 878-880. Scholte, E.J., Ng'habi, K., Kihonda, J., Takken, W., Paaijmans, K., Abdulla, S., Killeen, G.F. & Knols, B.G. (2005). An entomopathogenic fungus for control of adult African malaria mosquitoes. Science, 308(5728), 1641-1642. Sergeant, M., Jarrett, P., Ousley, M., & Morgan, J. A. W. (2003). Interactions of insecticidal toxin gene products from Xenorhabdus nematophilus PMFI296. Applied and Environmental Microbiology, 69(6), 3344-3349. Smith, T. M., & Stratton, G. W. (1986). Effects of synthetic pyrethroid insecticides on nontarget organisms. In Residue Reviews (pp. 93-120). Springer New York. Tetreau, G., Bayyareddy, K., Jones, C.M., Stalinski, R., Riaz, M.A., Paris, M., David, J.P., Adang, M.J. & Després, L. (2012). Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches. BMC Genomics, 13(1), 1. World Health Organization (2005). “Guidelines for laboratory and field testing of mosquito larvicides.” Document WHO/CDS/WHOPES/GCDPP/2005.13. Geneva, Switzerland.
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Zhuang, Z., Linser, P. J., & Harvey, W. R. (1999). Antibody to H (+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti). Journal of Experimental Biology, 202(18), 2449-2460.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϰ
Figure legends Fig.1. Weights of 2nd instar M. sexta after feeding on artificial diets treated with different concentrations of Xlt for 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. Data shown are mean weight ± SD. One-way ANOVA (p = 0.3776) and Tukey’s multiple comparisons test (p>0.05) indicated that the increase in weights of all Xlt treated larvae did not differ significantly from those of the untreated larvae. Fig.2. Nucleic acid staining of Xlt treated Ae. aegypti (Aag-2), D. melanogaster (S2) and M. sexta (GV1) cells using SYTOX® Green. Representative light and fluorescent microscopic images of untreated Aag-2 (A and B), S2 (C and D), and GV1 (E and F) and Xlt (2 ppm) treated Aag-2 (G and H), S2 (I and J) and GV1 (K and L) are shown. Images were captured 6 h post-treatment. Cytotoxicity induced by Xlt, indicated by green fluorescence, was observed in Xlt treated Aag-2 cells. The experiment was repeated four times with three replications in each experiment (400x magnification). Fig.3. Viability of Ae. aegypti (Aag-2), Ae. albopictus (C6/36), human skin fibroblast (Hs68), and human mast (HMC-1) cells treated with different concentrations of Xlt for 24 h. The percent cell viability is calculated as the number of cells in the treated samples divided by the number of cells in the untreated sample. Data are shown as the mean ± SD based on four independent replications. Tukey’s multiple comparisons test indicated that the viability of cells treated with every tested concentration of Xlt (1, 5, 10, 50, and 100 ppm) was significantly different from that
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϱ
of the untreated cells (***, p<0.05) for Aag-2 and C6/36 (A and B). For Hs68 (C), viability of cells treated with 50 and 100 ppm of Xlt was significantly different from that of the untreated cells (***, p<0.05) for Hs68 (C) and for HMC-1 (D), the viability of 10, 50 and 100 ppm of Xlt treated cells was significantly different from that of the untreated cells (***, p<0.05). Fig.4. pH changes in Ae. aegypti larval midguts induced by Xlt treatment. Early 4th instar Ae. aegypti were treated with Xlt (2 and 6 ppm) and stained with 0.04% w/v m-cresol purple to detect pH changes. Images were captured at 2 (A, C, E) and 6 h (B, D, F) post-treatment. Midguts from untreated larvae (A and B) or larvae treated with 2 ppm (C and D) or 6 ppm (E and F) of Xlt are shown. The experiment was repeated three times and panels show representatives of a total of ninety insects that were dissected and examined (Size bar = 0.8 mm). AM, anterior midgut; PM, posterior midgut. Fig.5. Bti endotoxin- and Xlt-treated late 3rd instar An. gambiae and Ae. aegypti stained with SYTOX® Green. Larvae were treated with 2 ppm of Bti and Xlt and observed after 2 h. Representative light and green fluorescence images of untreated (A and B), Bti (C and D), and Xlt (E and F) treated An. gambiae larva and Bti (G and H) and Xlt (I and J) treated Ae. aegypti larva are shown. Green fluorescence is not observed in untreated larva (B). Green fluorescence (indicated by the white arrow) is distributed throughout the midgut in the Bti treated An. gambiae and Ae. aegypti larva (D and H). Green fluorescence is highly localized in the anterior midgut in the Xlt treated An. gambiae and Ae. aegypti larva (F and J). The experiment was repeated five times and images
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϲ
are representative of fifty larvae per experiment that were examined (16 x magnification). AT, anterior ; PT, posterior. Fig.6. Confocal microscopy images of Xlt-treated late 3rd instar Ae. aegypti larval midguts. Representative confocal images of untreated anterior (A), central (B) and posterior (C) midgut and Xlt (2 ppm) treated anterior (D), central (E) and posterior (F) midguts are shown. Dissected midguts were treated with a 1:100 dilution of Xlt-specific antibody and a 1:200 dilution of Alexa- 555®, and then observed for the presence of immunoreactions, indicated by the red fluorescence. The experiment was repeated five times and a total of fifty larvae were dissected and examined (Size bar = 50 µ m). AM, anterior midgut; CM, central midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϳ
Table 1 Insecticidal activity of Xlt against various insect species.
Insect species Aedes aegypti larvaec Aedes aegypti pupae Aedes aegypti adult (female) Culex pipiens larvae Anopheles gambiae larvae Apis mellifera adult Leptinotarsa decemlineata adult Manduca sexta larvae
LC50 (ppm) (95% FL)a 1.81 (1.47-2.13) N/Td N/T 1.25 (0.32-3.09) 1.86 (0.93-2.72) N/T N/T N/T
Slope (SEM)b 4.11 (0.48) -e 0.78 (0.10) 2.27 (0.47) -
a 95% fiducial limits as determined by probit analysis b Slope and standard error (SEM) calculated by probit analysis c Late 3rd instars were used for mosquito bioassays d N/T: Not toxic (unable to determine LC50 using 100 ppm of Xlt as the highest concentration) e -: not applicable
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϴ
Fig.1. Weights of 2nd instar M. sexta after feeding on artificial diets treated with varying concentrations of Xlt for 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. Data shown are mean weights ± SD. One-way ANOVA (p = 0.3776) and Tukey’s multiple comparisons test (p>0.05) indicated that the increase in weights of all Xlt-treated larvae did not differ significantly from those of the untreated larvae.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϵ
Aag2 untreated
S2 untreated
GV1 untreated
A
C
E
B
D
F
Aag-2 Xlt treated
S2 Xlt treated
GV1 Xlt treated
G
I
K
H
J
L
Fig.2. Nucleic acid staining of Xlt treated Ae. aegypti (Aag-2), D. melanogaster (S2) and M. sexta (GV1) cells using SYTOX® Green. Representative light and fluorescent microscopic images of untreated Aag-2 (A and B), S2 (C and D), and GV1 (E and F) and Xlt (2 ppm) treated Aag-2 (G and H), S2 (I and J) and GV1 (K and L) are shown. Images were captured 6 h post-treatment. Cytotoxicity induced by Xlt, indicated by green fluorescence, was observed in Xlt treated Aag-2 cells. The experiment was repeated four times with three replications of each experiment (Size bar = 50 µm).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϬ
A
C
Aag-2
Hs68
B
C6/36
D
HMC-1
Fig.3. Viability of Ae. aegypti (Aag-2), Ae. albopictus (C6/36), human skin fibroblast (Hs68), and human mast (HMC-1) cells treated with varying concentrations of Xlt for 24 h. The percent cell viability is calculated as the number of cells in the treated samples divided by the number of cells in the untreated sample. Data are shown as the means ± SD based on four independent replications. Tukey’s multiple comparisons test indicated that the viability of cells treated with every tested concentration of Xlt (1, 5, 10, 50, and 100 ppm) was significantly different from that of the untreated cells (***, p<0.05) for Aag-2 and C6/36 (A and B). For Hs68 (C), viability of cells treated with 50 and 100 ppm of Xlt was significantly different from that of the untreated cells (***, p<0.05) for Hs68 (C) and for HMC-1 (D), the viability of 10, 50 and 100 ppm of Xlt treated cells was significantly different from that of the untreated cells (***, p<0.05).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϭ
Untreated
6 ppm treated
2 ppm treated
2h AM PM A
C
E
B
D
F
6h
Fig.4. pH changes in Ae. aegypti larval midguts induced by Xlt treatment. Early 4th instar Ae. aegypti were treated with Xlt (2 and 6 ppm) and stained with 0.04% w/v m-cresol purple to detect pH changes. Images were captured at 2 (A, C, E) and 6 h (B, D, F) post-treatment. Midguts from untreated larvae (A and B) or larvae treated with 2 ppm (C and D) or 6 ppm (E and F) of Xlt are shown. The experiment was repeated three times and panels show representatives of a total of ninety insects that were dissected and examined (Size bar = 0.8 mm). AM, anterior midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϮ
Untreated An. gambiae
Bti treated An. gambiae
Xlt treated An. gambiae
AM
A
PM
C
E
AM
PM B
D
F
Bti treated Ae. aegypti
Xlt treated Ae. aegypti
G
I
H
J
Fig.5. Bti endotoxin- and Xlt-treated late 3rd instar An. gambiae and Ae. aegypti stained with SYTOX® Green. Larvae were treated with 2 ppm of Bti and Xlt and observed after 2 h. Representative light and green fluorescence images of untreated (A and B), Bti (C and D), and Xlt (E and F) treated An. gambiae larva and Bti (G and H) and Xlt (I and J) treated Ae. aegypti larva are shown. Green fluorescence is not observed in untreated larva (B). Green fluorescence (indicated by the white arrow) is distributed throughout the midgut in the Bti treated An. gambiae and Ae. aegypti larva (D and H). Green fluorescence is highly localized in the anterior midgut in the Xlt treated An. gambiae and Ae. aegypti larva (F and J). The experiment was repeated five times and images are representative of fifty larvae per experiment that were examined (Size bar = 800 µm). AT, anterior; PT, posterior.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϯ
A
B
C AM
PM Untreated AM
D
CM
E
F AM
Xlt treated AM
CM
PM
Fig.6. Confocal microscopy images of Xlt-treated late 3rd instar Ae. aegypti midguts. Representative confocal images of untreated anterior (A), central (B) and posterior (C) midgut and Xlt (2 ppm) treated anterior (D), central (E) and posterior (F) midguts are shown. Dissected midguts were treated with a 1:100 dilution of Xlt-specific antibody and a 1:200 dilution of Alexa- 555®, and then observed for the presence of immunoreactions, indicated by the red fluorescence. The experiment was repeated five times and a total of fifty larvae were dissected and examined (Size bar = 200 µm). AM, anterior midgut; CM, central midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϰ
Highlights •
Potential for bacterial lipopeptide (Xlt) as a mosquito larval control agent
•
Lipopeptide produced by Xenorhabdus innexi is orally toxic to mosquito larvae
•
Xlt alters larval midgut pH and cause cytotoxicity in the anterior midgut
•
Xlt has a different mode of action from Cry toxins in its pathogenicity
Graphical abstract
S0022-2011(17)30096-4 http://dx.doi.org/10.1016/j.jip.2017.07.002 YJIPA 6970
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
25 March 2017 1 July 2017 10 July 2017
Please cite this article as: Kim, I-H., Ensign, J., Kim, D-Y., Jung, H-Y., Kim, N-R., Choi, B-H., Park, S-M., Lan, Q., Goodman, W.G., Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.07.002
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Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi
Il-Hwan Kim1,5, Jerald Ensign2, Do-Young Kim3, Hoe-Yune Jung3,4, Na-Ri Kim3, Bo-Hwa Choi3, Sun-Min Park3, Que Lan5,6 and Walter G. Goodman5
1
Vector Biology Section, Laboratory of Malaria and Vector Research; National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA 2
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, 53706, USA
3
Advanced Bio Convergence Center, Pohang Technopark, Jigok-dong, Pohang, Republic of
Korea 4
R&D Center, NovMetaPharma Co., Ltd., Jigok-dong, Pohang, Republic of Korea
5
Department of Entomology, University of Wisconsin-Madison, Madison, WI, 53706, USA
6
Deceased
Correspondence: e-mail: [email protected]; phone: (301)-219-0569
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Abstract Reduction of mosquito-borne diseases relies, in part, on the use of synthetic pesticides to control pest mosquitoes. This reliance has led to genetic resistance, environmental contamination and the nondiscriminatory elimination of both pest and non-pest species. To expand our options for control, we screened entomopathogenic bacteria for potential larvicidal activity. A lipopeptide from the bacterium, Xenorhabdus innexi, was discovered that displayed potent larvicidal activity. The LC50s of the lipopeptide towards Aedes aegypti, Culex pipiens and Anopheles gambiae larvae were 1.81, 1.25 and 1.86 parts-per-million, respectively. No mortality was observed in other insect species tested. The putative mode of action of the lipopeptide suggested that after orally ingestion, it bound to the apical membrane of anterior midgut cells and created pores in the cellular membranes. The rapid neutralization of midgut pH suggested the pores disabled the H+-V-ATPase on the basal membrane and led to epithelial cell death. Specificity and toxicity towards mosquito larvae and the unique mode of action makes this lipopeptide a potentially attractive bacterial insecticide for control of mosquitoes. Keywords: Aedes aegypti; Xenorhabdus innexi; bacterial secondary metabolites; biolarvicide
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1. Introduction Mosquitoes transmit life-threatening diseases such as malaria, dengue, Chikungunya (CHIK), West Nile (WNF) and yellow fever (Gratz, 1999). Recent outbreaks and emergence of Zika virus underscores the public health threat posed by certain mosquitoes (Malone et al., 2016). There are several strategies to reduce mosquito-borne disease; vaccinations, therapeutic drugs and mosquito population control (Scholte et al., 2005 and Hemingway et al., 2006). Where medical intervention is impractical, synthetic insecticides are often used to control mosquito populations. Chemical control measures, while effective, have significant drawbacks including genetic resistance, non-target effects, and environmental pollution (Hemingway & Ranson 2000, Smith & Stratton 1986, Casida & Quistad, 1998 and Hao et al., 2016a). Several non-Bacillus entomopathogenic bacterial species, Xenorhabdus and Photorhabdus, have been studied for their insecticidal activities against agricultural pest insects (Bowen & Ensign 1998 and Sergeant et al., 2003). Toxin complex proteins (Tc), produced by Photorhabdus luminescens, have modes of action that are different from Bacillus thuringiensis (Bt) toxins, and may be considered potential alternatives to Bt toxins in agricultural pest management systems (Meusch et al., 2014 and Dowling & Waterfield 2007). Isolates from the bacterium Xenorhabdus innexi display potent insecticidal activities against medically important mosquito species including Aedes, Culex, and Anopheles larvae (Ensign et al., 2014). Preliminary structural analysis of the purified compound (Xlt) indicated that the larvicidal toxin is a low-molecular weight lipopeptide and its molecular weight ranges between 1195 to 1478 (Ensign et al., 2014). The peptide component of Xlt
includes amino acids such as histidine, glycine,
asparagine/aspartate, diaminobutyric acid and serine (Ensign et al., 2014). Variation in its molecular weight is due to the differences in its fatty acid component and its lipid moiety
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includes at least one oxo-fatty acid of C8 to C20 (Ensign et al., 2014). In this study, Xlt target specificity and mode of action is investigated.
2. Materials and methods 2.1 Bacterial strain X. innexi strain PTA-6826 was grown in modified minimal media broth (50 mM Na2HPO4, 50 mM KH2PO4, 20 mM (NH4)2SO4, 1 mM MgSO4, 0.25% (w/v) yeast extract and 100 mM glucose) at 30 °C for 72 h. The mosquitocidal lipopeptide toxin, Xlt, was extracted and purified as described previously (Ensign et al., 2014). Purified Xlt, which has a molecular weight of 1350, was quantified using Nanodrop ND-1000 spectrophotometer (Thermo scientific, Waltham, MA).
2.2 Mosquitoes and other test insects Laboratory colonies of Aedes aegypti, Anopheles gambiae and Culex pipiens larvae were reared at 26 °C under a 14L: 10D photoperiod. Larvae were fed fish food (Spectrum Brands, Middleton, WI). Late 3rd and early 4th instars were used in bioassays. Manduca sexta (Madison strain) were reared at 25 °C under a 16L: 8D photoperiod. Larvae were fed a wheat-germ based artificial diet (MP Biomedicals, Aurora, OH). Second instars were used in bioassays. Apis mellifera adults were obtained from Dr. Jon Roll (Department of Bacteriology, University of WisconsinMadison). Leptinotarsa decemlineata adults were collected at Hancock Agricultural Experiment Station (Hancock, WI).
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2.3 Insect and human cell culture Aedes aegypti cell line-2 (Aag-2) was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS). Aedes albopictus cells (C6/36) were propagated in minimal essential medium (MEM) supplemented with 10% FBS. Both cell lines were maintained at 28 °C with 5% CO2 and passed every 7 days with a 1:10 dilution. Drosophila cells (S2) were grown in SF-900 serum-free medium at 28 °C and M. sexta cells (GV1) were cultured in modified Grace’s medium supplemented with 10% FBS at 28 °C. Human fibroblasts cells (Hs68) were grown in DMEM supplemented with 1% antibioticantimycotic (Gibco) and 10% FBS and maintained at 37 °C and 5% CO2. Cells between the 20th and 25th passages were sub-cultured following trypsinization and used for experiments. Human mast cells (HMC-1) were cultured in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 2 mM L-glutamine, 25 mM HEPES, 10% FBS and 1% penicillin and streptomycin, and incubated in a 95% humidity-controlled incubator with 5% CO2 at 37 °C.
2.3 Bioassay of Xlt on mosquito larvae, pupae and adults Larval bioassays were conducted following standard protocols outlined by the World Health Organization (2005). Late 3rd instars of Aedes aegypti, An. gambiae, and C. pipiens were placed in 12-well plastic plates (Becton Dickinson Labware, Franklin Lakes, NJ) containing 5 ml of double distilled water. Ten mosquito larvae were placed in a single well and each bioassay series tested at seven different concentrations of Xlt. Larval mortality was determined 24 h after treatment. Approximately 240 larvae were tested in three independent replications. The lethal concentration (LC50) for each species was calculated by probit analysis (SAS Institute, 2009;
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Huseth & Groves 2013). Newly emerged Ae. aegypti pupae were tested for susceptibility to Xlt following the protocol described for the larval bioassay. Ae. aegypti adult mosquitoes were maintained at 26 °C under a 14L: 10D photoperiod at 70-80% humidity. One day after emergence, ten female mosquitoes were transferred to a small cage and provided with a cotton ball soaked in a 10% (w/v) sucrose solution. After an equilibration period of 24 h, adults were provided with cotton balls soaked in 10% sucrose and varying concentrations of Xlt. Mortality was determined after 24 h and the LC50 calculated by probit analysis.
2.4 Bioassay of Xlt on A. mellifera, L. decemlineata, and M. sexta Between 20 and 30 adult A. mellifera were transferred to a cage and maintained at 26 °C under a 14L: 10D photoperiod at 70-80% humidity. A. mellifera fed on 2 M sucrose solution from 50 ml conical tube that contained small feeding holes. Different concentrations of Xlt (0, 50, 75 and 100 ppm) were dissolved separately in sucrose solutions and placed in the feeder. Mortality was checked at 24 h and 48 h. The bioassay was repeated three times and the LC50 was calculated by probit analysis. Field-collected L. decemlineata adults were maintained at 26 °C in 70-80% humidity under a 14L: 10D photoperiod. Adults were provided with fresh potato leaves. To conduct the feeding bioassay, three adults were placed in a petri dish (8.5 cm diameter) and provided with 1 cm2 of potato leaf treated with either Xlt (1, 5, 10, 50 or 100 ppm) or distilled water. A freshly Xlttreated or untreated potato leaf was provided every three hours and mortality assessed at 24 h and
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48 h. Nine beetles were tested per treatment in each bioassay series and each dosage was repeated three times. The LC50 was calculated by probit analysis. Second instar M. sexta were selected for feeding bioassays using methods described by Schesser et al. (1976). Individual M. sexta larvae were placed into a plastic cup and provided with an Xlttreated (0, 1, 5, 10, 25, 50, 75 or 100 ppm) cube (1 cm3) of artificial diet. Diets were changed every 12 h; body weights of larvae were measured after 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. The average body weight increase at 48 h was calculated and one-way ANOVA and Tukey’s multiple comparisons test was conducted to evaluate whether Xlt affected the weight increase.
2.5 SYTOX® Green nucleic acid stain of Xlt treated insect cell lines Aag-2, S2, or GV1 cells were cultured in 12-well plates (1 x 106 cells/well), and then treated with 2 ppm of Xlt or distilled water and 8 µM of SYTOX® Green nucleic acid dye (Invitrogen). Cells were incubated at 28 °C with 5% CO2 for 6 h. Cell images were captured using a brightfield and fluorescence microscope equipped with 488 nm argon-ion laser (excitation/emission: 504/523 nm). The experiment was repeated four times with three replications in each series.
2.6 Cell viability assay Cell viability was determined using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay with modification (Jung et al., 2014). Briefly, Aag-2, C6/36, Hs68 or HMC-1 cells cultured in 6-well plates (4 X 106 cells/well) were treated with five
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concentrations (1, 5, 10, 50, and 100 ppm) of Xlt and then incubated for 24 h in serum-free medium. Cells were then incubated with 1 mg/ml MTT for 2 h at 37 °C. MTT/formazan was extracted by overnight incubation at 37 °C with 1 ml extraction buffer (10% triton X-100, 89% isopropanol and 11% 1N HCl) and optical densities measured at 570 nm. The percent cell viability was calculated as the number of cells in the Xlt-treated samples divided by the number of cells in the control treatment based on the optical densities measured at 570 nm. Each experiment was repeated four times for Aag-2 and C6/36, and three times for Hs68 and DLD-1. Tukey’s multiple comparisons test was conducted to examine whether the Xlt-treated cell populations were significantly different from the untreated cells.
2.7 pH detection in Ae. aegypti larval midgut Larval bioassays were prepared following the protocol described above. Ten early 4th instar Ae. aegypti were placed in 5 ml of water in a well of 12-well plates, and treated with Xlt (2 or 6 ppm) or distilled water. m-cresol purple (0.04% ,w/v; Sigma-Aldrich, St. Louis, MO) was added to each well to detect pH changes in larval midguts. After 2 h or 6 h, treated larvae were dissected and the midgut imaged under bright-field microscope. The experiment was repeated three times.
2.8 SYTOX® Green stain of Bti endotoxins and Xlt fed mosquitoes SYTOX® Green stain was used to detect the location of cells undergoing apoptosis in Xlt-fed larval mosquito midguts. Mosquito larval bioassays were conducted as described above. Late 3rd instar An. gambiae and Ae. aegypti were treated with 2 ppm of Bti endotoxins (Summit Chemical, Baltimore, MD) or Xlt, followed by addition of SYTOX® Green stain to bring it to 8 µM in the
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well. After 2 h, images of larvae were taken with a compound microscope with 488 nm argonion laser (excitation/emission: 504/523 nm). The experiment was replicated five times and fifty larvae were examined in each treatment.
2.9 Confocal microscopy of Xlt treated Ae. aegypti larval midgut The late 3rd instar Ae. aegypti larvae were treated with 2 ppm of Xlt as described above. After 2 h, both Xlt treated and healthy larval midguts were dissected and fixed in 4% paraformaldehyde for 1 h. Fixed midguts were washed 3 times in 0.1M PBS-0.05% Tween 20 (PBS-T) for 10 minutes, and then blocked in 0.1% bovine serum albumin in PBS-T for 1 hour. Washed midguts were treated with a 1:100 dilution of Xlt-specific polyclonal antibody and incubated overnight on a shaking platform at 4 °C. After washing 3 times in PBS-T for 10 minutes, midguts were treated with a 1:200 dilution of Alexa-555® (Thermo Fisher Scientific, Waltham, MA) for 1 h. Midguts were then washed 3 times in PBS-T and once in 0.1M PBS for 10 minutes. Midguts were placed into depression microslides filled with mounting media. Antibody-treated midguts were observed under bright field and red fluorophore spectrum (561 nm laser line) and examined for immunofluorescence. The experiment was conducted five times and a total of fifty Ae. aegypti larvae were dissected and examined.
3. Results 3.1 Xlt insecticidal activity against larvae of various mosquito species
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Preliminary observations demonstrated that X. innexi culture supernatants have insecticidal activities against the larvae of the Yellow fever mosquito, Ae. aegypti. Xlt, was purified from X. innexi cultured in minimal medium as described previously (Ensign et al., 2014). Ae. aegypti, C. pipiens, and An. gambiae larvae were treated with varying doses of purified Xlt (1, 5, 10, 25, 50, 75, and 100 ppm) for 24 h to assess the median lethal concentrations (LC50). The LC50 for late 3rd instar Ae. aegypti was 1.81 ppm. Table 1 indicates that 3rd instar C. pipiens and An. gambiae were also as sensitive to Xlt as Ae. aegypti. The LC50 for C. pipiens and An. gambiae was 1.25 and 1.86 ppm, respectively. Bioassays were conducted on Ae. aegypti pupae and adult females to examine Xlt lethality in other developmental stages. Xlt did not induce acute toxicity in pupae or adults during a 24 h treatment. The LC50 for pupae and adults could not be determined since the highest concentration tested, 100 ppm, did not cause mortality (Table 1). The bioassay results suggested that Xlt is an effective mosquito larvicide at low concentrations and its toxicity is exclusively directed towards the larval stage.
Xlt lacks lethal or insect growth inhibitor To investigate the potential non-target effects of Xlt on other insects, we conducted bioassays on representative holometabolan species including L. decemlineata adults, A. mellifera adults and M. sexta larvae. These experiments were designed to measure short-term (acute) toxicity and growth inhibition. At any concentration tested, Xlt had no lethal effects on these species (Table 1). The LC50 for L. decemlineata, A. mellifera and M. sexta were not calculated since the highest concentrations, 100 ppm, did not cause mortality.
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We also examined the possibility that Xlt functions as an insect growth inhibitor. Body weight of Xlt-treated and untreated M. sexta were measured every 12 h for 2 days. Weight increases between untreated and Xlt treated larvae were not significantly different (p=0.3776 as determined by one-way ANOVA and p>0.05 by Tukey’s multiple comparisons test; Fig. 1). Although body weights of L. decemlineata larvae was not measured, we observed that the larvae rapidly consumed Xlt-treated potato leaves. Moreover, A. mellifera adults that repeatedly fed on 2 M sucrose solutions treated with Xlt displayed no short-term effects. Thus, Xlt is not lethal to L. decemlineata, A. mellifera nor does it affect M. sexta growth.
3.2 Xlt causes acute toxicity specific in mosquito cell lines To examine Xlt effects at the cellular level, different insect cell lines including Ae. aegypti (Aag2), D. melanogaster (S2) and M. sexta (GV1) cells were treated with 2 ppm of Xlt, which represents the LC50 concentration for mosquito larvae. Cells were first treated with Xlt and then stained with SYTOX® Green, a nucleic acid stain that does not penetrate live cells but fluoresces green when it interacts with nucleic acids of dying cells (Gaforio et al., 2002). Light microscope images showed that when S2 and GV1 cell were treated for 6 h with Xlt, cell morphology remained unchanged (Fig. 2I and 2K). However, Aag-2 cells underwent aggregation and apoptosis following a 6 h treatment (Fig. 2G). Green fluorescence was observed in Xlt-treated Aag-2 cells, which further confirmed that Xlt induced apoptosis (Fig. 2H). Neither S2 nor GV1 cells displayed green fluorescence, indicating a lack of acute cytotoxicity in response to Xlt treatment (Fig. 2J and 2L). This demonstrates that Xlt at low concentrations is not toxic to nontarget cells but is effective against mosquito cells.
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The toxicity of Xlt on human cell lines was tested using human skin fibroblast cells (Hs68) and human mast cells (HMC-1), and compared with two mosquito cell lines, Ae. aegypti (Aag-2) and Ae. albopictus (C6/36). At 24 h post-treatment, fewer than 20% of Aag-2 and C6/36 cells survived treatment with 1 ppm, consistent with their susceptibility to Xlt toxicity (Fig. 3A and 3B). Unlike mosquito cell lines, Hs68 cells were not affected until treated with 50 ppm of Xlt. Tukey’s multiple comparisons test indicated that a significant population decrease occurred in Hs68 cells treated with 50 ppm and 100 ppm of Xlt (Fig. 3C); however, the percent viability at these concentrations were significantly higher than that of Aag-2 and C6/36 cells. The population of HMC-1 cells increased significantly at 10 and 50 ppm of Xlt treatment. At 100 ppm, the HMC-1 cell viability decreased significantly with fewer than 20% of cells surviving (Fig. 3D). Thus, Xlt is acutely toxic to mosquito cells but much less so in human cells. 3.3 Xlt induces pH changes within mosquito larval midgut Xlt is orally toxic to mosquito larvae raising the possibility that Xlt disrupts mosquito larval midgut in a fashion similar to the Bti endotoxins (Bravo et al., 2007). To test this idea, early 4th instar Ae. aegypti were treated with 2 and 6 ppm of Xlt, and then stained with 0.04% m-cresol purple to observe potential pH changes in the midgut. Disruption of midgut integrity would be expected to reduce the midgut pH (12-13) of control mosquito larvae (Boudko et al., 2001). As expected, untreated larvae displayed a midgut pH of 12 or higher (Fig. 4A and 4B). When treated with 2 ppm of Xlt, the anterior and central midgut became more acidic within 2 h (Fig. 4C and 4D). Larvae subjected to 6 ppm displayed lower pH levels in the anterior and middle midguts (Fig. 4E). After 6 h of treatment with 6 ppm, Ae. aegypti midguts were yellow, indicating that the entire midgut pH fell below pH 7 (Fig. 4F).
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3.4 Xlt induced cell death at the anterior midgut To identify the initial sites of Xlt-induced toxicity in midgut epithelial cells, we stained Xlttreated mosquito larvae midguts with SYTOX® Green stain. As a comparison, we included larvae fed Bti endotoxins, which targets the posterior midgut (Chen et al., 2009a,b). Untreated An. gambiae midguts showed no green fluorescence indicating a lack of cytotoxicity (Fig. 5B). Larvae treated with 2 ppm of Bti endotoxin showed green fluorescence throughout the midgut 2 h after treatment (Fig. 5D), consistent with published observations (Chen et al., 2009a,b). By contrast, larvae treated with 2 ppm of Xlt displayed green fluorescence only in the anterior midgut (Fig. 5F). This suggests, unlike Bti endotoxins, Xlt induces cell death in the anterior midgut. Similar observations were made in Ae. aegypti larvae (Fig. 5H and 5J). These data indicate that while both Bti endotoxins and Xlt cause midgut epithelial cell apoptosis, their initial cellular targets are spatially distinct, possibly indicating different binding sites.
3.5 Xlt binds specifically in the anterior midgut region We used immunocytochemistry to locate Xlt within mosquito larval midguts. Late 3rd instar Ae. aegypti were treated with Xlt (2 ppm) for 2 h, and then their midguts were dissected and incubated with Xlt-specific antibody followed by Alexa-555® dye (red fluorescence signal) to probe the potential binding sites for Xlt. Confocal microscopy of untreated Ae. aegypti larval midguts indicated a lack of red fluorescence (Fig. 6A, 6B and 6C). By contrast, Xlt-treated midguts displayed areas of strong red fluorescence in the anterior midgut (Fig. 6D). No immunofluorescence was observed in the central mid- and posterior midgut of Xlt treated larvae
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(Fig. 6E and 6F). This agrees with our observation that Xlt-induced apoptosis occurs in the anterior regions of the larval midgut.
4. Discussion 4.1 Specificity of Xlt in whole organisms and in cell lines The LC50s for Xlt treated Ae. aegypti, An. gambiae, and C. pipiens indicated that Xlt is highly toxic to larvae of the three mosquito species tested. Xlt proved inactive against several other insect species from different orders including L. decemlineata, A. mellifera, and M. sexta. We also considered that Xlt may function as an insect growth regulator by examining the growth and development of Xlt-fed M. sexta larvae. The results strongly suggested that Xlt does not function as an insect growth inhibitor. Thus, Xlt appears to be specific and it does not affect other insect orders at the concentrations that kills mosquito larvae. Cells are sensitive to toxic stress and in vitro cytotoxicity assay using DNA-binding dye such as SYTOX® Green or propidium iodide is a frequently used method to measure the acute toxicity of a compound (Eisenbrand et al., 2002, Roth et al., 1997 and Hao et al., 2016b). Our study demonstrated that mosquito cells are highly susceptible to Xlt showing rapid cellular degradation. However, Xlt did not elicit cytotoxicity in D. melanogaster or M. sexta cells at comparable concentrations that killed mosquito cells. Since human toxicity is of critical importance to the deployment of this novel compound as a larvicide, we examined the toxicity of Xlt on several human cell lines. Human epithelial cells are often tested to evaluate drugs, chemicals, personal care products, and potential toxicants (Elmore et al., 1999). Although results indicated that human skin cells were affected by high
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concentrations of Xlt, the concentrations required to reduce viability were significantly higher than that in mosquito cell lines. Human mast cells were also tested for the potential cytotoxicity of Xlt. Mast cells serve an important role in immune surveillance and contribute to the human defense system. Various host and foreign stimuli are known to activate mast cells at different locations (Krishnaswamy et al., 2001). Unlike human skin cells, mast cell populations increased significantly when Xlt concentrations of 10 and 50 ppm were introduced. This may stem from the natural response of human mast cells to foreign stimuli since certain lipoproteins and peptides activate mast cells (Krishnaswamy et al., 2001 and Niyonsaba et al., 2002). However, at an elevated concentration of 100 ppm, fewer than 20% survived, suggesting that Xlt could be cytotoxic towards human mast cells if used at high levels.
4.2 Xlt modifies gut function After ingestion, Xlt appears to alter the midgut pH, changing it from a relatively alkaline condition to a neutral/acidic condition. In larval mosquito midguts, H+ V-ATPases contribute to luminal alkalization (Zhuang et al., 1999, Boudko et al., 2001, Patrick et al., 2006, Okech et al., 2007, and Onken et al., 2008). In the anterior midgut, H+ V-ATPases are located in the basal membranes of gut cells (Zhuang et al., 1999). High proton concentrations are found near the basal membrane and establish a gradient that promotes proton movement into the hemolymph (Boudko et al., 2001). By contrast, a neutral pH is observed in the posterior midgut where fewer H+ V-ATPases are located and leads to a lower concentration of protons moving into the lumen which maintains the pH level within the midgut (Zhuang et al., 1999, Boudko et al., 2001).
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SYTOX® Green stain of Xlt-treated midguts indicates that Xlt induces a region of localized cell death in the anterior midgut in both Ae. aegypti and An. gambiae larvae. Images from in vitro cytotoxicity assays showed that Xlt caused Aag-2 cells to undergo both a morphological change and a cellular membrane degradation which leads to their cell death. Thus, the localized cell death in the anterior midgut is likely due to the membrane disintegration of the apical membrane of midgut epithelial cells. As a result, the H+ V-ATPases on the basal membrane is disabled, thereby disrupting the proton gradient and leading to the neutralization of pH in the midgut lumen. This hypothesis is supported by our studies using the pH sensitive indicator, m-cresol purple. Cellular differences in the anatomy of the anterior and posterior midgut may also play an important role in localization of Xlt. Midgut epithelial cells of mosquito larvae display considerable divergence in cellular structure (Zhuang et al., 1999). Anterior midgut apical microvilli are short and broad whereas the posterior midgut apical microvilli are, long thin and numerous (Zhuang et al., 1999). The structure of the apical membranes in anterior midgut cells perhaps provides enhanced surface features that are compatible for interactions with Xlt. Using immunofluorescence, Xlt appeared to be localized in the anterior midgut whereas none was detected in the posterior midgut. We propose that Xlt, following oral ingestion, interacts with the apical membrane of anterior midgut cells and creates pores on the cellular membranes. This leads to epithelial cell death in the anterior midgut which disables the H+ V-ATPase on the basal membrane. The midgut epithelium, now breached, cannot maintain the alkaline pH. Cell deaths and loss of cellular functions in larval midgut leads to the mortality of mosquito larvae. It is yet unclear whether the nature of the Xlt binding is specific or non-specific.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϳ
A comparison of another family of bacterial-derived pesticides, the Bti delta endotoxins, may provide insight into the molecular mode of action of Xlt. Although not well understood, studies suggest that the Bti endotoxins interact with H+ V-ATPases in the posterior midgut of larval mosquitoes where H+ V-ATPases are located on the apical membrane (Bayyareddy et al., 2009, Likitvivatanavong et al., 2011 and Tetreau et al., 2012). Other membrane receptors of Bti endotoxins including alkaline phosphatase, aminopeptidase-N, and cadherin are also exclusively located in the posterior midgut of mosquito larvae (Fernandez et al., 2006 and Chen et al., 2009a,b). Since our observations indicate that Xlt is localized in the anterior midgut, it is evident that Xlt does not interact with known binding receptors of the Bti delta-endotoxins. Xlt may not require specific membrane receptors; the epithelial cell surface may facilitate the non-specific binding of Xlt. This mode of action may be similar to that observed for the Bti cytolytic deltaendotoxin, Cyt1A, which directly interacts with membrane lipids to create pores (Knowles et al., 1989 and Butko 2003).
5. Conclusion Xlt displays characteristics that are favorable for its deployment as a larvicide. It is stable to heat, UV irradiation and proteases (Ensign et al., 2014), characteristics not observed in other bacterial derived insect toxins. The relative stability observed in the lab suggests that the molecule will be more environmentally stable. Xlt appears to display a different mode of action from other biological insecticides such as the Bti delta-endotoxins. The synergistic effect of applying multiple insecticides with varying modes of action, such as Bti delta endotoxins and Xlt, could substantially enhance mosquito larvicidal activities and delay development of resistance in the
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϴ
field (Gill et al., 1992). Indeed, higher efficacy is observed when using mixtures of insecticides with varying modes of actions rather than a single compound (Poopathi 2012).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϭϵ
Acknowledgement We thank Dr. Jon Roll and Dr. Justin Clements for generously providing A. mellifera and L. decemlineata used in this study. We also thank Dr. Yeon Soo Han and Dr. Yong Hun Jo for their technical assistance in confocal microscopy. This study was supported by the College of Agricultural and Life Sciences, the Graduate School, University of Wisconsin-Madison and the National Science Foundation - East Asia and Pacific Summer Institutes for U.S. Graduate Students Fellowship.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϬ
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Zhuang, Z., Linser, P. J., & Harvey, W. R. (1999). Antibody to H (+) V-ATPase subunit E colocalizes with portasomes in alkaline larval midgut of a freshwater mosquito (Aedes aegypti). Journal of Experimental Biology, 202(18), 2449-2460.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϰ
Figure legends Fig.1. Weights of 2nd instar M. sexta after feeding on artificial diets treated with different concentrations of Xlt for 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. Data shown are mean weight ± SD. One-way ANOVA (p = 0.3776) and Tukey’s multiple comparisons test (p>0.05) indicated that the increase in weights of all Xlt treated larvae did not differ significantly from those of the untreated larvae. Fig.2. Nucleic acid staining of Xlt treated Ae. aegypti (Aag-2), D. melanogaster (S2) and M. sexta (GV1) cells using SYTOX® Green. Representative light and fluorescent microscopic images of untreated Aag-2 (A and B), S2 (C and D), and GV1 (E and F) and Xlt (2 ppm) treated Aag-2 (G and H), S2 (I and J) and GV1 (K and L) are shown. Images were captured 6 h post-treatment. Cytotoxicity induced by Xlt, indicated by green fluorescence, was observed in Xlt treated Aag-2 cells. The experiment was repeated four times with three replications in each experiment (400x magnification). Fig.3. Viability of Ae. aegypti (Aag-2), Ae. albopictus (C6/36), human skin fibroblast (Hs68), and human mast (HMC-1) cells treated with different concentrations of Xlt for 24 h. The percent cell viability is calculated as the number of cells in the treated samples divided by the number of cells in the untreated sample. Data are shown as the mean ± SD based on four independent replications. Tukey’s multiple comparisons test indicated that the viability of cells treated with every tested concentration of Xlt (1, 5, 10, 50, and 100 ppm) was significantly different from that
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϱ
of the untreated cells (***, p<0.05) for Aag-2 and C6/36 (A and B). For Hs68 (C), viability of cells treated with 50 and 100 ppm of Xlt was significantly different from that of the untreated cells (***, p<0.05) for Hs68 (C) and for HMC-1 (D), the viability of 10, 50 and 100 ppm of Xlt treated cells was significantly different from that of the untreated cells (***, p<0.05). Fig.4. pH changes in Ae. aegypti larval midguts induced by Xlt treatment. Early 4th instar Ae. aegypti were treated with Xlt (2 and 6 ppm) and stained with 0.04% w/v m-cresol purple to detect pH changes. Images were captured at 2 (A, C, E) and 6 h (B, D, F) post-treatment. Midguts from untreated larvae (A and B) or larvae treated with 2 ppm (C and D) or 6 ppm (E and F) of Xlt are shown. The experiment was repeated three times and panels show representatives of a total of ninety insects that were dissected and examined (Size bar = 0.8 mm). AM, anterior midgut; PM, posterior midgut. Fig.5. Bti endotoxin- and Xlt-treated late 3rd instar An. gambiae and Ae. aegypti stained with SYTOX® Green. Larvae were treated with 2 ppm of Bti and Xlt and observed after 2 h. Representative light and green fluorescence images of untreated (A and B), Bti (C and D), and Xlt (E and F) treated An. gambiae larva and Bti (G and H) and Xlt (I and J) treated Ae. aegypti larva are shown. Green fluorescence is not observed in untreated larva (B). Green fluorescence (indicated by the white arrow) is distributed throughout the midgut in the Bti treated An. gambiae and Ae. aegypti larva (D and H). Green fluorescence is highly localized in the anterior midgut in the Xlt treated An. gambiae and Ae. aegypti larva (F and J). The experiment was repeated five times and images
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϲ
are representative of fifty larvae per experiment that were examined (16 x magnification). AT, anterior ; PT, posterior. Fig.6. Confocal microscopy images of Xlt-treated late 3rd instar Ae. aegypti larval midguts. Representative confocal images of untreated anterior (A), central (B) and posterior (C) midgut and Xlt (2 ppm) treated anterior (D), central (E) and posterior (F) midguts are shown. Dissected midguts were treated with a 1:100 dilution of Xlt-specific antibody and a 1:200 dilution of Alexa- 555®, and then observed for the presence of immunoreactions, indicated by the red fluorescence. The experiment was repeated five times and a total of fifty larvae were dissected and examined (Size bar = 50 µ m). AM, anterior midgut; CM, central midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϳ
Table 1 Insecticidal activity of Xlt against various insect species.
Insect species Aedes aegypti larvaec Aedes aegypti pupae Aedes aegypti adult (female) Culex pipiens larvae Anopheles gambiae larvae Apis mellifera adult Leptinotarsa decemlineata adult Manduca sexta larvae
LC50 (ppm) (95% FL)a 1.81 (1.47-2.13) N/Td N/T 1.25 (0.32-3.09) 1.86 (0.93-2.72) N/T N/T N/T
Slope (SEM)b 4.11 (0.48) -e 0.78 (0.10) 2.27 (0.47) -
a 95% fiducial limits as determined by probit analysis b Slope and standard error (SEM) calculated by probit analysis c Late 3rd instars were used for mosquito bioassays d N/T: Not toxic (unable to determine LC50 using 100 ppm of Xlt as the highest concentration) e -: not applicable
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϴ
Fig.1. Weights of 2nd instar M. sexta after feeding on artificial diets treated with varying concentrations of Xlt for 48 h. Twelve M. sexta larvae were used per treatment in three independent replications. Data shown are mean weights ± SD. One-way ANOVA (p = 0.3776) and Tukey’s multiple comparisons test (p>0.05) indicated that the increase in weights of all Xlt-treated larvae did not differ significantly from those of the untreated larvae.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϮϵ
Aag2 untreated
S2 untreated
GV1 untreated
A
C
E
B
D
F
Aag-2 Xlt treated
S2 Xlt treated
GV1 Xlt treated
G
I
K
H
J
L
Fig.2. Nucleic acid staining of Xlt treated Ae. aegypti (Aag-2), D. melanogaster (S2) and M. sexta (GV1) cells using SYTOX® Green. Representative light and fluorescent microscopic images of untreated Aag-2 (A and B), S2 (C and D), and GV1 (E and F) and Xlt (2 ppm) treated Aag-2 (G and H), S2 (I and J) and GV1 (K and L) are shown. Images were captured 6 h post-treatment. Cytotoxicity induced by Xlt, indicated by green fluorescence, was observed in Xlt treated Aag-2 cells. The experiment was repeated four times with three replications of each experiment (Size bar = 50 µm).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϬ
A
C
Aag-2
Hs68
B
C6/36
D
HMC-1
Fig.3. Viability of Ae. aegypti (Aag-2), Ae. albopictus (C6/36), human skin fibroblast (Hs68), and human mast (HMC-1) cells treated with varying concentrations of Xlt for 24 h. The percent cell viability is calculated as the number of cells in the treated samples divided by the number of cells in the untreated sample. Data are shown as the means ± SD based on four independent replications. Tukey’s multiple comparisons test indicated that the viability of cells treated with every tested concentration of Xlt (1, 5, 10, 50, and 100 ppm) was significantly different from that of the untreated cells (***, p<0.05) for Aag-2 and C6/36 (A and B). For Hs68 (C), viability of cells treated with 50 and 100 ppm of Xlt was significantly different from that of the untreated cells (***, p<0.05) for Hs68 (C) and for HMC-1 (D), the viability of 10, 50 and 100 ppm of Xlt treated cells was significantly different from that of the untreated cells (***, p<0.05).
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϭ
Untreated
6 ppm treated
2 ppm treated
2h AM PM A
C
E
B
D
F
6h
Fig.4. pH changes in Ae. aegypti larval midguts induced by Xlt treatment. Early 4th instar Ae. aegypti were treated with Xlt (2 and 6 ppm) and stained with 0.04% w/v m-cresol purple to detect pH changes. Images were captured at 2 (A, C, E) and 6 h (B, D, F) post-treatment. Midguts from untreated larvae (A and B) or larvae treated with 2 ppm (C and D) or 6 ppm (E and F) of Xlt are shown. The experiment was repeated three times and panels show representatives of a total of ninety insects that were dissected and examined (Size bar = 0.8 mm). AM, anterior midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϮ
Untreated An. gambiae
Bti treated An. gambiae
Xlt treated An. gambiae
AM
A
PM
C
E
AM
PM B
D
F
Bti treated Ae. aegypti
Xlt treated Ae. aegypti
G
I
H
J
Fig.5. Bti endotoxin- and Xlt-treated late 3rd instar An. gambiae and Ae. aegypti stained with SYTOX® Green. Larvae were treated with 2 ppm of Bti and Xlt and observed after 2 h. Representative light and green fluorescence images of untreated (A and B), Bti (C and D), and Xlt (E and F) treated An. gambiae larva and Bti (G and H) and Xlt (I and J) treated Ae. aegypti larva are shown. Green fluorescence is not observed in untreated larva (B). Green fluorescence (indicated by the white arrow) is distributed throughout the midgut in the Bti treated An. gambiae and Ae. aegypti larva (D and H). Green fluorescence is highly localized in the anterior midgut in the Xlt treated An. gambiae and Ae. aegypti larva (F and J). The experiment was repeated five times and images are representative of fifty larvae per experiment that were examined (Size bar = 800 µm). AT, anterior; PT, posterior.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϯ
A
B
C AM
PM Untreated AM
D
CM
E
F AM
Xlt treated AM
CM
PM
Fig.6. Confocal microscopy images of Xlt-treated late 3rd instar Ae. aegypti midguts. Representative confocal images of untreated anterior (A), central (B) and posterior (C) midgut and Xlt (2 ppm) treated anterior (D), central (E) and posterior (F) midguts are shown. Dissected midguts were treated with a 1:100 dilution of Xlt-specific antibody and a 1:200 dilution of Alexa- 555®, and then observed for the presence of immunoreactions, indicated by the red fluorescence. The experiment was repeated five times and a total of fifty larvae were dissected and examined (Size bar = 200 µm). AM, anterior midgut; CM, central midgut; PM, posterior midgut.
<ŝŵĞƚĂů͘:ŽƵƌŶĂůŽĨ/ŶǀĞƌƚĞďƌĂƚĞWĂƚŚŽůŽŐLJϯϰ
Highlights •
Potential for bacterial lipopeptide (Xlt) as a mosquito larval control agent
•
Lipopeptide produced by Xenorhabdus innexi is orally toxic to mosquito larvae
•
Xlt alters larval midgut pH and cause cytotoxicity in the anterior midgut
•
Xlt has a different mode of action from Cry toxins in its pathogenicity
Graphical abstract