Lure-and-Kill Yeast Interfering RNA Larvicides Targeting Neural Genes in the Human Disease Vector Mosquito Aedes aegypti

New mosquito control strategies are vitally needed to address established arthropod-borne infectious diseases such as dengue and yellow fever and emerging diseases such as Zika and chikungunya, all of which are transmitted by the disease vector mosquito Aedes aegypti. In this investigation, Saccharomyces cerevisiae (baker’s yeast) was engineered to produce short hairpin RNAs (shRNAs) corresponding to the Aedes aegypti orthologs of fasciculation and elongation protein zeta 2 (fez2) and leukocyte receptor cluster (lrc) member, two genes identified in a recent screen for A. aegypti larval lethal genes. Feeding A. aegypti with the engineered yeasts resulted in silenced target gene expression, disrupted neural development, and highly significant larval mortality. Larvicidal activities were retained following heat inactivation and drying of the yeast into tabular formulations that induced >95% mortality and were found to attract adult females to oviposit. These ready-to-use inactivated yeast interfering RNA tablets may one day facilitate the seamless integration of this new class of lure-and-kill species-specific biorational mosquito larvicides into integrated mosquito control programs.

Larviciding is a key component of integrated control and disease prevention strategies targeting Aedes mosquitoes, which breed in water-filled containers located within or close to human dwellings 1 . Given the increase of reported insecticide resistance and rising concern for the negative effects of pesticides on non-target organisms, the current pesticide repertoire is faced with great challenges to sustainability. Due to the lack of vaccines, mosquito control is the primary means of disease control, and new biorational pesticides are vitally needed to address established arthropod-borne diseases such as dengue and emerging infectious diseases such as Zika. Although it is beginning to attract attention in agricultural biotechnology communities 2 , the use of RNA interference (RNAi) is a largely unexplored approach for control of disease vector mosquitoes. While most mosquito researchers use longer (300-400 bp) double stranded RNA (dsRNA) molecules for RNAi, the short length (21-25 bp) of custom small interfering RNAs (siRNAs) facilitates the design of interfering RNA that recognizes genes in targeted organisms, but not in non-target species. We have demonstrated that ingested siRNAs can be used to selectively target Aedes larval genes and have used this technology to characterize mosquito larval development [3][4][5][6][7][8] . These siR-NAs and their short hairpin RNA (shRNA) counterparts may represent a novel class of larvicides with untapped potential for sustainable mosquito control.
Two siRNA molecules, #52 and #101, which correspond to the Aedes aegypti fasciculation and elongation zeta 2 (fez2) and the receptor-encoding ortholog of leukocyte receptor cluster (lrc) member genes, respectively, were identified in recent screens for mosquito larval lethal genes, which will be described in their entirety elsewhere. These two interfering RNAs were prioritized, due to their high larvicidal activities and lack of conserved target sites in humans and other non-target organisms, for further characterization. This research investigation examines the hypothesis that Saccharomyces cerevisiae (baker's yeast) genetically engineered to express interfering

Results and Discussion
S. cerevisiae expressing shRNA targeting larval lethal genes effectively kills A. aegypti larvae.
Given the significant larvicidal capacity of interfering RNAs #52 and #101 observed in conjunction with multiple delivery systems, these RNAs were selected for evaluation in a yeast delivery system. Pichia pastoris has been used for delivery of recombinant DNA and dsRNA to Aedes 12,13 . S. cerevisiae was chosen for our investigation, as many genetic strains and plasmids are available for this genetic model organism. Furthermore, it is used very broadly for commercial applications, which might facilitate commercialization of this putative intervention in the future. shRNAs corresponding to the #52 and #101 target sequences were constitutively expressed from a non-integrating multi-copy yeast shuttle plasmid that was transformed into S. cerevisiae. These yeast strains, as well as control yeast expressing shRNA with no known target in A. aegypti, were fed to larvae in an agarose gel-covered formulation. While control-fed larvae survived (Fig. 1d,e), gel-coated live yeast expressing shRNA larvicides #52 and #101 killed 83 ± 7% (P = 0.0001 vs. control yeast interfering RNA treatment) and 93 ± 4% (P = 0.0001 vs. control yeast interfering RNA treatment) of mosquitoes, respectively (Fig. 1d). Longer pieces of dsRNA (~200 bp) targeting Drosophila suzukii genes have been used to silence target genes in S. cerevisiae 14 . Our discovery that short hairpins with 25 bp target sequences can induce larval death is useful, as it facilitates the design of interfering RNA pesticides with little potential to affect non-target species.
Whyard et al. 15 reported that dsRNA expressed in E. coli maintained its activity following heat-killing of the bacteria. The impact of heat-killing the yeast prior to delivery of shRNA larvicides, which is unknown, was therefore explored. Heat-inactivated gel-coated yeasts #52 and #101 induced 91 ± 3% (P = 0.0001 vs. control treatment) and 93 ± 3% (P = 0.0001 vs. control treatment) mortality, respectively (Fig. 1E). No significant differences were observed in the larvicidal capacity of live vs. heat-inactivated yeast interfering RNA larvicides (Fig. 1d,e). These results suggest that it will be possible to use heat-inactivated yeast interfering RNA larvicides, a finding that will greatly facilitate the potential use of these larvicides in the field, where the release of live yeast may not be desirable.

Dry inactivated yeast tablets retain larvicidal activity and function as oviposition attractants.
Although agarose-coated yeast larvicides induced high mortality rates (Fig. 1a,b), these wet and gummy formulations are not user-friendly. Moreover, water in containers treated with gel-coated yeast becomes cloudy, which could impact female oviposition behavior and would not be desirable for treatment of potable water. Dried tablets of inactivated S. cerevisiae (Fig. 1g) expressing shRNA (Fig. 1a), which are comparable in appearance to yeast nutritional tablet human dietary supplements, were prepared. This user-friendly formulation does not cloud the water and sinks to the bottom of treated containers, where A. aegypti larvae readily consume it. When used to treat 50 ml water in containers with 20 larvae, a 95 mg dried tablet of yeast larvicides #52 or #101 induced 88 ± 4% and 85 ± 5% larval mortality, respectively ( Fig. 1f; P = 1.0 × 10 −5 ). These tablets (LD50 #52 = 31.07 mg; LD50 #101 = 33.41 mg; Table 1a, Supplementary Fig. S2a,b), which effectively kill larvae in containers with larger volumes of water and varying larval densities (Table 1b), maintain significant residual activity after one week of submersion in water (Table 1c; P = 1.0 × 10 −5 ).
Active yeast, which releases carbon dioxide, is known to attract gravid female mosquitoes 9,10 , but the impact of dried inactivated yeast interfering RNA tablets on oviposition container choice is unknown. A. aegypti oviposition choice experiments demonstrated that in comparison to ovicups with water alone, gravid females deposit significantly more eggs in ovicups containing inactivated dry yeast tablets ( Fig. 1h; P = 0.00131). These data suggest that inactivated dry yeast could be used as lure-and-kill larvicides that attract both A. aegypti larvae and gravid adult females. Larval mortality induced by interfering RNA larvicides #52 and #101. (a) siRNA #52 was identified in a chitosan nanoparticle larvicide screen in which it induced significant larval death; control larvae that ingested siRNA nanoparticles with no known Aedes target site survived. The screen was performed in duplicate (10 animals/treatment). (b) siRNA larvicide #101 was identified in a soaking screen. Although control siRNA-treated animals lived, significant larval mortality was observed in larvae soaked in siRNA #101 or #52; experiments were performed in triplicate with 20 animals/treatment and evaluated with Fisher's exact test. (c) Significant mortality was observed in larvae fed with heat-inactivated bacteria expressing dsRNA corresponding to the siRNA #52 or #101 target sites, while animals fed a normal lab diet (WT) or control dsRNA bacteria survived (results were compiled from seven replicate experiments with 20 animals/treatment and assessed by ANOVA with Tukey's multiple comparison test). Significant larval death was observed in larvae fed with gelcoated active (d), gel-coated heat-inactivated (e), or heat-inactivated dried (f) yeast larvicides #52 and #101; WT larvae or larvae fed yeast expressing control shRNA survive. No significant differences were observed in larvae fed a WT vs. control interfering RNA diet (d-f; not shown in a-c) or in the larvicidal capacity of active vs. heat-inactivated or gel-coated vs. dry yeast interfering RNA larvicides #52 or #101 (d-f). Results compiled from two (d) or three (e,f) biological replicates with four replicate containers of 20 larvae/replicate were analyzed by ANOVA with Tukey's multiple comparison test. Ready-to-use dried and heat-inactivated yeast interfering RNA tablets (g, penny shown for scale) could be seamlessly integrated with current vector control strategies. (h) 10 gravid adult females deposited significantly more eggs in ovicups containing rain water with dried yeast interfering RNA tablets (470 ± 44 eggs laid) vs. rain water alone (248 ± 34 eggs laid); data (4 replications with 5 repetitions, n = 20) were analyzed with a paired t-test. Data are represented as mean % mortality, and error bars represent standard errors of the mean. ***P < 0.001 in comparison to control larvae (a-f) or vs. water alone (h); see text for exact P values.
No obvious external morphological defects or motor deficits were noted in larvicide-treated animals, which grew to normal size, as evidenced by the lack of body length differences in control vs. larvicide-treated animals (P = 0.116) in early L4, just prior to their death as fourth instar larvae ( Supplementary Fig. S2c,d). The prospective target of #52, fez2, is orthologous to unc-76, an evolutionarily conserved cytosolic protein that binds to the kinesin heavy chain. In D. melanogaster, Unc-76 functions in kinesin-mediated transport in neurons. Drosophila loss-of-function mutants display neural defects and die prior to pupation 16 . The prospective target of #101, lrc, is an ortholog of D. melanogaster CG6700, which encodes a conserved protein containing a C-terminal SAC3/ GANP domain that is a component of the Dystroglycan-Dystrophin (Dg-Dys) complex 17,18 and is required for viability 19 . CG6700 was recently included in a catalog of genes likely to function in synapse assembly and function 20 , although its role in this capacity remained to be tested. Given that the Drosophila orthologs of fez2 and lrc have been implicated in fly neural development and function, the impact of silencing these genes during larval development was assessed in the A. aegypti larval nervous system during early L4 just prior to the time at which larvicide treated animals die ( Supplementary Fig. S2c,d).
As observed in Drosophila 16 , loss of fez2 results in mortality in late A. aegypti larval development. Although Drosophila larvae lacking function of this gene display obvious locomotor defects 16 Table 1. Analysis of heat-inactivated dried yeast pellet activity. LD50 values and confidence intervals (a), larvicide activities in varying container sizes/larval densities (b), and larvicide residual activities (c) are shown for dried inactivated larvicide #52 and #101 yeast tablets vs. control interfering RNA tablets. For each experiment (treatments at the indicated water volumes/larval densities in b or residual activities at the indicated time points in c), means in the same column followed by different letters are significantly different (P = 1.0 × 10 −5 ). No differences in mortality were noted between control interfering RNA yeast-fed animals (a-c) vs. animals fed a normal laboratory diet that were reared in parallel (not shown). No significant differences were detected between containers treated with larvicides #52 vs. #101 in a-c. SEM = standard error of the mean. Information concerning n numbers for each experiment is detailed in the methods section.
A. aegypti larvae. However, it is possible that detailed tracing and analysis of larval swimming patterns could reveal more subtle defects that might be of higher consequence to wild mosquito larvae. It has been suggested that kinesin-mediated transport needs may increase in neurons during late larval development in flies, at which time both body size and neuron size increase rapidly. This may also explain why fez2-silenced Aedes larvae, like their Drosophila unc-76 mutant counterparts 16 , die during late larval development, at which time fez2-silenced A. aegypti larvae display a lack of synaptic active zones in the brain, a defect which is presumably secondary to a more generalized loss of kinesin-mediated microtubule transport. Likewise, in the absence of lrc, which was predicted to function at the Drosophila synapse 20 , a lack of synaptic active zones in the L4 larval brain is observed. Proteins with the SAC3/GANP domain found in Lrc have a wide variety of cellular functions 16 , and it will be interesting to assess the precise roles of Lrc at the neural synapse in future studies. The Drosophila Dg-Dys complex, like its vertebrate counterparts, is believed to function in the transmission of information from the extracellular matrix to the cytoskeleton, and so Lrc may function in this capacity in the mosquito brain. Furthermore, given that it has been suggested that the Drosophila Dg-Dys complex interacts with Semaphorins 17 , it is interesting to note that a reduction in active zones as evidenced by decreased nc82 staining levels was also reported in A. aegypti larvae in which semaphorin1a had been silenced 5 . Preparation and characterization of yeast interfering RNA larvicides with integrated inducible shRNA expression cassettes. The results obtained with transiently transformed yeast suggest that yeast interfering RNA larvicides could be valuable tools for control of mosquitoes in the field (Fig. 1, Table 1). In preparation for field trials, #52, #101, and control shRNA expression cassettes were stably integrated at both the TRP1 and URA3 loci in the S. cerevisiae genome (Fig. 3a). Generation of these stable transformants eliminates the use of plasmids with antibiotic resistance markers and the potential for horizontal transfer of shRNA expression cassettes. In these strains, the #52, #101, and control shRNA expression cassettes were placed under control of an inducible Gal1 promoter (Fig. 3a), allowing for induction of shRNA expression through galactose inclusion in the media, followed by heat inactivation and harvesting of the yeast. Dry inactivated yeast tablets prepared from these stably transformed #52 and #101 yeast strains induced 97 ± 4% (P = 0.0001 vs. control yeast interfering RNA treatment) and 95 ± 5% (P = 0.0001 vs. control yeast interfering RNA treatment) mortality, respectively, in A. aegypti laboratory strain larvae (Fig. 3b). High levels of mortality were also induced following treatment of larvae hatched from the eggs of an A. aegypti strain recently established from mosquitoes collected in Trinidad (Fig. 3c). In Trinidad larvae, #52 and #101 yeast interfering RNA tablets prepared from stable yeast transformants induced 83 ± 3% mortality ( Fig. 3d; p = 0.000001 vs. control treatment; LD50 = 27.49 mg) and 85 ± 2% mortality ( Fig. 3e; p = 0.000001 vs. control treatment; LD50 = 27.21 mg), respectively. Semi-field trials with these stably transformed inactivated yeast interfering RNA larvicides (Fig. 3) will demonstrate proof of concept of this new tool for mosquito control. These studies will evaluate efficacy, feasibility, as well as consumer attitudes toward the potential of introducing biorational yeast interfering RNA larvicides into integrated vector mosquito control programs. The field stability of yeast interfering RNA larvicides will of course need to be evaluated in these studies. However, dsRNA stability has been reported to be quite high 22,23 and was found to be acceptable in our laboratory trials with heat-inactivated dried yeast tablets (Table 1c). Yeast interfering RNA larvicides that induce high levels of mortality in Anopheles mosquitoes have been identified (M. Duman-Scheel, unpublished). Thus, it is anticipated that the yeast interfering RNA larvicide approach could be extended to Anopheles mosquitoes and other mosquito vectors of disease. Furthermore, expression of dsRNA in S. cerevisiae was recently used to target genes in the agricultural pest Drosophila suzukii 14 . Combined, these results suggest that yeast interfering RNA technology could potentially be used for biorational control of a wide variety of insect pests.
We anticipate that ready-to-use inactivated formulations of biorational yeast interfering RNA larvicides could seamlessly integrate, with minimal training and educational campaigns, into existing mosquito control programs to combat resistance to current pesticides and concerns for their potential impacts on non-target organisms. For example, Bacillus thuringiensis israelensis (Bti) and methoprene briquettes are currently used for treatment of Aedes breeding sites, as are granular formulations of temephos [24][25][26][27] . However, resistance to these pesticides has begun to emerge [28][29][30][31][32][33][34] . Furthermore, regulatory approval for temephos was not renewed in the United States last year 35 , and methoprene can be extremely toxic to non-target invertebrate species 25 . The results of this investigation demonstrate that dried inactive yeast interfering RNA tablets can function as lure-and-kill larvicides that offer biorational alternatives to these existing pesticides. Moreover, if resistance were to develop due to a mutation in any one shRNA target site in A. aegypti, an alternative target site could be used. Thus, by building an arsenal of different yeast interfering RNA larvicide strains, we can start to combat resistance before these larvicides are tested in the field. Baker's yeast is manufactured in large-scale industrial cultures in a variety of active and inactive formulations that are readily packaged and shipped, making this technology versatile, scalable, and distributable. Moreover, yeasts have been cultivated worldwide for thousands of years, and this technology can therefore be adapted in resource-limited countries with constrained infrastructures.

Methods
Animal rearing. The A. aegypti Liverpool-IB12 (LVP-IB12) strain from which the genome sequence 36 was generated was used in all the studies described, except for the trials described in Figs 3c-e and S2c, in which F6 animals of a strain recently established from eggs collected in ovitraps in Trinidad 37 were used. Mosquitoes were maintained as described 38 , except that an artificial membrane feeding system was used for delivery of sheep blood (HemoStat Laboratories, Dixon, CA) to adult females. The mosquitoes were reared in an insectary at 26 °C, at ~80% humidity, and under a 12 hr light/12 hr dark cycle with 1 hr crepuscular periods at the beginning and end of each light cycle.
For testing of siRNA #52, chitosan/siRNA nanoparticles prepared with #52 or control siRNA were mixed with larval food and fed to 10 mosquito larvae/condition using previously described methods 8 . Larvae were then reared and assessed per World Health Organization (WHO) larvicide testing guidelines. Control vs. #52 treatment data were assessed with Fisher's exact test. These experiments were part of a larger screen performed in duplicate (M. Duman-Scheel, unpublished) that will be described in its entirety elsewhere. Larval soaking experiments were performed in triplicate with 20 L1 larvae soaked at a concentration of 0.25 ug/ul for 4 hrs. with control vs. larvicide RNA per the methodology of Singh et al. 39 . For microinjection experiments, ~10 pmol custom screening siRNA were injected in a 30 nL volume per larva (n = 30/condition/replicate X two biological replicates) using previously described methodology 40,41 . Following siRNA treatment, larvae were reared and assessed per the WHO larvicide testing guidelines. Data were assessed using Fisher's exact test. Soaking and microinjection experiments were performed in conjunction with larval lethal screens (M. Duman-Scheel, unpublished) that will be described in their entirety elsewhere.
Bacteria interfering RNA larvicides. Heat-killed orally delivered non-pathogenic E. coli expressing dsRNA were constructed and fed to larvae beginning in the L1 stage as described by Whyard et al. 15 .
Strain HT115-DE3, obtained from the Caenorhabditis Genetics Center (CGC, which is funded by the NIH Office of Research Infrastructure Programs, P40 OD010440), was used. The strain was transformed with the dsRNA transcription plasmid pL4440 (deposited at Addgene by Andrew Fire; plasmid # 1654), which contains forward and reverse T7 polymerase binding sites that flank a multiple cloning site in which DNA corresponding to the siRNA #52 or #101 target sequences had been cloned. This allowed for inducible expression of dsRNA in bacteria, which were prepared and fed to larvae per the Whyard et al. 15 protocol. E. coli transformed with GFP::L4440, which was deposited at Addgene by Guy Caldwell (plasmid # 11335), were used as a control feeding strain that expressed dsRNA corresponding to GFP. Larvicide assays were conducted per WHO guidelines. Seven replicate experiments, each with 20 larvae per treatment, were performed. The percentage of mortality was transformed to arcsine values for ANOVA comparisons (per WHO recommendations) followed by Tukey's multiple comparison test.
Generation of yeast interfering RNA larvicide strains. shRNA-encoding DNA oligonucleotides corresponding to the A. aegypti siRNA #52 target sequence in fez2, the siRNA #101 target sequence in lrc, and a control hairpin with no known Aedes target site 7 were custom synthesized by Invitrogen Life Technologies. Two types of yeast transformants were generated: Transient transformation. For transient transformation assays, the shRNA expression cassettes were cloned into pRS426 GPD, a non-integrating bacteria-yeast shuttle vector with a URA3 marker that permits constitutive expression of inserts cloned downstream of a GPD promoter 42 . Following sequencing to confirm the inserts, the plasmids were transformed into S. cerevisiae strain BY4742 43 , genotype MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0. Transformants were selected by growth on minimal media lacking uracil.
Yeast culturing. Following selection as described above, yeasts were grown under standard conditions in synthetic media to an OD 600 of 3.0. For galactose induction experiments, yeasts were cultured in 20 ml SCD medium containing 20 g/L glucose to early stationary growth phase. Cells were harvested by centrifugation and transferred into 200 ml of fresh SCD medium containing 20 g/L galactose along with 2 g/L glucose. This was cultured at 30 °C and 250 rpm for 18 h (OD600 ~ 3.0).

Preparation of yeast larvicide formulations.
For initial experiments, yeast-agarose tablets were prepared for feeding assays from 50 ml of liquid yeast culture as described in the Whyard et al. 15 procedure for production of both live and heat-inactivated bacterial pellets. For preparation of dried tablet formulations, yeast cultures were grown as indicated above. The culture was then transferred to 50 ml conical tubes, each containing 50 ml of culture for transient transformants or 40 ml of culture for stable transformants, and pelleted by centrifugation for 20 min at 4000 rpm (Eppendorf, 5810 R plus). The supernatant (media) was discarded before placing the pellet in a 70 °C water bath for 5 mins. The yeast pellet was then placed into a 2 ml tube containing 10 mg of liver powder (MP Biomedicals, LLC, Solon, OH) as a nutritional supplement and centrifuged for 1 min at ~13.2 rpm (Eppendorf, 5415D). The supernatant was again discarded, and the tubes were left open in an incubator at 30 °C for 48 hrs to evaporate remaining media. The tablets were allowed to dry before storage in capped microfuge tubes at −20 °C. The final weight of each tablet averaged 95 mg for transiently-transformed yeast and 82 mg for stable yeast transformant tablets, each including 85 mg and 72 mg of yeast (~1.6 × 10 10 cells) respectively, as well as 10 mg of liver powder.
Larval feedings with yeast. Agarose gel coated formulations were divided into three portions, with one portion fed daily for three days to 20 first instar larvae (L1) per replicate experiment. For dried tabular formulations, one control or experimental tablet was placed in each container with 20 L1 animals at the start of each experiment. Larval bioassays were performed in 500 ml plastic cups containing 50 ml of distilled water and conformed to the WHO larvicide testing guidelines 48 . Larvae fed with larvicides #52, #101, and the control larvicide formulations were evaluated in parallel in at least three biological replicate experiments, each with at least three replicates per condition. The ages of larvae (days post-hatching and larval instar) at the time of death were recorded. Body lengths of control and larvicide-treated animals were also assessed using Fiji ImageJ software by measuring the distance between the head to the 8 th abdominal segment in treated early L4 larvae that had been fixed and photographed; body length data were statistically analyzed with ANOVA. Dried tablet formulations were also assessed in 33 cm × 23 cm × 5 cm aluminum pans and 18.9 L white buckets containing 1.5 L and 15.75 L SCIENTIfIC RepORTS | 7: 13223 | DOI:10.1038/s41598-017-13566-y of distilled water, respectively and evaluated in parallel in three biological replicate experiments, with three replicates in 0.050 L water, and two replicates in 1.5 L and 15.75 L water. For statistical analysis of larval mortality assays, the percentage of mortality was transformed to arcsine values for ANOVA (per the WHO 48 recommendations) followed by Tukey's multiple comparison test.
Lethal dose determination. Initially, mosquitoes were exposed to various amounts of control, #52, and #101 yeast to determine the activity range. Control yeast was mixed with #52 and #101 yeast to various concentrations to generate the dried inactivated yeast tablets. First instar larvae were then fed and assessed as described above. Four and three biological replicate experiments with at least four replicate containers per concentration were run for #52 and #101, respectively. Abbot's formula was used to account for mortality in control animals, as discussed in the WHO guidelines 48 . Data from all replicates were pooled for analysis. LD50 with 95% confidence intervals were calculated from a log dosage-probit mortality regression line using SPSS computer software.
Assessment of residual activity. Following preparation of dried inactivated yeast tablets prepared using the aforementioned protocol, the tablets were placed in 50 ml conical tubes, flooded with 50 ml of distilled water, and used immediately or stored in the insectary for one to two weeks prior to use. Prior to larval feeding, the yeast and water mixture was transferred into plastic cups and allowed to precipitate (for ~5 hours) before adding the larvae, which were then assessed per WHO guidelines 48 . Larvae fed with larvicides #52, #101, and the control larvicide formulations were evaluated in parallel in three biological replicate experiments with three replicates for each week. The weekly percentages of mortality were transformed to arcsine values for ANOVA (per WHO 48 ) followed by Tukey's multiple comparison test.

Whole mount in situ hybridization and immunohistochemistry. Riboprobes corresponding to fez2
and lrc were synthesized according to the Patel 49 protocol, and in situ hybridization experiments were performed in duplicate as previously described 50 . Living animals were fixed for these assays. Tissues were then mounted and imaged with a Zeiss Axioimager equipped with a Spot Flex camera. For transcript quantification analyses, mean gray values (average signal intensity over the selected area) were calculated for digoxigenin-labeled transcript signal in control or experimental brains combined from two biological replicate experiments, each with 20 animals in each of four replicate containers for each condition. Transcript quantification data were statistically analyzed using an unpaired two tailed t-test. Immunohistochemical staining experiments were performed as previously described 51,52 using mAb nc82 anti-Bruchpilot 21 (DSHB Hybridoma Product nc82, which was deposited to the DSHB by E. Buchner), anti-HRP (Jackson Immunoresearch, West Grove, PA) and TO-PRO-3 iodide (Molecular Probes, Eugene, OR). Two biological replicate experiments, each with 20 animals in each of four replicate containers per condition, were processed. Living larvae were fixed for these experiments. After processing, tissues were mounted and imaged on a Zeiss 710 confocal microscope using Zen software. Images were analyzed with FIJI ImageJ and Adobe Photoshop CC 2014 software. For antibody staining intensity analyses, mean gray values were calculated as described 6 for control or experimental brains combined from two replicate experiments. Data were statistically analyzed using one-way ANOVA followed by the Kruskal-Wallis post hoc test.
Oviposition response assays. The impact of yeast interfering RNA larvicides on the attraction of gravid females was assessed. Heat-inactivated yeast was evaluated in these trials, which were conducted in laboratory assays performed in the insectary. Oviposition was measured with 10 gravid females released in a 30 cm × 30 cm × 30 cm cage containing two ovicups. Ovicups consisted of a 300 ml plastic cup lined with white paper towel filled with 100 ml of rainwater with and without a dried yeast tablet containing heat-inactivated control interfering yeast larvicide. Eggs were collected for four days during each assay. Four biological replicate experiments, each with five cage replicates, were assessed. Data were evaluated with a paired two tailed t-test. After a four-day egg laying period, a paired two tailed t-test was used to compare the number of eggs laid in the two treatments (4 replications and 5 repetitions, n = 20).