The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

Allatostatins (ASTs) are multifunctional neuropeptides that generally act in an inhibitory fashion. ASTs were identified as inhibitors of juvenile hormone biosynthesis. Juvenile hormone regulates insect metamorphosis, reproduction, food intake, growth, and development. Drosophila melanogaster RNAi lines of PheGlyLeu-amide-ASTs (FGLa/ASTs) and their cognate receptor, Dar-1, were used to characterize roles these neuropeptides and their respective receptor may play in behavior and physiology. Dar-1 and FGLa/AST RNAi lines showed a significant reduction in larval foraging in the presence of food. The larval foraging defect is not observed in the absence of food. These RNAi lines have decreased for transcript levels which encodes cGMP- dependent protein kinase. A reduction in the for transcript is known to be associated with a naturally occuring allelic variation that creates a sitter phenotype in contrast to the rover phenotype which is caused by a for allele associated with increased for activity. The sitting phenotype of FGLa/AST and Dar-1 RNAi lines is similar to the phenotype of a deletion mutant of an AST/galanin-like receptor (NPR-9) in Caenorhabditis elegans. Associated with the foraging defect in C. elegans npr-9 mutants is accumulation of intestinal lipid. Lipid accumulation was not a phenotype associated with the FGLa/AST and Dar-1 RNAi lines.

A Caenorhabditis elegans AST-like peptide receptor, NPR-9, was identified in a BLAST search as the closest related GPCR to Dar-1 [18]. Analysis of a deletion mutant of npr-9 revealed enhanced local search behavior and increased pivoting only in the presence of food. Mutant npr-9 animals also displayed an increased level of intestinal fat. The foraging phenotype of npr-9 is similar to a D. melanogaster mutation known as 'sitter' [19]. The sitter phenotype is due to a naturally occuring polymorphism in the foraging (for) gene. Two different alleles at the for locus characterize two different food-search behavioral phenotypes; for S = sitter, for R = rover.
Rovers travel significantly greater distances when feeding as compared to the sitters, but no locomotor differences were seen between the two strains on non-nutrient media, suggesting that pathlength differences on food are not the result of a general locomotory defect [20]. The alleles differ by natural polymorphisms at the dg2 (for) locus [21]. The for gene encodes a cGMP dependent protein kinase (PKG) and differences in PKG activity and for transcript levels are attributable to the differences in foraging behavior, where rovers have a significantly greater level of PKG activity and for transcript level when compared to sitters.
In this manuscript, we have identified an alteration in foraging behavior due to reduction in FGLa/ASTs or their receptor Dar-1. This is the first identified functional role for FGLa/ASTs or receptor Dar-1 in D. melanogaster.

Materials and Methods
Animals D. melanogaster stocks were reared at 22uC, 12 hr light/dark cycle and 70%65% relative humidity on standard medium containing 0.94% agar, 0.01% molasses, 8.2% cornmeal, 3.4% killed yeast, 0.18% benzoic acid, 0.66% proprionic acid. Gal 4-UAS RNAi transgenic lines were obtained from Vienna Drosophila RNAi Center (VDRC, Vienna, Austria) [22]. These lines were created by transformation of isogenic strain w 1118 which was used as a control in our experiments. Homozygous viable RNAi lines Dar-1 48496 and 101395 contained an insertion on chromosomes 1 and 2, respectively. Isogenic homozygous viable FGLa/AST RNAi lines 103215 and 14397 contained insertions on chromosomes 2 and 3, respectively. Dar-2 RNAi lines 1326 and 1327 were not used in this study as they showed slow developmental growth and feeding defects. The ubiquitous driver line daughterless (Da)Gal 4 and tissue specific driver 6986 were obtained from the Bloomington Stock center. The driver line 6986 expresses primarily in larval ring gland but also expresses in histoblasts, gut, Malpighian tubules, male accessory glands, testis sheath, and cyst cells [23].

Larval Foraging Behavioral Assay
Third instar larval foraging assays from each cross (RNAi lines and w 1118 control crossed to DaGal4 or 6986 drivers) were examined using a modified procedure described [24], which is briefly outlined here. Third instar larvae (approximately 72 hours post-hatching) reared at 25uC were collected and washed with distilled water. Black rectangular Plexiglas plates (25 cm width637 cm length60.5 cm height) with 6 circular wells (0.5 mm deep with a 4.25 cm radius) were provided courtesy of the Sokolowski Lab (University of Toronto at Mississauga). Larvae were placed into the center of each of the 6 circular wells, which were filled with a thin layer of homogenized yeast paste (distilled water and Fleischmann's Bakers' Yeast; approximately 3:1 ratio by weight). Wells were then covered with standard Petri dish lids. After 5 minutes the foraging path lengths made within the yeast were traced, scanned, and quantified using the ImageJ program (http://rsb.info.nih.gov/ij/).
Third instar larvae foraging behavior was also analyzed off food. Standard Petri dishes were used and filled with a thin layer of 2% agar containing neutral red dye. Larvae were placed into the center of one of these agar filled Petri dishes and the foraging path lengths traced after 5 minutes and analyzed the same way as the on food foraging assay.

Triglyceride Extraction and Quantification
Triglyceride and proteins were extracted from third instar larvae as described [25]. Three independent extractions were used for quantification of triglyceride and protein. The triglyceride levels from the extracts were quantified using the TRIGs Kit (Randox). Protein levels were quantified using the BCA Protein Assay (ThermoScientific). Triglyceride levels were normalized to protein levels.

RNA Extraction and Reverse Transcription
RNA was extracted from 30 mg of D. melanogaster third instar larvae from each RNAi, DaGal4 or 6898 driver control, and w 1118 lines using an RNeasy Kit (Qiagen). RNA was eluted with 30 ml of RNAse free water as opposed to the 50 ml suggested in the manual to concentrate the RNA extract further for reverse transcription. Remaining genomic DNA contamination was removed through the use of a DNA-free kit (Ambion), according to kit protocol.
All reverse transcription reactions were made with 8.0 ml volume of total isolated RNA at appropriate initial concentrations, as well as 83 mM dNTPs, 42 ng/ml oligo d(T), 3 ml of 56 RT Buffer, 40 U RNaseOUT (Invitrogen), 10.5 mM DTT, and 150 U of SuperScript II Reverse transcriptase (Invitrogen) into a final reaction volume of 15 ml. The following procedures and conditions were used for all reaction: first step was incubating RNA with a primer mix of oligo-d(T) and dNTPs at 65uC for 5 minutes, 3 minutes on ice and then a master mix of 56 RT Buffer, RNaseOUT, DTT was added, followed by an incubation step for 2 minutes at 42uC. SuperScript II reverse transcriptase was added to the mixture and a final incubation session for 50 minutes at 42uC was carried out. The reaction was then terminated by heat inactivation at 70uC for 15 minutes and chilled on ice for 3 minutes. Immediately after, 60 ml of autoclaved water was added to each reaction tube, resulting in a 5 fold-dilution of all cDNA samples.

Quantitative PCR and Standard Curves using PCR Products
For all qPCR experiments, primers were set at a 700 nM concentration and added with 5 ml of cDNA and 16volume of 26 qPCR MasterMix Plus for SYBR Green I Low ROX (Eurogentec) to bring the final volume to 25 ml for each reaction. A mastermix of SYBR green, primers, and water was prepared to minimize variation. All qPCR reactions were performed in an Applied Biosystems 7500 Real Time PCR System (Foster City, USA) under the following conditions: pre-PCR denaturation and polymerase activation step for 15 minutes at 95uC, followed by 45 cycles of 15 second denaturation at 95uC, 1 minute hybridization step at variable temperatures (see hybridization temperatures of select primers below), and a 36 second elongation step at 72uC. Dissociation curve analyses were done to confirm the amplification of a single PCR product, under the following real time conditions: 15 second denaturation step at 95uC, followed by 1 minute at 60uC, and 15 seconds at 95uC. The forward for primer 59-ATTGTCGGGAGCGAAGGTC-39 and the reverse primer 59-ATGATGGTCTGAAAGCACTGG-39 were used at a hybridization temperature of 62uC. The forward and reverse sequences for the Dar-1 primers were 59-GCAGCCACTTATCGGT-CATT-39and 59-CTTCCACACCAGACCACCTT-39, respectively and the hybridization temperature used was 62uC. The forward FGLa/AST primer 59-CTACGACCAGGACAAC-GAGA-39 and the reverse primer 59-CCCAGGCCAAAGTT-GAAGG-39 were used at a hybridization temperature of 62uC.The forward and reverse sequences for ribosomal protein gene Rp49 were 59-GACGCTTCAAGGGACAGTATCTG-39 and 59-AAACGCGGTTCTGCATGAG-39, respectively and were used at a hybridization temperature of 56.8uC. Transcript levels for each gene were normalized to gene Rp49 and presented as relative expression levels compared to a control except for for transcript levels which are presented as for expression levels normalized to Rp49.

Statistical Analysis
For larval foraging behavioral assays, Image J was used to quantify foraging path lengths that were traced and scanned. Ttests assuming unequal variance were performed in Graphpad Prism to determine the statistical significance of the foraging path lengths, for transcript levels, and triglyceride levels between RNAi strains and their controls.

Confirmation of RNAi Knockdown in Dar-1 and FGLa/AST RNAi
To assess the level of mRNA reduction/knockdown in Dar-1 and FGLa/AST RNAi lines (VDRC transformant IDs: 48496 , 101395; and 103215, 14397 respectively), each were crossed to driver lines (DaGal4 or 6896) or to w 1118 . The relative transcript levels of each gene in the third instar larval stage was quantitated by real-time qPCR. Each Dar-1 and FGLa/AST RNAi line crossed to w 1118 ( Figure 1A, white bars) was compared DaGal4 crossed to w 1118 ( Figure 1A, black bar). In the absence of being crossed to DaGal4, the Dar-1 and FGLa/AST lines had significantly reduced RNA expression levels which ranged from approximately 12-18% ( Figure 1A) suggesting that these RNAi lines without a driver exhibit some leaky gene knock down activity.

Larval Foraging Assays forDaGal4 driven Dar-1 and FGLa/ AST RNAi
In the larval foraging assay, a self-cross of non-transformed w 1118 was compared to the driver DaGal4 X w 1118 to confirm that no statistical difference in foraging resulted from the introduction of the DaGal4 driver ( Figure 2). A cross of each RNAi line with the DaGal4 driver (eg. 48496 Dar-1 RNAi) was then compared to a cross of each RNAi line with w 1118 in which RNAi should not be expressed (Figure 2A). On food, Dar-1 and FGLa/AST RNAi lines crossed to the DaGal4 driver showed significantly decreased foraging distances compared to the same RNAi lines crossed to w 1118 (Figure 2A). Similarly, on food foraging of Dar-1 and FGLa/ AST RNAi lines crossed to the DaGal4 driver was significantly reduced relative to DaGal4 X w 1118 and the w 1118 self-cross ( Figure 2A).
When tested in the absence of food, no significant difference in foraging behavior was found between self-crossed w 1118 and the DaGal4 driver line crossed to w 1118 . In the absence of food, no significant difference was found when RNAi lines for either Dar-1 or FGLa/AST crossed to the DaGal4 driver line were compared to their respective RNAi lines crossed to w 1118 ( Figure 2B).

Larval Foraging Assays for 6896 driven Dar-1 and FGLa/ AST RNAi
The foraging assay was repeated using third instar larvae from Dar-1 and FGLa/AST RNAi lines crossed to the tissue selective driver 6896. No significant reduction in foraging path length in the presence ( Figure 3A) or absence ( Figure 3B) of food was noted when 6896 X Dar 1 or FGLa/AST RNAi lines were compared to their respective RNAi lines crossed to w 1118 for Transcript Levels of Dar-1 and FGLa/AST Third Instar Larvae RNAi Sitter and rover phenotypes differ in PKG activity and for transcript level, where sitters have lower PKG activity and for transcript levels compared to their rover counterparts [21]. This suggested that foraging defects in Dar-1 and FGLa/AST RNAi lines may result from reduced PKG due to alterations in the for transcript level. To examine this, we measured for transcript levels in each RNAi line.  in DaGal4 x w 1118 and RNA levels in RNAi lines crossed to either DaGal4 (patterned bar) or w 1118 (white bar) were measured relative to this control. B. The same comparisons as in A. with driver line 6896 X w 1118 serving as the control. The number associated with each RNAi stock is the Transformation ID established by the Vienna Drosophila RNAi Center. Each bar represents two independent RNA extractions that were each assayed by qPCR three times. Thirty third instar larvae were used for each extraction. Expression levels were normalized using RP49 (ribosomal protein) as a standard. Asterisks indicate significant difference * = P,0.05; **P,0.001; ***P,0.0001. doi:10.1371/journal.pone.0036059.g001 The for transcript levels of Dar-1 and FGLa/AST RNAi lines crossed to DaGal4 driver line were significantly reduced compared to the same RNAi lines crossed to w 1118 (Figure 4)  RNAi lines did not have any significant alteration in for mRNA levels relative to w 1118 self-cross ( Figure 4). This is consistent with 6986 crosses failing to influence foraging behavior ( Figure 3).

Triglyceride Levels of Dar-1 and FGLa/AST Third Instar Larvae RNAi
C. elegans npr-9 mutants showed both local search behavior defects and an increase in intestinal lipid accumulation compared to wild type worms [18]. Dar-1 and FGLa/AST RNAi lines showed foraging defects similar to that of npr-9 mutants, which would suggest that foraging defects may be tied into decreased metabolic rate or increased food uptake efficiency. In order to assess this we measured the levels of total triglyceride in third instar larvae RNAi lines that showed foraging defects.
In contrast to our hypothesis, there was no significant difference in triglyceride levels between the Dar-1 and FGLa/AST RNAi lines crossed to DaGal4 or w 1118 ( Figure 5).

Discussion
FGLa/AST peptides were first identified as JH biosynthesis inhibitors from brain extracts of Diploptera punctata [8,26]. In D. punctata, FGLa/ASTs have been shown to elicit myoinhibitory effects in the hindgut and activate midgut a-amylase secretion [12,27]. Injection of FGLa/ASTs into Blattella germanica females inhibited food consumption, linking FGLa/ASTs with the regulation of digestive or feeding processes [28]. In D. melanogaster, FGLa/AST-specific antibodies reveal expression in interneurons, motorneurons and endocrine cells in the midgut [7]. FGLa/ASTs do not inhibit JH biosynthesis or innervate the ring gland/corpora allata in Diptera [3]. Based on immunohistochemical localization and mRNA expression, FGLa/ASTs are referred to as brain-gut peptides [29] but their function remains unclear. Work in C. elegans provided a testable hypothesis that D. melanogaster FGLa/ASTs and their CNS-localized receptor Dar-1 may be associated with foraging behavior [18]. Foraging behaviors in D. melanogaster, has been associated with naturally occurring variations in the for gene that encodes PKG [19,30]. On food, the rover phenotype (for R ) exhibits greater mobility than the sitter phenotype (for S ), however, both genotypes move at similar speeds in the absence of food [31,32]. PKG has an evloutionary conserved function in the regulation of foraging behavior in fruit flies, the honeybee Apis mellifera, red harvester ant Pogonomyrmex barbatus, and the nemato-deC. elegans [33][34][35]. Nurse honey bees were found to have lower PKG activity levels and lower Amfor RNA levels (ortholog of for) than forager honey bees, as well, nurse honey bees can change to foragers when fed an activator of PKG [33]. Interestingly, the difference in PKG activity is reversed when comparing dwellers to roamers in C. elegans, where a loss-of-function mutation in PKG (egl-4) caused an increase in roaming behavior in the presence of food. This suggests that PKG has a conserved function among these organisms even though the effect it has may differ between them.
Our results show that the ubiquitous expression of FGLa/AST RNAi or Dar-1 RNAi is related to a decrease in foraging behavior of D. melanogaster third instar larvae in the presence of food. Foraging behavior, under these conditions, is not altered in the absence of food. This alteration in foraging behavior appears to be related to a decrease in for transcript levels of Dar-1 and FGLa/ AST RNAi lines crossed to DaGal4 compared to both RNAi lines crossed to w 1118 . This suggests that D. melanogaster Dar-1 and its FGLa/AST ligand directly or indirectly activates or stabilizes for . Each bar represents three independent RNA extractions that were each assayed by qPCR three times. Thirty third instar larvae were used for each extraction. Expression levels were normalized using RP49 as a standard. Asterisks indicate a significant difference * = p,0.05 and ** = p,0.001. Only the significance in comparison with w 1118 self-cross is indicated although comparison with DaGal4 X w 1118 was equivalent. doi:10.1371/journal.pone.0036059.g004 Figure 5. The triglyceride levels of third instar larvae in Dar-1 and FGLa/AST RNAi x DaGal4 and controls DaGal4 x w 1118 and w 1118 self-cross. Each bar represents the mean of three independent triglyceride assays. Triglycerides were extracted from five third instar larvae. Triglyceride levels were normalized using protein levels as a standard. doi:10.1371/journal.pone.0036059.g005 gene expression, as reduction of Dar-1 or its FGLa/AST ligand significantly reduces for mRNA levels causing a reduced foraging (for S ) phenotype. RNAi lines crossed to w 1118 (i.e. no driver) appeared to have reduced for transcripts relative to controls DalGal4 X w 1118 and the w 1118 self-cross, however, this reduction was only significant in the case of 14397 FGLa/AST X w 1118 and may be explained, in part, by all RNAi lines X w 1118 having 'leaky' expression which led to a significant reduction in their respective gene expression. The decrease in foraging behavior on food was not found when the reduction in FGLa/AST and Dar-1 mRNA levels were reduced in a tissue selective manner. This is consistent with the observation that the tissue specific RNAi did not reduce Dar-1 or FGLa/AST gene expression to the levels caused by the ubiquitously expressed DaGal4 driver. Expression of Dar-1 and FGLa/AST RNAi under the direction of the tissue selective driver 6986 did not alter for mRNA levels relative to controls. It is also likely that foraging behavior is only affected when FGLa/ASTs interact with Dar-1 and alter for expression in select cellular localizations.
C. elegans npr-9 mutants showed a significant increase in intestinal lipid accumulation compared to N2 wild type worms, which would suggest that the increased local search behavior seen in these mutant worms may also increase food uptake efficiency [18]. Since Dar-1 and FGLa/AST RNAi lines showed foraging defects similar to that of npr-9 mutants, we hypothesized that similar increase in triglycerides would also be seen. However, our results show no significant difference in triglyceride levels between the Dar-1 and FGLa/AST RNAi lines crossed to either the DaGal4 driver or w 1118 control. The lack of lipid accumulation when Dar-1 or FGLa/AST levels are reduced is similar to lipid accumulation in D. melanogaster for S larvae. . In the presence of food, for R larvae ingest less food, exhibit higher glucose absorption and preferential glucose allocation to lipids rather than sugars. for R larvae thus have higher lipid levels than for S larvae [36,37]. This contrasts with A. mellifera, where foraging bees have reduced lipid levels in comparison to nurse bees [38].
FGLa/ASTs and receptor Dar-1 do not participate in the regulation of juvenile hormone biosynthesis in D. melanogaster [39]. The alteration in foraging behavior and direct or indirect influence on the for transcript is the first function assigned to the D. melanogaster FGLa/ASTs and its receptor Dar-1. Future work will be directed at defining where FGLa/ASTs interact with Dar-1 and how this interaction influences for gene expression.