Lyophilized yeast-encapsulated orange oil (YEOO) was prepared as previously described12. Briefly, Citrus sinensis essential oil (orange oil, California origin, Sigma- Aldrich, St. Louis, USA) was encapsulated into S. cerevisiae (Red Star fresh baker’s yeast).
Orange oil, water, and yeast were combined in a baffled flask and agitated for 24 h at 40˚C. The resulting mixture was then centrifuged, the supernatant discarded, and the remaining precipitate was subsequently washed three times to remove excess oil and lyophilized prior to storage. The dried larvicide was reconstituted in water between 4.4 – 5% oil in solution prior to bioassay application.
Larval strains and bioassays
Larvae used in our studies were obtained from mosquito colonies maintained at Uniformed Services University of the Health Sciences (USUHS). Ae. aegypti (Liverpool strain – AAE-L and Washington, D.C. strain – AAE-DC strains) and Anopheles gambiae (G3 strain - AGA) were tested in our bioassays and the AAE-L strain was used in all other experiments. Larvae were kept in DI water at 28°C and were fed with fish food ad libitum. Larvae bioassays were performed according to the standard WHOPES protocol18. Briefly, 25 third instar (L3) larvae were placed into cups with 100 ml of DI water. After a 30 min acclimation, different quantities of YEOO larvicide were added to each cup to reach concentrations ranging from 2.5 mg/L to 60 mg/L, and lethality was assessed after 24 hours. In the experiments described here, concentrations of 5, 10, and 30 mg/L of YEOO correspond to LC20, LC50, and LC90, respectively, and were based on our previous results12 using L3 stage AAE-L. The effects of YEOO on AAE-L larval swimming behavior and lethality can be seen in Supplemental Videos 1-3.
Gene Expression Analyses. Individual live whole larvae or guts and carcasses dissected from AAE-L L3 were collected following 1, 4, 8, and 24 h exposure to 10 or 30 mg/L (equivalent to LC50 or LC90, respectively) of YEOO. Samples were placed into 30 µl of RNAlater (Thermo Scientific, Waltham, MA), and frozen at -80°C until RNA extraction. Genes associated with apoptosis (IAP1 and IAP2), autophagy (ATG1, ATG6, and ATG8) and innate immune response genes (Vein, Pirk, Serpin-1, Serpin-2, IMP2, CASPS7 and CASPS8) were assessed in two independent experiments using RNA samples (in triplicate) obtained from pools of three L3 dissected guts or carcasses for each of the four time points. Here, gut samples were composed of foregut and midgut whereas carcasses included the hindgut, the Malpighian tubules and all remaining larvae-matched body parts such as head, thorax, and abdomen. In contrast, for cytochrome P450 gene expression analysis, one whole L3 individual was used per sample (up to eight samples) for each of the three independent experiments (i.e., three replicates) for both 5 mg/L (LC20) and 10 mg/L (LC50) YEOO concentrations tested after 4 h exposure. Regarding P450, we assessed genes representative of families CYP6 (CYP6M11, CYP6N12, and CYP6Z8) and CYP9 (CYP9J10 and CYP9M9). Individual whole larvae were used to determine the systemic insecticide resistance response to YEOO to determine individual gene response variation. In addition, for the P450 analysis specifically, we were not interested in ascertaining spatial expression profiles.
RNA extraction and cDNA Synthesis. AAE-L total RNA was isolated using the RNeasy tissue kit (Qiagen, Hilden, Germany) followed by DNase treatment using TURBO DNA-free kit (Invitrogen, Carlsbad, CA). Each RNA sample was quantified on NanoDrop (Thermo Scientific, Waltham, MA) and assessed on 1% agarose-5% formaldehyde in 1x MOPS. RNA samples were stored at -80°C until use. First-strand cDNAs were obtained using Superscript III reverse transcription kit (Invitrogen) following manufacture protocols with oligo dT12-20 primers and 200 ng of each RNA.
Quantitative Real-Time PCR. mRNA levels were quantified with PowerUp SYBR Green Supermix (Applied Biosystems, Foster City, CA) using 10 ng of cDNA with a 50°C for 2 min and 95°C for 2 min hot start initialization, followed by 40 cycles of 60°C for 5 sec and 95°C for 15 sec using a ABI7500 FAST (Applied Biosystems, Foster City, CA). Relative fold changes were assessed using the ∆∆Ct method19 and calibrated against the expression of the housekeeping gene Actin6 (Supplemental Table 1) in L3 larvae control samples fed on blank yeast. Primer sequences for each gene measured are listed in Supplemental Table 1.
Confocal Microscopy. Confocal microscopy was used to assess the distribution of YEOO throughout the larva alimentary canal. Whole guts from AAE-L L3 larvae were dissected after exposure to either 30 mg/L YEOO or blank yeast control (30 mg/L) in PBS and fixed for 15-30 min at room temperature with Zamboni’s Fixative20 (4% paraformaldehyde). For experiments focused on the permeability of the type 2 peritrophic matrix (PM2), 20 L3 larvae were exposed to blank yeast control (30 mg/L) or 30 mg/L YEOO in 10 ml DI water at 28°C supplemented with FITC-dextran (Cell Signaling Technology, Danvers, MA) (Molecular weights of 150, 500, or 2,000 kDa) at 0.5 mg/ml for 4 h. Fixed guts were washed 3x in PBS, stained for 5 min with 10 µg/ml of DAPI (Invitrogen, Carlsbad, CA) and washed 3x in PBS before imaging on a ZEISS 710 Two Photon (Carl Zeiss AG, Hoberkochen, Germany) confocal microscope. The permeability of larval PM2 was qualitatively assessed by presence/absence of fluorescein signal in the caeca of dissected guts/larvae21. For microscopy, entire alimentary canals were used for viewing clarity.
TUNEL Analysis. TUNEL was used to assess apoptosis via DNA fragmentation. AAE-L L3 larvae were exposed to either YEOO (10 mg/L) or yeast blank for 24 h. Guts dissected from insects were fixed for 15-30 min at room temperature with Zamboni’s Fixative, washed three times (5 min each) in PBS, twice (2 min each) in PBS containing 0.3% Triton X-100 (PBST), and stained for TUNEL analysis (Roche, Basel, Switzerland) following manufacturer’s protocol. Samples were counterstained with 10 µg/mL of DAPI (Invitrogen, Carlsbad, CA), mounted onto slides in Vectashield→ (Vector Laboratories, Burlingame, CA), and subsequently imaged on a ZEISS 710 Two Photon (Carl Zeiss AG, Hoberkochen, Germany) confocal microscope.
Transmission Electron Microscopy. TEM was used to assess cellular damage caused by YEOO ingestion and to verify the structural integrity of YEOO. AAE-L larvae were exposed to either blank yeast control (30 mg/L) or 30 mg/L YEOO. Following 4 h exposure, guts were dissected and fixed overnight at room temperature in freshly prepared 2% formaldehyde and 2% EM grade glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.1M cacodylate buffer, pH 7.2. Fixed guts were washed three times in cacodylate buffer (without aldehydes) and incubated in 2% OsO4 in 0.1M cacodylate buffer for 1 h, After another round of three washes 10 min each in cacodylate buffer, guts were dehydrated in a graduated series of ethanol (10 min each in 30%, 50%, 70%, and 95% ethanol and 2 x 10 min in 100% ethanol), infiltrated in a graduated series of Spurr's epoxy resin (Electron Microscopy Sciences), and then polymerized at 70°C for 11 h. Polymerized blocks were sectioned in a Leica UC6 ultramicrotome. Thin sections of guts cut transversely and longitudinally were collected on 3 mm copper grids. Grids were post-stained in a Leica EM AC20 grid stainer and then examined on a JEOL JEM-1011 transmission electron microscope (JEOL USA, Peabody, MA). Images were collected on an Advanced Microscopy Techniques (AMT Corp., Woburn, MA) digital camera.
Statistical Analysis. Larvae bioassay data to determine LC50 and LC90 YEOO effective concentrations were determine with Nonlinear or Probit regression analyses22. Gene expression data of dissected guts and carcasses were subjected to two-way analysis of variance (ANOVA) with repeated measures, with Dunnett’s multiple comparisons tests and p-values evaluated at α = 0.05, 0.01, and 0.001. In whole larvae P450 gene expression analyses, data were subjected to one-way ANOVA, and Tukey’s Post-Hoc tests with p-values evaluated at α = 0.05, 0.01, and 0.001. Statistics were carried out in SAS 9.4 (Cary, NC) or GraphPad Prism8 (San Diego, CA).