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The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

The FGLamide-Allatostatins Influence Foraging Behavior in Drosophila melanogaster

  • Christine Wang, 
  • Ian Chin-Sang, 
  • William G. Bendena
PLOS
x

Abstract

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.

Introduction

In insects, three differing allatostatin (AST) peptide structures have been isolated that inhibit juvenile hormone (JH) biosynthesis. These include the FGLamide (FGLa)/ASTs, the W(X)6Wamide/ASTs and the PISCF/ASTs. Each unique peptide structure appears to inhibit JH biosynthesis in select insect species [1][3]. All three types of ASTs have been identified in D. melanogaster, but none have been identified as a regulator of JH biosynthesis [4][7].

The FGLa/ASTs are related to vertebrate somatostatin, galanin and opioid peptides and inhibit JH biosynthesis only in cockroaches, crickets, and termites [8][10]. In cockroaches, FGLa/ASTs also function to regulate gut contraction [11][13]. In D.melanogaster and other Diptera the FGLa/ASTs do not inhibit JH biosynthesis [7], [14]and their function has yet to be determined. D. melanogaster FGLa/ASTs functionally interact with two galanin-like receptors Dar-1 and Dar-2 [4], [15], [16]. Dar-1 is primarily expressed in the larval CNS whereas Dar-2 appears to be expressed in the crop, midgut and hindgut [17].

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; forS = sitter, forR = 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 22°C, 12 hr light/dark cycle and 70%±5% 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 w1118 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 w1118 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 25°C were collected and washed with distilled water. Black rectangular Plexiglas plates (25 cm width×37 cm length×0.5 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 w1118 lines using an RNeasy Kit (Qiagen). RNA was eluted with 30 µl of RNAse free water as opposed to the 50 µl 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 µl volume of total isolated RNA at appropriate initial concentrations, as well as 83 µM dNTPs, 42 ng/µl oligo d(T), 3 µl of 5× 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 µl. 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 65°C for 5 minutes, 3 minutes on ice and then a master mix of 5× RT Buffer, RNaseOUT, DTT was added, followed by an incubation step for 2 minutes at 42°C. SuperScript II reverse transcriptase was added to the mixture and a final incubation session for 50 minutes at 42°C was carried out. The reaction was then terminated by heat inactivation at 70°C for 15 minutes and chilled on ice for 3 minutes. Immediately after, 60 µl 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 µl of cDNA and 1× volume of 2× qPCR MasterMix Plus for SYBR Green I Low ROX (Eurogentec) to bring the final volume to 25 µl 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 95°C, followed by 45 cycles of 15 second denaturation at 95°C, 1 minute hybridization step at variable temperatures (see hybridization temperatures of select primers below), and a 36 second elongation step at 72°C. 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 95°C, followed by 1 minute at 60°C, and 15 seconds at 95°C. The forward for primer 5′-ATTGTCGGGAGCGAAGGTC-3′ and the reverse primer 5′- ATGATGGTCTGAAAGCACTGG-3′ were used at a hybridization temperature of 62°C. The forward and reverse sequences for the Dar-1 primers were 5′-GCAGCCACTTATCGGTCATT-3′and 5′-CTTCCACACCAGACCACCTT-3′, respectively and the hybridization temperature used was 62°C. The forward FGLa/AST primer 5′- CTACGACCAGGACAACGAGA-3′ and the reverse primer 5′- CCCAGGCCAAAGTTGAAGG-3′ were used at a hybridization temperature of 62°C.The forward and reverse sequences for ribosomal protein gene Rp49 were 5′-GACGCTTCAAGGGACAGTATCTG-3′ and 5′-AAACGCGGTTCTGCATGAG-3′, respectively and were used at a hybridization temperature of 56.8°C. 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. T-tests 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.

Results

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 w1118 . 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 w1118 (Figure 1A, white bars) was compared DaGal4 crossed to w1118 (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. When crossed to the DaGal4 each RNAi line exhibited a further significant suppression of mRNA levels. Relative to their respective RNAi lines crossed to w1118, DaGal 4 expression in 48496 and 101395 Dar-1 RNAi lines resulted in mRNA suppression by 70 and 56%, respectively. DaGal4 expression with FGLa/AST RNAi lines 14397 and 103215 resulted in mRNA levels being suppressed 76 and 70% respectively. Dar 1 and FGLa/AST RNAi were also crossed to a tissue selective driver line 6896 and each displayed a reduction in their respective target gene mRNA levels of approximately 20% relative to the same RNAi lines crossed to w1118 (Figure 1B).

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Figure 1. Relative mRNA expression levels of individual D. melanogaster stocks expressing RNAi to gene sequences for Dar-1 and FGLa/AST.

A. Dar-1 or FGLa/AST mRNA levels were measured in DaGal4 x w1118 and RNA levels in RNAi lines crossed to either DaGal4 (patterned bar) or w1118(white bar) were measured relative to this control. B. The same comparisons as in A. with driver line 6896 X w1118 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.

https://doi.org/10.1371/journal.pone.0036059.g001

Larval Foraging Assays forDaGal4 driven Dar-1 and FGLa/AST RNAi

In the larval foraging assay, a self-cross of non-transformed w1118 was compared to the driver DaGal4 X w1118 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 w1118 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 w1118(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 w1118 and the w1118 self-cross (Figure 2A).

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Figure 2. The foraging distance of third instar larvae (path length) A. in the presence of food and B. absence of food for 5 mins was measured for controls DaGal4 x w1118(black bar), w1118 self-cross (grey bar) and Dar-1and FGLa/AST RNAi lines crossed to DaGal4 (patterned bar) or w1118 (white bar); N = 30–34.

Asterisks indicate a significant difference * = p<0.05;** = p<0.001 and *** = p<0.0001. Only the significance in comparison with w1118 self-cross is indicated although comparison with DaGal4 X w1118 was equivalent.

https://doi.org/10.1371/journal.pone.0036059.g002

When tested in the absence of food, no significant difference in foraging behavior was found between self-crossed w1118 and the DaGal4 driver line crossed to w1118. 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 w1118 (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 w1118

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Figure 3. The foraging distance of third instar larvae (path length) A. in the presence of food and B. absence of food for 5 mins was measured for controls 6896 x w1118(black bar), w1118 self-cross (grey bar) and Dar-1and FGLa/AST RNAi lines crossed to 6896 (patterned bar) or w1118 (white bar); N = 30–34.

https://doi.org/10.1371/journal.pone.0036059.g003

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.

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 w1118 (Figure 4). RNA extracted from larvae of Dar-1 and FGLa/AST RNAi lines crossed to w1118 have reduced for mRNA levels, with a significant reduction in the 14397 FGLa/AST RNAi line relative to DaGal4 X w1118 and self-crossed w1118 controls (Figure 4) which is consistent the RNAi lines without the drivers showing some knock down of the the Dar-1 and FGLa/AST genes. The tissue selective driver 6986 when crossed to w1118, 101395 Dar-1 RNAi, or 103215 FGLa/AST RNAi lines did not have any significant alteration in for mRNA levels relative to w1118self-cross (Figure 4). This is consistent with 6986 crosses failing to influence foraging behavior (Figure 3).

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Figure 4. for mRNA expression levels of controls DaGal4 x w1118 (black bar), w1118 self-cross (grey bar), 6896 X w1118 (black bar with white diagonal stripes) and experimental Dar-1 and FGLa/AST RNAi lines x DaGal4 (patterned bars) or w1118(white bars) and Dar1 and FGLa/AST RNAi lines x 6896 (white bars with black diagonal stripes).

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 w1118 self-cross is indicated although comparison with DaGal4 X w1118 was equivalent.

https://doi.org/10.1371/journal.pone.0036059.g004

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 w1118 (Figure 5).

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Figure 5. The triglyceride levels of third instar larvae in Dar-1 and FGLa/AST RNAi x DaGal4 and controls DaGal4 x w1118 and w1118 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.

https://doi.org/10.1371/journal.pone.0036059.g005

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 α-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 (forR) exhibits greater mobility than the sitter phenotype (forS), 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 nematodeC. elegans [33][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 w1118. This suggests that D. melanogaster Dar-1 and its FGLa/AST ligand directly or indirectly activates or stabilizes for gene expression, as reduction of Dar-1 or its FGLa/AST ligand significantly reduces for mRNA levels causing a reduced foraging (forS) phenotype. RNAi lines crossed to w1118 (i.e. no driver) appeared to have reduced for transcripts relative to controls DalGal4 X w1118 and the w1118self-cross, however, this reduction was only significant in the case of 14397 FGLa/AST X w1118 and may be explained, in part, by all RNAi lines X w1118 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 w1118 control. The lack of lipid accumulation when Dar-1 or FGLa/AST levels are reduced is similar to lipid accumulation in D. melanogaster forS larvae. . In the presence of food, forR larvae ingest less food, exhibit higher glucose absorption and preferential glucose allocation to lipids rather than sugars. forR larvae thus have higher lipid levels than forS 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.

Author Contributions

Conceived and designed the experiments: CW WGB. Performed the experiments: CW. Analyzed the data: CW ICS WGB. Contributed reagents/materials/analysis tools: WGB. Wrote the paper: CW WGB.

References

  1. 1. Bendena WG, Garside CS, Yu CG, Tobe SS (1997) Allatostatins: Diversity in structure and function of an insect neuropeptide family. Ann N Y Acad Sci 814: 53–66.WG BendenaCS GarsideCG YuSS Tobe1997Allatostatins: Diversity in structure and function of an insect neuropeptide family.Ann N Y Acad Sci8145366
  2. 2. Bendena WG, Donly BC, Tobe SS (1999) Allatostatins: A growing family of neuropeptides with structural and functional diversity. Ann N Y Acad Sci 897: 311–329.WG BendenaBC DonlySS Tobe1999Allatostatins: A growing family of neuropeptides with structural and functional diversity.Ann N Y Acad Sci897311329
  3. 3. Stay B, Tobe SS (2007) The role of allatostatins in juvenile hormone synthesis in insects and crustaceans. Annu Rev Entomol 52: 277–299.B. StaySS Tobe2007The role of allatostatins in juvenile hormone synthesis in insects and crustaceans.Annu Rev Entomol52277299
  4. 4. Birgul N, Weise C, Kreienkamp HJ, Richter D (1999) Reverse physiology in Drosophila: Identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. EMBO J 18(21): 5892–5900.N. BirgulC. WeiseHJ KreienkampD. Richter1999Reverse physiology in Drosophila: Identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family.EMBO J182158925900
  5. 5. Williamson M, Lenz C, Winther AM, Nassel DR, Grimmelikhuijzen CJ (2001) Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster. Biochem Biophys Res Commun 281(2): 544–550.M. WilliamsonC. LenzAM WintherDR NasselCJ Grimmelikhuijzen2001Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster.Biochem Biophys Res Commun2812544550
  6. 6. Williamson M, Lenz C, Winther AME, Nassel DR, Grimmelikhuijzen CJP (2001) Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster. Biochem Biophys Res Commun 282(1): 124–130.M. WilliamsonC. LenzAME WintherDR NasselCJP Grimmelikhuijzen2001Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster.Biochem Biophys Res Commun2821124130
  7. 7. Yoon JG, Stay B (1995) Immunocytochemical localization of Diploptera punctata allatostatin-like peptide in Drosophila melanogaster. J Comp Neurol 363(3): 475–488.JG YoonB. Stay1995Immunocytochemical localization of Diploptera punctata allatostatin-like peptide in Drosophila melanogaster.J Comp Neurol3633475488
  8. 8. Woodhead AP, Stay B, Seidel SL, Khan MA, Tobe SS (1989) Primary structure of four allatostatins: Neuropeptide inhibitors of juvenile hormone synthesis. Proc Natl Acad Sci U S A 86(15): 5997–6001.AP WoodheadB. StaySL SeidelMA KhanSS Tobe1989Primary structure of four allatostatins: Neuropeptide inhibitors of juvenile hormone synthesis.Proc Natl Acad Sci U S A861559976001
  9. 9. Lorenz MW, Kellner R, Hoffmann KH (1995) Identification of two allatostatins from the cricket, Gryllus bimaculatus de geer (ensifera, gryllidae): Additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis. Regul Pept 57(3): 227–236.MW LorenzR. KellnerKH Hoffmann1995Identification of two allatostatins from the cricket, Gryllus bimaculatus de geer (ensifera, gryllidae): Additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis.Regul Pept573227236
  10. 10. Yagi KJ, Kwok R, Chan KK, Setter RR, Myles TG, et al. (2005) Phe-gly-leu-amide allatostatin in the termite Reticulitermes flavipes: Content in brain and corpus allatum and effect on juvenile hormone synthesis. J Insect Physiol 51(4): 357–365.KJ YagiR. KwokKK ChanRR SetterTG Myles2005Phe-gly-leu-amide allatostatin in the termite Reticulitermes flavipes: Content in brain and corpus allatum and effect on juvenile hormone synthesis.J Insect Physiol514357365
  11. 11. Reichwald K, Unnithan GC, Davis NT, Agricola H, Feyereisen R (1994) Expression of the allatostatin gene in endocrine cells of the cockroach midgut. Proc Natl Acad Sci U S A 91(25): 11894–11898.K. ReichwaldGC UnnithanNT DavisH. AgricolaR. Feyereisen1994Expression of the allatostatin gene in endocrine cells of the cockroach midgut.Proc Natl Acad Sci U S A91251189411898
  12. 12. Lange AB, Bendena WG, Tobe SS (1995) The effect of the 13 dip-allatostatins on myogenic and induced contractions of the cockroach (Diploptera punctata) hindgut. J Insect Physiol 41(7): 581–588.AB LangeWG BendenaSS Tobe1995The effect of the 13 dip-allatostatins on myogenic and induced contractions of the cockroach (Diploptera punctata) hindgut.J Insect Physiol417581588
  13. 13. Yu CG, Stay B, Ding Q, Bendena WG, Tobe SS (1995) Immunochemical identification and expression of allatostatins in the gut of Diploptera punctata. J Insect Physiol 41(12): 1035–1043.CG YuB. StayQ. DingWG BendenaSS Tobe1995Immunochemical identification and expression of allatostatins in the gut of Diploptera punctata.J Insect Physiol411210351043
  14. 14. Duve H, Johnsen AH, Scott AG, Yu CG, Yagi KJ, et al. (1993) Callatostatins: Neuropeptides from the blowfly Calliphora vomitoria with sequence homology to cockroach allatostatins. Proc Natl Acad Sci U S A 90(6): 2456–2460.H. DuveAH JohnsenAG ScottCG YuKJ Yagi1993Callatostatins: Neuropeptides from the blowfly Calliphora vomitoria with sequence homology to cockroach allatostatins.Proc Natl Acad Sci U S A90624562460
  15. 15. Lenz C, Sondergaard L, Grimmelikhuijzen CJ (2000) Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors. Biochem Biophys Res Commun 269(1): 91–96.C. LenzL. SondergaardCJ Grimmelikhuijzen2000Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors.Biochem Biophys Res Commun26919196
  16. 16. Lenz C, Williamson M, Grimmelikhuijzen CJ (2000) Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster. Biochem Biophys Res Commun 273(2): 571–577.C. LenzM. WilliamsonCJ Grimmelikhuijzen2000Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster.Biochem Biophys Res Commun2732571577
  17. 17. Chintapalli VR, Wang J, Dow JAT (2007) Using FlyAtlas to identify better drosophila melanogaster models of human disease. Nat Genet 39(6): 715–720.VR ChintapalliJ. WangJAT Dow2007Using FlyAtlas to identify better drosophila melanogaster models of human disease.Nat Genet396715720
  18. 18. Bendena WG, Boudreau JR, Papanicolaou T, Maltby M, Tobe SS, et al. (2008) A Caenorhabditis elegans allatostatin/galanin-like receptor NPR-9 inhibits local search behavior in response to feeding cues. Proc Natl Acad Sci U S A 105(4): 1339–1342.WG BendenaJR BoudreauT. PapanicolaouM. MaltbySS Tobe2008A Caenorhabditis elegans allatostatin/galanin-like receptor NPR-9 inhibits local search behavior in response to feeding cues.Proc Natl Acad Sci U S A10541339134210.1073/pnas.0709492105. 10.1073/pnas.0709492105.
  19. 19. Pereira HS, Sokolowski MB (1993) Mutations in the larval foraging gene affect adult locomotory behavior after feeding in Drosophila melanogaster. Proc Natl Acad Sci U S A 90(11): 5044–5046.HS PereiraMB Sokolowski1993Mutations in the larval foraging gene affect adult locomotory behavior after feeding in Drosophila melanogaster.Proc Natl Acad Sci U S A901150445046
  20. 20. Sokolowski MB, Hansell KP (1992) The foraging locus - behavioral-tests for normal muscle movement in rover and sitter Drosophila melanogaster larvae. Genetica 85(3): 205–209.MB SokolowskiKP Hansell1992The foraging locus - behavioral-tests for normal muscle movement in rover and sitter Drosophila melanogaster larvae.Genetica853205209
  21. 21. Osborne KA, Robichon A, Burgess E, Butland S, Shaw RA, et al. (1997) Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science 277(5327): 834–836.KA OsborneA. RobichonE. BurgessS. ButlandRA Shaw1997Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila.Science2775327834836
  22. 22. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, et al. (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448(7150): 151–156.G. DietzlD. ChenF. SchnorrerKC SuY. Barinova2007A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.Nature4487150151156
  23. 23. Manseau L, Baradaran A, Brower D, Budhu A, Elefant F, et al. (1997) GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of drosophila. Dev Dyn 209(3): 310–322.L. ManseauA. BaradaranD. BrowerA. BudhuF. Elefant1997GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of drosophila.Dev Dyn2093310322
  24. 24. Pereira HS, Macdonald DE, Hilliker AJ, Sokolowski MB (1995) Chaser (csr), a new gene affecting larval foraging behavior in Drosophila melanogaster. Genetics 141(1): 263–270.HS PereiraDE MacdonaldAJ HillikerMB Sokolowski1995Chaser (csr), a new gene affecting larval foraging behavior in Drosophila melanogaster.Genetics1411263270
  25. 25. Gronke S, Beller M, Fellert S, Ramakrishnan H, Jackle H, et al. (2003) Control of fat storage by a Drosophila PAT domain protein. Current Biology 13(7): 603–606.S. GronkeM. BellerS. FellertH. RamakrishnanH. Jackle2003Control of fat storage by a Drosophila PAT domain protein.Current Biology137603606
  26. 26. Pratt GE, Farnsworth DE, Siegel NR, Fok KF, Feyereisen R (1989) Identification of an allatostatin from adult Diploptera punctata. Biochem Biophys Res Commun 163(3): 1243–1247.GE PrattDE FarnsworthNR SiegelKF FokR. Feyereisen1989Identification of an allatostatin from adult Diploptera punctata.Biochem Biophys Res Commun163312431247
  27. 27. Fuse M, Zhang JR, Partridge E, Nachman RJ, Orchard I, et al. (1999) Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata. Peptides 20(11): 1285–1293.M. FuseJR ZhangE. PartridgeRJ NachmanI. Orchard1999Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata.Peptides201112851293
  28. 28. Aguilar R, Maestro JL, Vilaplana L, Pascual N, Piulachs MD, et al. (2003) Allatostatin gene expression in brain and midgut, and activity of synthetic allatostatins on feeding-related processes in the cockroach Blattella germanica. Regul Pept 115(3): 171–177.R. AguilarJL MaestroL. VilaplanaN. PascualMD Piulachs2003Allatostatin gene expression in brain and midgut, and activity of synthetic allatostatins on feeding-related processes in the cockroach Blattella germanica.Regul Pept1153171177
  29. 29. Lenz C, Williamson M, Grimmelikhuijzen CJ (2000) Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster. Biochem Biophys Res Commun 273(3): 1126–1131.C. LenzM. WilliamsonCJ Grimmelikhuijzen2000Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster.Biochem Biophys Res Commun273311261131
  30. 30. De Belle JS, Sokolowski MB (1987) Heredity of rover-sitter alternative foraging strategies of Drosophila-melanogaster larvae. Heredity 59(1): 73–84.JS De BelleMB Sokolowski1987Heredity of rover-sitter alternative foraging strategies of Drosophila-melanogaster larvae.Heredity5917384
  31. 31. Sokolowski MB (1980) Foraging strategies of Drosophila melanogaster - a chromosomal analysis. Behav Genet 10(3): 291–302.MB Sokolowski1980Foraging strategies of Drosophila melanogaster - a chromosomal analysis.Behav Genet103291302
  32. 32. Sokolowski MB (2001) Drosophila: Genetics meets behaviour. Nature Reviews Genetics 2(11): 879–890.MB Sokolowski2001Drosophila: Genetics meets behaviour.Nature Reviews Genetics211879890
  33. 33. Ben-Shahar Y, Robichon A, Sokolowski MB, Robinson GE (2002) Influence of gene action across different time scales on behavior. Science 296(5568): 741–744.Y. Ben-ShaharA. RobichonMB SokolowskiGE Robinson2002Influence of gene action across different time scales on behavior.Science2965568741744
  34. 34. Fujiwara M, Sengupta P, McIntire SL (2002) Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase. Neuron 36(6): 1091–1102.M. FujiwaraP. SenguptaSL McIntire2002Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase.Neuron36610911102
  35. 35. Ingram KK, Oefner P, Gordon DM (2005) Task-specific expression of the foraging gene in harvester ants. Mol Ecol 14(3): 813–818.KK IngramP. OefnerDM Gordon2005Task-specific expression of the foraging gene in harvester ants.Mol Ecol143813818
  36. 36. Kaun KR, Riedl CAL, Chakaborty-Chatterjee M, Belay AT, Douglas SJ, et al. (2007) Natural variation in food acquisition mediated via a Drosophila cGMP-dependent protein kinase. J Exp Biol 210(20): 3547–3558.KR KaunCAL RiedlM. Chakaborty-ChatterjeeAT BelaySJ Douglas2007Natural variation in food acquisition mediated via a Drosophila cGMP-dependent protein kinase.J Exp Biol2102035473558
  37. 37. Kaun KR, Sokolowski MB (2009) cGMP-dependent protein kinase: Linking foraging to energy homeostasis. Genome 52(1): 1–7.KR KaunMB Sokolowski2009cGMP-dependent protein kinase: Linking foraging to energy homeostasis.Genome52117
  38. 38. Toth AL, Robinson GE (2005) Worker nutrition and division of labour in honeybees. Anim Behav 69(2): 427–435.AL TothGE Robinson2005Worker nutrition and division of labour in honeybees.Anim Behav692427435
  39. 39. Wang C, Zhang J, Tobe SS, Bendena WG (2012) Defining the contribution of select neuropeptides and their receptors in regulating sesquiterpenoid biosynthesis by Drosophila melanogaster ring gland/corpus allatum through RNAi analysis. Gen Comp Endocrinol. C. WangJ. ZhangSS TobeWG Bendena2012Defining the contribution of select neuropeptides and their receptors in regulating sesquiterpenoid biosynthesis by Drosophila melanogaster ring gland/corpus allatum through RNAi analysis.Gen Comp Endocrinolhttp://dx.doi.org/10.1016/j.ygcen.2011.12.039. http://dx.doi.org/10.1016/j.ygcen.2011.12.039.