A Hawaii laboratory strain of medfly was reared at 65% relative humidity and 25°C throughout life cycle. Eggs were collected within 6 h after oviposition and were seeded in the fatty acid-supplemented diet (solid mill feed diet; diet A) and the fatty acid-deficient diet (liquid diet; diet B) [20]–[23] until pupation.

Upon pupation, 100 pupae were collected from each of four lots (400 pupae in total) and placed in a petri dish. The dish was then placed over with a black plastic polyvinyl chloride tube (20 cm in length and 8.5 cm in diameter) of which the inside wall was coated with talcum powder (Hawaii Chemical Co., Honolulu, Hawaii) to prevent emerged flies from crawling, instead of flying, out of the tube. Two pieces of folded cardstock (12×1 cm) were placed inside the tube with the pupae to give emerging adults a room to expand their wings [24]. The tubes were placed in a lighted flight cage (120×120×60 cm) without food or water until all flies had emerged and died, usually 24 d after egg seeding or 4 d after adult emergence. Flies that flied out of the tubes were trapped and killed by the sticky paper hanging above the tubes. Flies being incapable of flying were dead inside the petri dish. Flies were categorized into five groups: 1) unemerged, 2) partially emerged (part of adult body stuck to the puparium), 3) flies with deformed wings, 4) nonflying flies (flies that look normal, but are not capable of flying), and 5) flies capable of flying. Three independent experiments were performed and each experiment had 4 replicates.

Five-day old medfly pupae were collected, rinsed with proteomics grade water (Bio-Rad, Hercules, CA), and were then stored at −80°C for later use. Pupae (3×8 g; 8 g were approximately 200 pupae) were incubated with 15 mL of lysis buffer [40 mM Tris-HCl, pH 7.4, 5 mM dithiotreitol (DTT), 1 mM, phenylmethylsufonyl fluoride] and with 750 µL of a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) at ambient temperature for 15 min. Lysates were homogenized on ice using an ultraturrax homogenizer (Cole Parmer, Vernon Hills, IL) for 3 min at an interval of 30 s. The homogenate was then centrifuged at 17,000 g for 30 min and the supernatant was obtained by passing through a membrane Econofilter (0.2-mm×25-µm, Agilent, Palo Alto, CA). The supernatant was used for identification of proteins and determination of L-leucine content. Protein concentrations were determined with Coomassie PlusTM protein assay kit (Pierce, Rockford, IL). Images of Coomassie stained gels were obtained on a Bio-Rad densitometer GS-800. The gels were analyzed with PD-Quest (Bio-Rad) to compare protein content from pupae A and pupae B. Statistical analyses were performed with GraphPad software (Bio-Rad).

An aliquot of protein extracts from pupae emerged from larvae grown on diet A (1.5 µL) and diet B (2.0 µL) (both 25 µg protein equivalents) was mixed with SDS-PAGE sample buffer (3.0 µL and 4.0 µL, respectively) and heated at 100°C for 5 min. The denatured proteins were separated on 10–20% gradient SDS-PAGE mini gels (9×10 cm, PAGE Gold Precast Gel, Cambrex Bioscience, Rockland, ME) followed by Coomassie dye G-250 staining. Precision plus protein standards (10 µL) (Bio-Rad) were used as protein markers. Each gel lane was cut into 20 even slices, destained with 50% (v/v) acetonitrile (ACN) in 25 mM NH₄HCO₃, and then completely dried in a speed-vacuum centrifuge (Eppendorf, Hamburg, Germany) after dehydration with ACN. The dried gel slices were reduced in 50 µL of 10 mM DTT for 45 min at 56°C, alkylated in 50 µL of 55 mM iodoacetamide for 45 min at ambient temperature in the dark. The gel slices were dehydrated with ACN followed by drying in the speed-vacuum centrifuge. After addition of 20 µL of sequencing-grade modified porcine trypsin (20 ng/µL in 50 mM NH₄HCO₃), samples were incubated at 37°C overnight. Tryptic digestion was stopped by adding 5 µL of 2% trifluoroacetic acid (TFA). The digested peptides were extracted from each gel slice with 30 µL of water/ACN/TFA (93∶5∶2, v/v/v) twice by sonication for 10 min on ice.

2D GE was conducted with ReadyStrip IPG (Bio-Rad) using 11 cm pH 3–10 IPG strips, and 10–20% Tris-HCl precast acrylamide gels. Pupae (3×8 g) were ground in liquid N₂ using a mortar and pestle. An amount of 100 mg of the powdered pupae was dissolved in 1 mL of lysis buffer (4% CHAPS, 8 M urea, 10% Triton-100, 2% bio-lyte, 1 M DTT, a trace amount (2 µL) of DNase I and RNase A, and protease inhibitor mixture) using an ultrasonic cell disruptor (Misonix, Farmingdale, NY) and mixed with 200 µL of rehydration buffer. After centrifugation at 29,774 g for 15 min at 4°C, an aliquot of the supernatant (10 µg of solubilized protein) was applied to isoelectric focusing (IEF) using immobiline dry strips (immobilized pH gradient, pH 3–10, 11 cm, linear) according to the instruction (Bio-Rad). IEF of the rehydrated strips was performed in a Protein® IEF Cell (Bio-Rad) with linear ramping of voltage (50 V for 10 h, 100 V for 3 h, 8,000 V for 1 h, 800 V for 10 h for a total of 50,000 Vh). After IEF, strips were equilibrated in 0.375 M Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 20% glycerol with 130 mM DTT for 15 min, and then for 15 min in the same buffer without DTT but with 135 mM iodoacetamide. Equilibrated strips were placed on the top of 20% PAGEs and fixed with 0.5% agarose in a concentrating buffer (62.5 mM Tris-HCl, pH 6.8, 0.1% SDS). The second dimension was run at 300 V and 30 mA/gel for 8 h.

The digested peptides of which the proteins were separated on 1D gel were analyzed on a Dionex UltiMate™ 3000 nano LC interfaced with an esquireHCT^ultra ion trap mass spectrometer (Bruker Daltonics, Billerica, ME) in nanoelectrospray mode with a PicoTip Emitter (360 µm O.D., 20 µm I.D., 10 µm tip I.D., New Objective, Woburn, MA) according to the procedure previously published [24], [25]. The nano-LC column was C₁₈ PepMap 100 (3 µm film thickness, 75 µm I.D. ×15 cm, Dionex Corp., Sunnyvale, CA) at a flow of ca. 180 nL/min. MS/MS spectra were interpreted with Mascot (Matrix Science, London, UK) via Biotools 2.2 software (Bruker). Peak lists for protein identification were created by Compass 1.3 of Bruker for esquireHCT^ultra. Peptide mass fingerprint (PMF) searches and sequence alignments were performed in Swiss-Prot through the Mascot sever with database of Drosophila melanogaster. UniProt classification was used to search cellular functions of identified proteins. Peptides were assumed to be monoisotopic, oxidized at methionine residues and carbamidomethylated at cysteine residues. Up to one missed trypsin cleavage was allowed, although matches that contained any missed cleavages were not noticed. Peptide mass and MS/MS tolerances were set at ±1.0 and ±0.8 Da, respectively. Probability-based molecular weight search (MOWSE) scores were estimated and were reported as: 10×log₁₀ (p), where p is the absolute probability. Scores in Mascot larger than the MOWSE score at p = 0.05 were considered statistically significant, meaning that the probability of the match being a random event is lower than 0.05. The false-positive rate (FPR) was estimated according to the method of Elias et al. [26] and was smaller than 1% [FPR  =  FP/(FP + TP), where FP is the number of FPR hits; TP is the number of true-positive hits]. Only proteins identified with at least two peptide hits, with each peptide containing two tryptic termini, were accepted. In addition, the MS/MS spectra of all positively identified peptides were manually confirmed twice. Proteins of pupae A and B were identified in triplicate and are summarized (Tables S1 and S2).

MALDI TOF/TOF MS was used to verify proteins identified with LC-ITMS. Protein spots on 2D GE were excised manually with a one touch spot picker (The Gel Company, San Francisco, CA), followed by on-gel digestion of proteins in the same matter as proteins on 1D SDS-PAGE. Trypsin-digested peptides (1 µL) from each spot were mixed with an equal volume of α-cyano-4-hydroxycinnamic acid, and then were applied directly onto a metal MTP 386 target plate. The samples were analyzed on a Bruker UltraflexIII MALDI-TOF/TOF mass spectrometer equipped with a Smartbeam™ laser.

Proteins detected in pupae B were compared with those in pupae A (control). Detection of a protein in pupae B but not in pupae A is referred to as over-expressed or over-represented, whereas a protein undetected in pupae B but detected in pupae A is referred to as under-expressed or under-represented.

Proteins identified based on mascot search with D. melanogaster database were interpreted as human ortholog in IPA (Ingenuity System, Redwood City, CA). Accession numbers of detected proteins (UniProt/SwissProt Ids) were listed in MS Excel and imported into IPA to create canonical pathways and networks of interacting proteins. Protein IDs uploaded into IPA were converted to the corresponding human Entrez Gene symbols for display in pathways and networks. The data were analyzed in the context of humans (IPA ID No.: 7571344).

L-Leucine was analyzed on an Agilent 1100 LC-MSD system equipped with a binary pump and an Atlantis HILIC silica column (4.6×250 mm, 5 µm; Waters, Milford, MA) housed in a thermostatic compartment at 20°C. The mobile phase was set linear gradient starting from 100% ACN and ending at 30% 6.5 mM ammonium acetate pH 5.5 and 70% ACN after 40 min at a constant flow rate of 0.4 mL/min. The injection volume was 10 µL. The MS was operated in electrospray ionization mode. The drying gas was nitrogen at a flow rate of 10.0 L/min at 350°C, creating a nebulizing pressure of 40 psi. The capillary voltage was set at 5 kV. Mass spectra were collected in positive ion mode with selected ion monitoring of m/z fragments at 131.2 amu with a fragmentation voltage of 150 V. Leucine in pupal samples was identified by comparison of retention times and MS spectra against L-leucine standard. Leucine concentrations were measured with calibration curves of a mixture of L- and D-leucine standards at concentrations of 2–200 µg/mL. All standards and samples were diluted with ACN/water (1/1, v/v) as appropriate.

When medfly larvae were reared on a conventional mill feed diet (designated as diet A) and a fatty acid deficient liquid diet (designated as diet B), full flight ability was displayed in 97±1% adult flies from diet A, but only 20±8% from diet B (Proc ANOVA; P<0.0001, df = 1, 7, F = 96.40) (Figure 1A, B). The results signify the diet changes affect flight ability of medfly. Recent studies showed essential dietary fatty acids are necessary for normal development of the eggs, larvae [27], and adult flies of Bactrocera dorsalis [28].

The LC-ITMS analyses showed a large difference in protein profiles between pupae A (normal) reared on diet A and pupae B (abnormal) reared on diet B (Figure 1C–E). A total of 400 proteins (Table S1) and 406 proteins (Table S2) were detected in pupae A and B, respectively, while 167 of these proteins were detected in both pupae A and B. Mascot search and differential comparison of the proteins showed that 239 proteins (Table S3) were detected in pupae B, but not in pupae A (referred to as over-expressed proteins in pupae B), while 233 proteins (Table S4) were detected in pupae A, but not in pupae B (referred to as under-expressed proteins in pupae B).

Fli-I, LRR-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B (Figure S1 and Table S3). They were detected with LC-ITMS in the second gel slice with MWs between approximately 144 and 185 KD (Figure S1 III). However, they were not detected in pupae A. Five tryptic peptides were identified and matched with the fli-I amino acid sequence in the database with 20% coverage and a Mascot score of 36 (Table S2). These five tryptic peptides were further fragmented in MS/MS mode to confirm their amino acid sequences (Figure S1, II). Differential detection of fli-I in pupae B, but not pupae A was also confirmed with 2D GE (Figure S2). The analysis of the 2D GE images showed large difference in the protein scatter plot between pupae A and B (75% of match rate, not shown). In addition to fli-I, over-expression of Hsp70Ab, paramysosin, LRR protein soc-2, LRR containing G protein-coupled receptor 2, E3 ubiquitin-protein ligase Su and protein wing apart-like was found in pupae B with 2D GE and MALDI-TOF/TOF MS analysis (Figure S2, Table S6), which agreed with the results of LC-ITMS (Table S3). The results indicate that induction of fli-I in pupae B by nutritional effects may link with medfly flightlessness. It is known that over-expression of fli-I causes flightlessness in D. melanogaster [6]–[12]. The results of the present study showed that the fatty acid deficient liquid diet caused the medfly flightlessness, which was related to fli-I over-expression in pupae B. To our knowledge, this is the first observation of relationships between nutritional deficiency, fli-I over-presentation and medfly flightlessness.

Out of the 233, 239 and 167 of correspondingly under-expressed, over-expressed and overlapped proteins, 98, 89 and 82 proteins (Figure 1, Table S5), respectively, were mapped in 13 biological networks and 17 pathways of interacting protein clusters according to the identifiers' HomoloGene to the ortholog information in the Ingenuity Knowledge Base (IKB). The biological network of differentially expressed proteins shows inter-relationships and relevant signaling pathways. Medfly proteins were interpreted as in human ortholog in IPA and the resulted networks would be for humans. One overall network was merged from the 4 highest scored networks of gene expression, cellular assembly and organization, cell morphology, and cell cycle (Figure 2). Thirty seven hub proteins in small circles interconnected groups of proteins to form protein clusters of pathways (Figure 2).

Among 13 over-expressed protein hubs (Figure 2, Table S5), v-ral simian leukemia viral oncogene homolog A (RALA) functions as a negative regulation of JNK cascade. Insulin-like growth factor 1 receptor (IGF1R) is a transmembrane receptor in plasma membrane. Bone morphogenetic protein 2 (BMP2) is associated with BMP signaling pathway. Fer tyrosine kinase (FER) is actin filament bundle assembly. N-Ethylmaleimide-sensitive factor (NSF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are related to glycolysis. Mediator complex subunit 1 (MED1) regulates transcription from RNA polymerase II promoter. DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (DDX17) is related to RNA-mediated gene silencing. General transcription factor IIF (CTF2F1) acts positive regulation of transcription. Over-expressed proteins that include FLII (i.e., fli-I), topoisomerase 1 (TOP1), sterile α and TIR motif containing 1 (SARM1), eye-specific diacylglycerol kniase (DGKZ), E3 ubiquitin-protein ligase nedd-4 (NEDD4) in pupae B have direct relationships with the nuclear factor of kappa light polypeptide gene enhancer of activated B cell (NF-kB) complex that was detected in both pupae A and B. NF-kB plays a key role in regulating the immune response to infection since kappa light chains are components of immunoglobulins [32].

Among 11 under-expressed hub proteins (Figure 2, Table S5), integrin alpha V (ITGAV) is related to cell adhesion. Vav 1 guanine nucleotide exchange factor (VAV1) is associated with the actin filament organization. Inositol 1,4,5-trisphosphate receptor (ITPR1) acts as the second messenger that mediates the release of intracellular calcium or calcium transport. Checkpoint kinase 1 (CHEK1), Cyclin E (CCNE1), cell division cycle 37 homolog (CDC37) and TATA box binding protein (TBP)-associated factor (TAF1) are associated with cell cycle. Clock homolog (CLOCK) is related to cellular response to the circadian regulation of gene expression. Rap guanine nucleotide exchange factor 1 (RAPGEF1) is related to the Ras protein signal transduction. Suppressor of zeste 12 homolog (SUZ12) is related to the dendrite morphogenesis. Minichromosome maintenance complex component 10 (MCM10) is related to DNA replication. Eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3) is related to stress response. Poly (ADP-ribose) polymerase 1 (PARP1) has a function on chromatin modification. Cyclin-T 1 (CCNT1) is related to the actin filament organization. Transformation/transcription domain-associated protein (TRRAP) and suppressor of Ty 16 homolog (SUPT16H) have a role of transcription regulation. DEAH (Asp-Glu-Ala-His) box polypeptide 9 (DHX9) is related to axon extension. Structure specific recognition protein 1 (SSRP1) is related to DNA damage or DNA repair.

Axin (AXIN1), protocadherin-like wing polarity protein stan (CELSR1), bifunctional heparan sulfate N-deacetylase (NDST2), RuvB-like helicase 2 (RUVBL2), protein Wnt-5 (WNT5B), exostosin-1 (EXT1) and protein split ends (SPEN), which are related to the Wnt signaling pathway, were detected in pupae A and mapped in the network (Figure 2). However, these proteins were not observed in pupae B. Exostosin-2 and protein BCL9 were detected in pupae B even though they were not mapped into the network (Table S3). Apolipophorins (RFABG) and frizzled-2 (FZ2) were detected in both pupae A and B. Both proteins are related to the Wnt/ß-catenin signaling pathway (Figure 3), which comprises a large family of highly conserved growth factors that are responsible for important developmental and homeostatic processes throughout the animal kingdom [33]. In general, Wnt signaling regulates cell proliferation. Notch-inducible signaling molecules modulate cell proliferation in wings. Wnt signaling is required to support cell survival and to promote cell proliferation during the rapid phase of wing disc growth. Cells deprived of Wnt signaling die as a result of cell competition and apoptosis. In the absence of cell competition, loss of Wnt signaling still results in cell death, thus supporting the idea that Wnt signaling functions as a survival factor to the cells in wings [34]. Misregulation of the Wnt pathway can lead to a variety of abnormalities and degenerative diseases. Zinc finger protein 2 (imaginal disc-derived wing morphogenesis), G protein-coupled receptor kinase 1 (specifically phosphorylates the activated forms of G protein-coupled receptors) and putative gustatory receptor 98a (G-protein coupled receptor protein signaling pathway) were under-expressed in pupae B (Table S4).

Wnt signaling is mediated by ß-catenin (Figure 3). ß-Catenin dependent transcription is known to be activated by exogenous fli-I LRR associated protein 1 (FLAP-1) and depressed by fli-I [33]. Over-expression of fli-I can cause developmental defects, such as medfly flightlessness (Figure 1, Figure S1), by causing deregulation of the Wnt/ß-catenin pathway [35]. The finding suggests that over-expression of fli-I depressed Wnt signaling and subsequently may result in flightlessness.

In addition to the differential detection of fli-I in pupae B but not in pupae A, the concentrations of L-leucine determined by LC-MSD were 0.21±0.01 and 0.23±0.01 mg/g (wet pupae body weight) in pupae A and B, respectively, which were confirmed with Fourier Transform infrared spectroscopy (Figure S3) and LC-fluorescence analyses (Figure S4). It is noteworthy that an LRR domain is part of fli-I [13]–[15]. Interestingly, adult medflies whose pupae had a high leucine content had an increased flightlessness rate (Figure S4). The fli-I in humans encodes fli-I and is located within the SMS region on chromosome 17 [19]. Human fli-I is similar to a D. melanogasters protein involved in early embryogenesis and the structural organization of indirect flight muscle. It is not known whether the phenomenon of fatty acid deficiency-induced fli-I over-expression and incapable flying observed in the present study would be species-specific to medfly or not.