Validamycin A Delays Development and Prevents Flight in Aedes aegypti (Diptera: Culicidae)

Abstract

Trehalose is a disaccharide that is the major sugar found in insect hemolymph fluid. Trehalose provides energy, and promotes growth, metamorphosis, stress recovery, chitin synthesis, and insect flight. The hydrolysis of trehalose is under the enzymatic control of the enzyme trehalase. Trehalase is critical to the role of trehalose in insect physiology, and is required for the regulation of metabolism and glucose generation. Trehalase inhibitors represent a novel class of insecticides that have not been fully developed. Here, we tested the ability of trehalose analogues to function as larvacides or adulticides in an important disease vector—Aedes aegypti. We show that validamycin A, but not 5-thiotrehalose, delays larval and pupal development and prevents flight of adult mosquitoes. Larval mosquitoes treated with validamycin A were hypoglycemic and pupae had increased levels of trehalose. Treatment also skewed the sex ratio toward male mosquitoes. These data reveal that validamycin A is a mosquito adulticide that can impair normal development of an important disease vector.

Trehalose is a nonreducing disaccharide that is the major sugar found in insect hemolymph fluid (Wyatt and Kale 1957). Trehalose provides energy, and promotes growth, metamorphosis, stress recovery, chitin synthesis, and insect flight (Katagiri et al. 1998; Xia et al. 2002; Wegener et al. 2003, 2010; Liebl et al. 2010; Thorat et al. 2012; Tatun et al. 2014; Shukla et al. 2015; Tang et al. 2017; Wolber et al. 2017; Zhang et al. 2017). The hydrolysis of trehalose is under the enzymatic control of the enzyme trehalase. Trehalase is critical to the role of trehalose in insect physiology, and is required for the regulation of metabolism and glucose generation (Shukla et al. 2015).

Previous research has shown that trehalose is synthesized in the fat body by conserved enzymes (Murphy and Wyatt 1964, Becker et al. 1996). This stored energy reserve is hydrolyzed by trehalase to meet the energy demands for development and flight (Wegener et al. 2010). There are two forms of insect trehalase. Trehalase 1 (Tre-1) is soluble and has been purified from hemolymph, midgut goblet cells, and eggs. Trehalase 2 (Tre-2) is a membrane bound form and has been identified in flight muscle, follicle cells, ovary cells, spermatophore, midgut, and brain tissue (Shukla et al. 2015). Trehalase is the only enzyme responsible for the hydrolysis of trehalose, which makes this an attractive molecular target.

Trehalose is a critical sugar and inhibiting its metabolism is deadly for insects (Shukla et al. 2015). RNAi studies have shown that silencing of trehalase leads to weight loss, defects in normal growth and development, and death in brown plant hopper (Laodelphax striatellus) (Hemiptera: Delphacidae) (Chen et al. 2010b). RNAi studies also led to abnormal development in beet armyworm (Spondoptera exigua) (Lepidoptera: Noctuidae) (Chen et al. 2010a). Injection of trehalase inhibitors such as validamycin A and trehazolin into insect larvae also led to unsuccessful pupation and lethal metamorphosis into the adult (Xia et al. 2002; Liebl et al. 2010; Wegener et al. 2003, 2010). Inhibition of trehalose metabolism also prevented oogenesis in silkworm (Bombyx mori) (Lepidoptera: Bombycidae) (Katagiri et al. 1998). Inhibition of trehalose metabolism led to hypoglycemia in flight muscles, and interfered with normal chitin synthesis in Locusta migratoria (Orthoptera: Acrididae) (Wegener et al. 2003, 2010; Liebl et al. 2010). Drosophila melanogaster (Diptera: Drosophilidae) and mosquitoes also use trehalose as a stress protectant (Thorat et al. 2012), and validamycin A has been shown to inhibit flight of Aedes aegypti when treated as larvae (Logan 2005).

Trehalose metabolism is an obvious target for the development of new pesticides. This sugar is not synthesized by humans or other vertebrates, and trehalase deficiency is a mild disease that can be resolved through small changes in diet such as not eating mushrooms (Richards et al. 2002). Trehalase inhibitors represent a targeted strategy that will have minimal to nonexistent effects in humans and other vertebrates. Trehalose is actively used by bacteria, fungi, and arthropods (Arguelles 2014). Validamycin A is a trehalase inhibitor that functions as an antibiotic and fungicide that has been used against soil-borne diseases such as controlling Rhizoctonia solani (Cantharellales: Ceratobasidiaceae) in rice, potatoes, and vegetables as well as damping off diseases in vegetable seedlings (Asano et al. 1987). The larger ecological impact of trehalase inhibitors on ecological systems has not been evaluated.

Several trehalose mimetics and analogues have been proposed as insecticides due to the critical role of trehalose in insect physiology (Shukla et al. 2015, O’Neill et al. 2017). These inhibitors are chemically iminosugars or pseudosaccharides. They are each competitive inhibitors of the catalytic site of trehalase, which can block trehalase activity in the micromolar to submicromolar range (Shukla et al. 2015). A promising trehalose mimetic is 5-thiotrehalose, which was effective at preventing trehalose utilization in Clostridium difficile (Danielson et al. 2019). We hypothesized that trehalose mimetics would be effective as larvacides or adulticides in a disease vector.

Aedes aegypti is the primary vector of a number of important global human pathogens including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) (Conway et al. 2014). There are no targeted antivirals for these viruses and the currently licensed DENV vaccine is restricted to a small subset of the human population due to the potential of eliciting antibody-dependent enhancement (ADE) (Conway et al. 2014, Sridhar et al. 2018). There is a need for new and safe insecticides. Emerging resistance to carbamates, organochlorines, organophosphates, and pyrethroids has been detected in the Americas, Africa, and Asia (Moyes et al. 2017).

Here we show that trehalase inhibitor validamycin A, but not 5-thiotrehalose, delays Ae. aegypti larval and pupal development and prevents flight of adult mosquitoes. Validamycin A treatment reduced glucose levels in larvae and increased trehalose levels in pupae suggesting that the delay in development and flight was at least partially due to hypoglycemia. Validamycin A treatment was most effective when mosquitoes were treated at the egg or early larval stage and ineffective once mosquitoes developed into pupae. Validamycin A also skewed the sex ratio toward male mosquitoes perhaps due to the increased energy demand of producing female mosquitoes. These data indicate that trehalase inhibitors such as validamycin A can be used as adulticides that can be applied directly to the larval environment and prevent the development of an important disease vector.

Materials and Methods

Mosquitoes and Reagents

Aedes aegypti (Rockefeller strain) was provided by the Connecticut Agricultural Experiment Station (New Haven, CT). Adult mosquitoes were maintained using a sugar solution at 27°C and 80% humidity according to standard rearing procedures, which included 12:12 (L:D) h photoperiods. Mosquitoes fed on anesthetized retired breeder mice for egg production (Central Michigan University IACUC approval # 18-30). Eggs were collected on filter paper and stored for 2 wk prior to use in experiments. Validamycin A was obtained from Cayman Chemicals (Ann Arbor, MI). Trehalose was obtained from Millipore Sigma (Burlington, MA). Validamycin A is soluble in water up to 140 g/liter. Trehalose is soluble in water up to 50 g/liter.

Egg, Pupation, and Eclosure Assays

Three containers of approximately 100 eggs were hatched in 200 ml rearing media for each experimental condition. Rearing media was prepared by making a 2% solution of 3:2 liver powder (Perfect supplements):active dry yeast (Red Star) in reverse osmosis water. Experimental conditions included complete rearing media, rearing media with liver powder alone, complete rearing media with 0.1, 0.2, and 0.5 mg/ml validamycin A, complete rearing media with 0.2 mg/ml trehalose, and complete rearing media with 0.1 mg/ml 5-thiotrehalose. One experiment hatched eggs and fed larvae with rearing media with liver powder alone. Larvae were treated with the above experimental conditions until they developed into pupae. In addition, larvae were fed with 5 ml of 1% complete rearing media every other day until all larvae developed into pupae. Larvae and pupae developed in the same environment as described for adult mosquitoes including 27°C and 80% humidity and 12:12 (L:D) h photoperiods. Percent hatch was determined for up to 7 d for each experimental condition. Percent hatch was calculated by dividing the total number of larvae at any developmental stage by the total number of eggs added to the rearing media. Percent pupation was determined until all larvae developed into pupae or until the remaining larvae died. Percent pupation was calculated by dividing the total number of pupae that developed by the total number of larvae that developed in a container. Percent eclosure was determined until all pupae developed into adults. Percent eclosure was calculated by dividing the total number of adults that developed by the total number of pupae that developed in a container. Percent of flying mosquitoes was determined by collecting and counting flying mosquitoes and confirming this observation by counting the number of adults that remained on the surface of the water.

Synthesis of 5-Deoxy-5-Thio-α,α-D-Trehalose (5-ThioTre)

The synthesis of 5-ThioTre was carried out using TreT catalysis, essentially as previously described (Danielson et al. 2019). To a 15 ml conical tube was added 20 mM 5-thio-D-glucose (15.4 mg, CarboSynth), 40 mM UDP-glucose (Abcam, Cambridge, MA), and 20 mM MgCl₂. TreT in Tris buffer (50 mM Tris, 300 mM NaCl, pH 8.0), plus additional Tris buffer if needed, were added to achieve a final volume of 4 ml and a final protein concentration of 10 µM. The reaction was incubated at 70°C with shaking at 300 rpm for 1 h, then the tube was cooled by placing it on ice. An Amicon Ultra-15 centrifugal filter unit (nominal molecular weight limit 10 kDa) was prerinsed with 3 ml deionized (DI) water three times by centrifugation at 3,900 × g for 20 min to remove trace glycerol in the membrane. After transferring the cooled enzymatic reaction mixture to the prerinsed centrifugal filter unit, it was spun at 3,900 × g for 20 min. The upper chamber of the centrifugal filter unit was rinsed two times with 3 ml of DI water and centrifuged again using the same speed and time. After discarding the upper chamber of the centrifugal filter unit, mixed-bed ion-exchange resin (3 g of Bio-Rad Bio-Rex RG 501-X8) was added to the tube and stirred for 1 h at room temperature. Next, the supernatant was filtered. The remaining resin was rinsed two times with 5 ml of DI water and the supernatant was filtered and combined with the rest of the product. Thin later chromatography was performed using n-butanol/EtOH/DI water 5:3:2 to assess conversion of the glucose analogue starting material to the trehalose analogue product. The purified product was concentrated by rotary evaporation to give 5-ThioTre (21.8 mg, 77%). ¹H and ¹³C NMR spectra of the product matched the literature.¹ HR ESI MS negative mode: calcd. for C₁₂H₂₂ClO₁₀S [M+Cl]⁻m/z, 393.0622; found, 393.0606. The water solubility of 5-ThioTre is predicted to be very close to trehalose and stocks were generated at 100 mM.

Glucose and Trehalose Assay

Eggs were hatched in complete rearing media in the presence of 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. Three pools of three 4-d-old larvae and recently developed pupae were made for each condition by drying insects a filter paper, and then adding the insects to 100 µl phosphate-buffered saline (PBS). Each pool was homogenized using an Argos Technologies Pestle Motor Mixer, and then passed through a Qiashredder (Qiagen, Hilden, Germany) to remove insoluble debris. The presence of glucose and trehalose in small volumes of crude lysates was assessed using a commercially available trehalose assay kit (Megazyme, Chicago, IL). In this assay, trehalase-catalyzed breakdown of trehalose yields two moles of glucose, which are converted by hexokinase to glucose-6-phosphate (G6P), which in turn is oxidized by G6P dehydrogenase to gluconate-6-phosphate. The latter step involves concomitant reduction of cofactor NADP⁺ to NADPH, which can be spectrophotometrically measured by the increase of absorbance at 340 nm. The assays were performed in clear, flat-bottom 96-well microplates according to the kit instructions with minor modifications. Equal volumes of crude lysate samples were incubated with all assay kit components except trehalase for 1 h at 37°C and absorbance at 340 nm was measured to establish baseline levels of glucose and G6P present in the samples (‘G’ in Fig. 6). Next, trehalase was added to the same samples, which were subsequently incubated at 37°C for 1 h and absorbance at 340 was measured (‘T’ in Fig. 6). Any increase in absorbance after addition of trehalase is due to the presence of trehalose. A PBS control lacking crude lysate was included in the analysis (‘Neg’ in Fig. 6). Absorbance at 340 nm was measured using a Tecan plate reader (Infinite F200 or M200 PRO operated by Tecan iControl software). Linearity of the assay under the conditions used was confirmed.

Fig. 6.

Validamycin A decreased glucose and increased trehalose levels in Aedes aegypti larvae and pupae. (A) 4-d-old larvae and (B) pupae were harvested after treatment with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. Crude lysates were assessed for glucose (G) and trehalose (T) levels using a spectrophotometric assay with the understanding that both glucose and trehalose are detected upon addition of trehalase. A PBS-only negative control (Neg) without crude lysate was included. (C) mM glucose per 4-d-old larva after treatment with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. (D) mM trehalose per pupa after treatment with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. The experiment was performed in triplicate using pools of three larvae or pupae. Student’s t-tests were performed between groups to assess statistical significance (P < 0.05).

Results

Validamycin A Delays Aedes aegypti Development and Prevents Flight

In order to test if validamycin A had larvicidal or adulticidal activity in Ae. aegypti, we treated batches of approximately 100 Ae. aegypti eggs with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A and then quantified percent hatch, pupation, and eclosure. Validamycin A delayed egg hatch, pupation, and eclosure in a dose-dependent manner, and was slightly larvacidal (Fig. 1A–C). No larval mortality was observed at 0 mg/ml validamycin A. In contrast, 7% larval mortality was observed at 0.1 and 0.2 mg/ml validamycin A and 9% larval mortality was observed at 0.5 mg/ml validamycin A. Validamycin A significantly reduced the percent of adult mosquitoes that could fly in a dose-dependent manner and 100% of the mosquitoes reared at 0.5 mg/ml were unable to fly (Fig. 1D and E). Interestingly, the sex ratio of validamycin A treated flying adults was skewed toward males, and no females were identified when mosquitoes were treated with 0.2 mg/ml validamycin A (Fig. 1F).

Fig. 1.

Validamycin A delays development, prevents flight, and skews sex ratio of Aedes aegypti. (A) Percent hatch, (B) percent pupation, and (C) percent eclosure were determined for Ae. aegypti that were treated with 0 (circles), 0.1 (squares), 0.2 (triangles), and 0.5 (inverted triangles) mg/ml validamycin A. Life stages were determined until all mosquitoes completed development or died. (D) Percent of mosquitoes that could fly after treatment with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. Student’s t-tests were performed between groups to determine statistical significance. (E) Representative image of eclosed mosquitoes after treatment with 0 and 0.5 mg/ml validamycin A. (F) Female:male sex ratios observed in adults that could fly after treatment with 0, 0.1, and 0.2 mg/ml validamycin A. There were no flying mosquitoes to quantify after treatment with 0.5 mg/ml. Experiments were performed in triplicate and at least 20 pupae were present in each group.

We then determined if validamycin A was effective after treatment of larvae versus pupae. We reasoned that efficacy against a larger number of developmental stages would increase the utility of validamycin A as an insecticide. Batches of Ae. aegypti eggs were hatched in the absence of validamycin A and then 2-d-old larvae or pupae were treated with 0 and 0.2 mg/ml validamycin A for the duration of their development into adult mosquitoes. We then allowed larvae and pupae to develop into adult mosquitoes and quantified the percent of adults that could fly. Treatment of 2-d-old larvae was effective at preventing flight (Fig. 2A). Treatment of recently developed pupae was not effective at preventing flight (Fig. 2B).

Fig. 2.

Validamycin A is effective during treatment of larvae but not pupae in Aedes aegypti. Percent of Ae. aegypti that could fly when (A) 2-d-old larvae or (B) pupae were treated with 0 and 0.2 mg/ml validamycin A. The experiment was performed in triplicate and at least 20 pupae were present in each group.

Validamycin A is an antifungal and an important nutritional supplement used in our rearing media was Saccharomyces cerevisiae. We tested if the adulticidal activity of validamycin A was due to the killing of S. cerevisiae by rearing batches of eggs with rearing media with liver powder alone or complete rearing media. Larvae were also fed with either rearing media with liver powder alone or complete rearing media. The percent of mosquitoes that underwent pupation and eclosure were slightly delayed when larvae were fed rearing media with liver powder alone, although 100% of adult mosquitoes were able to fly under both conditions (Fig. 3A–D).

Fig. 3.

Removal of yeast nutritional supplement did not delay development or prevent flight in Aedes aegypti. (A) Percent hatch, (B) percent pupation, and (C) percent eclosure were determined for Ae. aegypti that were fed with liver power alone (circles) or liver powder and yeast (squares). Life stages were determined until all mosquitoes completed development or died. (D) Percent of mosquitoes that could fly after being fed with liver powder alone (LP) or liver powder and yeast (LP + Y). The experiment was performed in triplicate and at least 20 pupae were present in each group.

5-Thiotrehalose Does Not Delay Aedes aegypti Development or Prevent Flight

Validamycin A is a potent inhibitor of trehalase in a number of species but failed to block utilization of trehalose by C. difficile. A novel degradation-resistant 5-thiotrehalose compound was able to block utilization of trehalose in C. difficile (Danielson et al. 2019). 5-Thiotrehalose is nearly identical to trehalose other than replacement of a single oxygen with sulfur. We hypothesized that 5-thiotrehalose would be a more effective trehalase inhibitor in Ae. aegypti.

We treated batches of Ae. aegypti eggs with 0 and 0.1 mg/ml 5-thiotrehalose for the duration of development into adult mosquitoes and then quantified the percent of mosquitoes that underwent hatch, pupation, and eclosure. 5-Thiotrehalose had no impact on percent hatch (Fig. 4A). Percent pupation and eclosure were slightly increased when larvae were treated with 0.1 mg/ml 5-thiotrehalose and 100% of adult mosquitoes were able to fly under both conditions (Fig. 4B–D). We also tested trehalose as a negative control and found no difference in the percent of mosquitoes that underwent hatch, pupation, eclosure, the number of adult mosquitoes that could fly (Fig. 5A–D).

Fig. 4.

5-Thiotrehalose did not modify development or flight in Aedes aegypti. (A) Percent hatch, (B) percent pupation, and (C) percent eclosure were determined for Ae. aegypti that were treated with 0 (squares) or 0.1 (circles) mg/ml 5-thiotrehalose. Life stages were determined until all mosquitoes completed development or died. (D) Percent of mosquitoes that could fly after treatment with 0 or 0.1 mg/ml 5-thiotrehalose. The experiment was performed in triplicate and at least 20 pupae were present in each group.

Fig. 5.

Trehalose did not modify development or flight in Aedes aegypti. (A) Percent hatch, (B) percent pupation, and (C) percent eclosure were determined for Ae. aegypti that were treated with 0 (squares) or 0.2 (circles) mg/ml trehalose. Life stages were determined until all mosquitoes completed development or died. (D) Percent of mosquitoes that could fly after treatment with 0 or 0.2 mg/ml trehalose. The experiment was performed in triplicate and at least 20 pupae were present in each group.

Validamycin A Treatment Reduces Glucose and Increases Trehalose in Developing Aedes aegypti

Validamycin A is a potent trehalase inhibitor and we hypothesized that it induces hypoglycemia in developing mosquitoes. We tested the impact of validamycin A treatment on glucose and trehalose concentration in 4-d-old larvae and recently developed pupae by treating batches of Ae. aegypti eggs with 0, 0.1, 0.2, and 0.5 mg/ml validamycin A. Three pools of three 4-d-old larvae and recently developed pupae were made for each condition by drying insects on filter paper, and then adding the insects to 100 µl PBS. Each pool was homogenized, and then passed through a Qiashredder to remove insoluble debris. The Megazyme Trehalose Assay Kit was adapted for using small volumes of crude lysates, and relative concentrations of glucose and trehalose were determined in equivalent volumes of crude lysates. In this assay, trehalase-catalyzed breakdown of trehalose yields two moles of glucose, which are converted by hexokinase to glucose-6-phosphate (G6P), which in turn is oxidized by G6P dehydrogenase to gluconate-6-phosphate. The latter step involves concomitant reduction of cofactor NADP⁺ to NADPH, which can be spectrophotometrically measured by the increase of absorbance at 340 nm. Therefore, this assay detects glucose if no trehalase is added and both glucose and trehalose if trehalase is added. Trehalose can only be detected if the absorbance after trehalase treatment is higher than the absorbance that develops from endogenous glucose. Glucose levels significantly decreased in 4-d-old larvae at all three concentrations (Fig. 6A and C). We were unable to determine trehalose concentration in untreated 4-d-old larvae with confidence because untreated larvae had high levels of endogenous glucose and addition of trehalase did not significantly alter the amount of glucose that was detected (Fig. 6A). Trehalose was detected in treated larvae due to decreases in glucose levels (Fig. 6A). Trehalose was detected in treated pupae and was significantly increased above the untreated control (Fig. 6B and D).

We then converted the difference in raw glucose and trehalose absorbance values to mM in order to determine if the concentration of trehalose that we detected in Ae. aegypti larvae and pupae was consistent with previous literature, which states that trehalose content varies between 5 and 50 mM depending on environmental conditions, physiological state, and nutrition (Shukla et al. 2015). A caveat was that this analysis was only possible in validamycin A-treated mosquitoes because there was no change in absorbance upon addition of trehalase to untreated mosquito lysates. Individual validamycin A-treated larvae and pupae contained an average of 1.2–1.5 mM trehalose.

Discussion

Control of Aedes spp. populations using insecticides has become more difficult due to the emergence of resistance to all four insecticide classes: carbamates, organochlorines, organophosphates, and pyrethroids (Moyes et al. 2017). Aedes spp. have selected mutations that reduce the penetration of insecticides into the insect and enhance enzymatic biodegradation or sequestration (Moyes et al. 2017). Novel insecticides with unique mechanisms of action are needed to integrate into vector control strategies (Shaw and Catteruccia 2019).

Trehalase inhibitors are a novel class of putative insecticides that target a critical enzyme involved in the metabolism of trehalose. Trehalose is the major sugar in hemolymph fluid and is required for mosquito development and flight (Asano et al. 1987; Becker et al. 1996; Katagiri et al. 1998; Chen et al. 2010a,b; Liebl et al. 2010; Shukla et al. 2015). We hypothesized that trehalase inhibitors such as validamycin A would have larvicidal or adulticidal activity.

We showed that validamycin A at concentrations of 0.1–0.5 mg/ml (0.2–1.0 mM) had dose-dependent activity delaying egg hatch, pupation, and eclosure of Ae. aegypti. Validamycin A treatment also had dose-dependent activity on flight of adult mosquitoes, and flight was completely prevented when mosquitoes were reared in 0.5 mg/ml validamycin A. Treated mosquitoes that did fly had a skewed sex ratio toward males, and female mosquitoes were absent when treated with 0.2 mg/ml validamycin A (Hickey and Craig 1966). It is possible that the hypoglycemic conditions induced by validamycin A prevents emergence of females, which are larger than males and may require more energy during eclosure. Previous literature supports that poor nutrient availability can skew the sex ratio toward males in Culex spp. (Alto et al. 2012; Kassim et al. 2012). However, while there was dose-dependent activity on mosquito development, the concentrations of glucose and trehalose in larvae and pupae treated with all three concentrations of validamycin A remained similar. This suggests that another mechanism in addition to hypoglycemia is involved.

Trehalase is expressed in bacteria, fungi, and arthropods (Shukla et al. 2015). This suggests that validamycin A treatment may have negatively influenced mosquito development through promoting dysbiosis of the mosquito microbiome. Trehalase inhibition can impact a wide array of physiological functions in insects including chitin synthesis, stress protection, larval and pupal development (Asano et al. 1987; Katagiri et al. 1998; Wegener et al. 2003, 2010; Chen et al. 2010a; Liebl et al. 2010; Thorat et al. 2012, Tatun et al. 2014, Zhang et al. 2017). Future research is required to determine if multiple biological mechanisms are responsible for validamycin A activity in Ae. aegypti.

We also tested 5-thiotrehalose as a putative insecticide. 5-Thiotrehalose is more structurally similar to trehalose than validamycin A and was effective at preventing trehalose utilization in C. difficile (Danielson et al. 2019). We hypothesized that 5-thiotrehalose, like validamycin A, would also be effective in Ae. aegypti; however, we found that 5-thiotrehalose lacked activity in Ae. aegypti at the concentration tested. It is important to note that while trehalase is highly specific to trehalose, there are differences in affinity of trehalase inhibitors to the trehalase enzymes from different species (Shukla et al. 2015, Danielson et al. 2019). This suggests that improvements can be made on current trehalose mimetics to optimize affinity and delivery to target species.

These data reveal that trehalase inhibitors such as validamycin A negatively impact mosquito development at multiple levels when applied directly to the larval environment. These data are bolstered by the U.S. EPA classification as Toxicity Class IV—practically nontoxic. Further research is needed to optimize trehalose inhibitors for disease vectors and determine how to integrate this new class of insecticides into vector control strategies.