Molecular characterization of neuropeptide F from the eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae)

Peptides 31 (2010) 419–428

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Molecular characterization of neuropeptide F from the eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) Andrew B. Nuss a,*, Brian T. Forschler a, Joe W. Crim b, Victoria TeBrugge c, Jan Pohl d, Mark R. Brown a a

Department of Entomology, 413 Biological Sciences Building, University of Georgia, Athens, GA 30602-2603, USA Department of Cellular Biology, 724 Biological Sciences Building, University of Georgia, Athens, GA 30602-2603, USA c Department of Biology, University of Toronto at Mississauga, South Building Room 3016B, 3359 Mississauga Rd North, Mississauga, Ontario, L5L-1C6 Canada d Biotechnology Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 April 2009 Received in revised form 1 September 2009 Accepted 1 September 2009 Available online 9 September 2009

Neuropeptide F (NPF)-like immunoreactivity was previously found to be abundant in the eastern subterranean termite, Reticulitermes flavipes. Purification of the NPF from a whole body extract of worker termites was accomplished in the current study by HPLC and heterologous radioimmunoassay for an NPF-related peptide, Helicoverpa zea Midgut Peptide-I. A partial amino acid sequence allowed determination of the corresponding cDNA that encoded an open reading frame deduced for authentic R. flavipes NPF (Ref NPF): KPSDPEQLADTLKYLEELDRFYSQVARPRFa. Effects of synthetic NPFs on muscle contractions were investigated for isolated foreguts and hindguts of workers, with Drm NPF inhibiting spontaneous contractions of hindguts. Phylogenetic analysis of invertebrate NPF sequences reveals two separate groupings, with Ref NPF occurring within a clade composed exclusively of arthropods. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Neuropeptide Y Insect Gut motility

1. Introduction The neuropeptide Y (NPY)/neuropeptide F (NPF) superfamily exhibits remarkable conservation across platyhelminths, mollusks, arthropods, and chordates [6,8,32]. Gene duplications resulted in three homologous peptides for mammals: NPY, peptide YY, and pancreatic polypeptide (PP) [4]. Numerous studies have focused on NPY, the most abundant neuropeptide in the mammalian brain [36]. Distributed in a brain–gut endocrine axis, the mammalian peptides coordinate appetite, digestion, and metabolism, as well as nociception [36]. In contrast, a single corresponding neuropeptide F (NPF) gene is thought to typify invertebrate species [39]. Across all life stages of insects, NPF exhibits a similar brain–gut distribution [6,18,46], but the functional significance of this axis in invertebrates is largely unknown. For transgenic Drosophila melanogaster, behavioral roles for NPF signaling [10,24,49] include regulation of food acquisition by larvae [30,50,51]. In vitro bioassays also show that NPF inhibits motility of gut regions from larval mosquitoes, Aedes aegypti [35], and kissing bugs, Rhodnius prolixus [16]. Suggestions of NPY/PP-like peptides in insects arose with immunocytochemical studies in the 1980s (see [5,33,41]). The first authentic insect NPF was structurally characterized following isolation from a whole body extract of adult D. melanogaster in 1999 [6]. Isolation of Drm NPF was monitored with a heterologous

* Corresponding author. Tel.: +1 706 542 4079; fax: +1 706 542 9755. E-mail address: [email protected] (A.B. Nuss). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.09.001

radioimmunoassay (RIA) based on an antiserum to a truncated NPF-like peptide (QAARPRFa; Hez MP-I) extracted from midguts of the corn earworm, Helicoverpa zea [17]. Similarly, NPF was isolated and characterized from the yellow fever mosquito, Ae. aegypti, with a RIA based on a Drm NPF antiserum [46]. Subsequent bioinformatics and related molecular techniques revealed NPF sequences for another mosquito species [14,38], locust [8], honeybee [19], and silkworm [39], but no such peptide has been identified in a beetle, Tribolium castanaeum [28]. Small peptides (5–9 amino acids) identified in various invertebrates exhibit an NPF-like Cterminus [17,21,42,44,45]. For squid, locust, and prawn, such sequences were subsequently shown to derive from full-length NPFs [7,8,47]. Other of these smaller peptides are likely short NPFs (sNPFs) encoded as multiple peptides within a single gene that is structurally distinct from the NPF gene [25]. Neuropeptide endocrinology is relatively unexplored in termites [2,29,43,52,53]. The social structure of termites includes a queen and king and castes of neotenics, nymphs, workers, and soldiers [23]. Behavioral and physiological bases of this complex hierarchy likely involve neuroendocrine regulation. A widespread distribution of NPF-like material in the nervous system and gut was detected with the Hez MP-I antiserum in different castes of the eastern subterranean termite, Reticulitermes flavipes [34]. Immunostaining occurred in cells in the brain and ventral nerve cord and in axons over the foregut, anterior midgut, and rectum, along with numerous isolated endocrine cells in the midgut. These findings suggested that NPF-immunoreactive material was abundant and distributed in a brain–gut axis in this termite species.

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The present study involved purification of NPF from a mass extract of workers. A partial amino acid sequence allowed determination of cDNA sequences for R. flavipes NPF. Expression of NPF transcripts in tissues of termites was examined and potential functions were explored in bioassays with synthetic NPF. The deduced sequence of termite NPF was compared to other invertebrate NPFs and found to reside in a distinctive arthropod clade. 2. Materials and methods 2.1. Whole body peptide extraction R. flavipes were collected from infested logs (Whitehall Forest, Athens, GA) [12]. Workers (117,000 estimated by weight; total 349.3 g) were separated from the other castes with forceps and frozen at 80 8C. Frozen termites were boiled in 3% acetic acid in batches of 100 g/500 ml for 10 min. Extracts were cooled and centrifuged at 4 8C for 15 min at 14,000  g. Supernatants were pooled, frozen and lyophilized to reduce volume to 1 l. Lipids were separated from the water extract in two batches by mixing with 200 ml hexane/500 ml extract. The water extract was loaded onto conditioned C18 cartridges (Varian Mega Bond Elut 60 cm3/10 g, Varian Co., Harbor City, CA) and step eluted with 5, 10, and 80% CH3CN in 0.1% TFA, and 100% CH3CN (60 ml each). The 80% CH3CN eluate was lyophilized and rehydrated in 1–2 ml of the initial solvent for the first HPLC step (see below). 2.2. Radioimmunoassay Chromatographic isolation of the immunoreactive peptide was monitored by heterologous RIA with the Hez MP-I antiserum and [125I]YQAARPRFa [6,17]. Aliquots from HPLC fractions were lyophilized and later rehydrated with buffer (0.05 M Tris–HCl, pH 7.2; 0.1% bovine serum albumin; 0.02% sodium azide) for RIA as described previously [6,17,46]. Successively greater numbers of termite equivalents (1–600) were necessary as some immunoreactive material was lost during each purification step. Immunoreactive peptide amounts were calculated from a standard line obtained by linear regression of bound/free counts/minute ratios and log fmol of Hez MP-I peptide standards (1–25 fmol). 2.3. HPLC Samples were loaded on a Beckman chromatography 126/166 unit for eight successive steps of reversed phase (RP) or cationexchange HPLC needed to isolate the immunoreactive peptide from the starting extract (Fig. 1A). For RP-HPLC, the ion pairing agent was either heptafluorobutyric acid (HFBA) or trifluoroacetic acid (TFA). Steps containing TFA were monitored at 206 or 215 nm and those with HFBA or ammonium acetate at 275 nm. Aliquots were taken from all fractions at each step and quantified for immunoreactivity by RIA.

extracted (RNeasy1 kit, Qiagen Inc.) from worker midguts (5) and heads (10), held overnight at 4 8C in RNAlater1 (Ambion, Inc.), and treated with DNase. Degenerate forward primers (Integrated DNA Technologies, Coralville, IA) were designed from the isolated peptide sequence (Table 1: Retic 1.1, 2, and 3.2) for the first PCR step. Products were amplified with the touchdown PCR procedure using the primers Retic 1.1 and NotI d(T) (Table 1) and head and midgut cDNA (10 cycles at 94 8C, 20 s; 65 8C for the first cycle and decreased by 1 8C each subsequent cycle, 20 s; 72 8C, 1 min followed by 30 cycles; 94 8C, 20 s; 56 8C, 20 s; 72 8C, 1 min). Products were amplified with primers Retic 2/NotI, and Retic 3.2/NotI from an aliquot (0.1 ml) of the first head and midgut PCR mixture (40 cycles; 94 8C, 20 s; 61 8C, 20 s; 72 8C, 1 min) for the second ‘‘nested’’ PCR step. After separation in a 1.2% agarose gel, two bands of the predicted size were purified (GenEluteTM Minus EtBr Spin Column, Sigma1) and sequenced (Molecular Cloning Laboratories, San Francisco, CA) or cloned (Sigma TOPO4TM vector kit and Nucleospin Plus Plasmid Miniprep Kit, Qiagen1) for sequencing. A consensus sequence of 350 bp from the above PCR products was used to design reverse primers near the 30 end (Table 1: NPF1, NPF2, NPF Rev) to determine the 50 sequence. Primer NPF1 (Table 1), and head and midgut total RNA were mixed with dNTPs to make cDNA by reverse transcription (Advantage RT for PCR; 70 8C, 2 min, add dNTPs then 42 8C, 1 h and 94 8C for 5 min). A poly A tail was added to the 30 end of the purified cDNA (High Pure PCR Product Purification Kit, Roche1) by incubating with dATP in TdT Reaction buffer (0.2 M potassium cacodylate, 25 mM Tris–HCl, 0.25 mg/ml bovine serum albumin; 3 min, 94 8C, then chilled on ice) followed by terminal transferase in 5 mM CoCl2 solution (Roche; 37 8C, 30 min; then inactivated at 70 8C, 10 min, then iced). Products were amplified from this cDNA mixture by PCR with the primers NPF2/ NotI d(T) (10 cycles; 94 8C, 20 s; 59.8 8C, 20 s; 72 8C, 40 s; then 10 cycles; 94 8C, 20 s; 59.8 8C, 20 s; 72 8C, 40 s [+20 s additional each subsequent cycle]). Products were amplified for ‘‘nested’’ PCR from the above PCR mixture with the primers NPF Rev/NotI (35 cycles; 94 8C, 20 s; 60.5 8C, 20 s; 72 8C, 1.5 min). After gel electrophoresis, PCR products of the expected size were purified and cloned (StratacloneTM PCR cloning kit) for sequencing in both directions (Molecular Cloning Laboratories, San Francisco, CA). A different 50 primer (UPNPF 1, Table 1) was used with NPF Rev for PCR with head and midgut cDNA (35 cycles; 94 8C, 20 s; 60.5 8C, 20 s; 72 8C, 1.5 min) to confirm the NPF cDNA sequence. Resulting products were cloned and sequenced (five each from head and midgut cDNA), and their sequences aligned for a consensus sequence. Table 1 Primer sequences used to amplify the Ref NPF cDNA sequence by PCR. Sequences in bold indicate degenerate primers based upon the partial R. flavipes NPF amino acid sequence. Letters below each primer are the corresponding amino acids. Nucleic acid abbreviations: I = inosine; N = A, G, C, T; R = A, G; H = A, C, T; B = C, G, T; Y = C, T. Forward primers/corresponding amino acids

2.4. Peptide analysis The immunoreactive peptide isolated in the last HPLC step (sample ‘B’, Fig. 1B) was analyzed by matrix-assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectrometry. A partial amino acid sequence of sample ‘B’ was obtained by Edman degradation using a model Procise-cLC (Applied Biosystems) sequencer [46]. 2.5. NPF cDNA cDNA libraries were generated with the Advantage1 RT-for-PCR kit (Clontech) and an oligo d(T) primer from total RNA separately

UPNPF1: 50 -ACACGACGGACTTTGTTGCC-30 F1 Bact: 50 -CCGTGACTTGACCGACTACC-30 Reverse primers NotI: 50 -AACTGGAAGAATTCGCGGCCGCAGGAA-30 NotI d(T): 50 -AACTGGAAGAATTCGCGGCCGCAGGAAT(18)-30 NPF 1: 50 -GCCGAAAATTTTTATTTAATTCTTAATGC-30 NPF 2: 50 -CAATGTGTTTCTGAAGACGAAGG-30 NPF Rev: 50 -CACTGTAATTGTTGGGAATTGCGG-30 R1 Bact: 50 -GAACAGAGCCTCAGGACAGC-30

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Fig. 1. Ref NPF purification. (A) Flowchart of HPLC steps. Hez MP-I immunoreactive fractions listed in bold were pooled. TFA and HFBA were at 0.1% unless otherwise noted. Solvent A contained water and the ion pairing agent except for step 3 where solvent A was 0.02 M CH3COONH4 with 10% CH3CN. (B) Chromatograph (l = 215 nm) of the final step of purification of sample ‘B’ of Reticulitermes flavipes worker extract by RP-HPLC (step 8). Amount of NPF-like material (gray bars) in each fraction was estimated by Hez MP-I RIA of aliquots equivalent to 600 workers.

2.6. Alignments and cladograms Sequences of invertebrate NPFs were obtained from published sources (cf. Tables 2a and 2b), and from BLAST searches of EST and

genomic public databases as described previously [38]. For sequences obtained in silico, translated NPFs were assumed to be a minimum of 36 amino acids in length, to standardize alignments. Following alignment with Clustal X, sequences were

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Table 2a Arthropod NPF-like sequences determined by Edman degradation, cloning, and bioinformatics. Phylum

Class

Order

Species

Sequence

Reference/accession number

Arthropoda (Subphylum Atelocerata)

Hexopoda

Orthoptera Isoptera Phthiraptera

Locusta migratoria NPF Reticulitermes flavipes NPF Pediculus humanus NPF 1 Pediculus humanus NPF 2 Rhodnius prolixus NPF Acyrthosiphon pisum NPF Eoxenos laboulbenei NPF Aedes aegypti NPF Anopheles gambiae NPF Armigeres subalbatus NPF Chironomus tentans NPF Culex quinquefasciatus NPF Drosophila grimshawi NPF Drosophila melanogaster NPFd Drosophila mojavensis NPF Drosophila virilis NPF Drosophila willistoni NPF Drosophila yakuba NPFe Antheraea assama NPF Bombyx mori NPF 1 Bombyx mori NPF 2 Danaus plexippus NPF Ostrinia nubilalis NPF 1 Ostrinia nubilalis NPF 2 Plodia interpunctella NPF Apis mellifera NPF1 Nasonia vitripennis NPF 1 Nasonia vitripennis NPF 2

AEAQQADGNKLEGLADALKYLQELDRFYSQVARPRFa VPSVWAKPSDPEQLADTLKYLEELDRFYSQVARPRFa TSTAETDQRKMKSMAEVLQILQNLDKYYTQAARPRFa NMLRPTRPKIFSSPNDFKSYLEDLGNFYAIAGRPRFa AMARPARPKSFASPDDLRTYLDQLGQYYAVAGRPRFa SIARPTRPKTFGSPDELRSYLDQLGQYLAVVSRPRFa MPDMVKQLKNLDQIDRLFAKFSRPRFa SFTDARPQDDPTSVAEAIRLLQELETKHAQHARPRFa LVAARPQDSDAASVAAAIRYLQELETKHAQHARPRFa VFTEARPQDDPTSVAEAIRLLQELESKHAQHARPRFa ANPNPDHSGDPVNVVDALRYLQDIESKHAQFARPRFa CLTEARPQDDPTSVAEAIRLLQELETKHAQHARPRFa SKARPPRNNEISNMADALKYLQDLDVYYGDRARVRFa SNSRPPRKNDVNTMADAYKFLQDLDTYYGDRARVRFa SKTRPPRNNEINTMADALKYLQDLDVYYGDRARVRFa SKARPPRNNEISTMADALKYLQDLDVYYGDRARVRFa SNSRPPRNNEINTMADAYKFLQDLDTYYGDRARVRFa SNSRPPRKNDVNTMADAYKFLQDLDSYYGDRARVRFa VFLAEAREEGPHDMADALRMLQELDRYYTQAARPRFa VCMAEAREEGPNNVAEALRILQLLDNYYTQAARPRFa QYPRPRRPERFDTAEQISNYLKELQEYYSVHGRGRYa VCLAEAREEGPHDMADALRMLQELDRLYTQASRPRFa ICLAEAREEGPHDMADALRMLQELDRYYTQAARPRFa QYPRPRRPERLDTAEQISNYLRELQEYYSVHGRGRYa ICMSEAREEGPHDMADAIRMLQELDRLYTQAARPRFa EPEPMARPTRPEIFTSPEELRRYIDHVSDYYLLSGKARYa EPEPMARPTRPKVFESPEELRQYLDLVKEYYSLSGKARFa VYYRNSNDVLQRKLHSVLQALERLENKHTVSPRPR__

[8]a; CO854418 This studyb,c DS235877.1a EEB15547a ACPB01036200a XM_001944830a CD492374a [46]b,c; AF474405 [38]a; AY579077 EU209590a DV187985a NZ_AAWU01035129a AAPT01020141.1a [6]b,c; AF117896 AAPU01011127.1a AANI01017194.1a AAQB01008982.1a AAEU02000024.1a FG203991a [39]c,a; AB362224 [39]c,a; AB298926 EY261386a GH998815a EL929496a EB823988a [19]a; GB16364-RA AAZX01003210a XM_001602217a

Hemiptera Strepsiptera Diptera

Lepidoptera

Hymenoptera

Arthropoda (Subphylum Crustacea)

Branchiopoda Maxillopoda Malacostraca

Cladocera Siphonostomatoida Decapoda

Daphnia magna NPF Lepeophtheirus salmonis NPF Marsupenaeus japonicus NPF

DGDVMGGGEGGEMTAMADAIKYLQGLDKVYGQAARPRFa QYDRPKERRVGNNMASALKKLSQIKDFYNEAGRPRFa KPDPSQLANMAEALKYLQELDKYYSQVSRPRFa

[7]a; EG565358 FK933794a [7]a; CI998017

a

Arthropod NPF-like sequences determined by bioinformatics. Arthropod NPF-like sequences determined by Edman degradation Arthropod NPF-like sequences determined by cloning. d Amino acid sequence is identical to D. ananassae (AAPP01019539.1), D. persimilis (AAIZ01005973.1), D. pseudoobscura (AAFS01000191.1), D. sechellia (AAKO01002387.1) and D. simulans (AAGH01000398.1) NPFs. e Amino acid sequence is identical to D. erecta (AAPQ01006453.1) NPF. b c

trimmed and gaps removed. Cladograms of resulting sequences were generated within GCG (Version 11.1, Accelrys Inc., San Diego, CA) by Jukes-Cantor distance correction method for UPGMA phylogenetic tree construction as described previously [14].

2.7. NPF expression in termite tissues Workers were collected from logs as above and maintained in the laboratory 2–4 weeks before assay (1500 workers/

Table 2b Non-arthropod invertebrate NPF-like sequences determined by Edman degradation, cloning, and bioinformatics. Phylum

Class

Order

Species

Annelida

Clitellata Polychaeta

Haplotaxida Capitellida Terebellida

Lumbricus rubellus NPF Capitella sp. NPF Alvinella pompejana NPF

ADGPPVRPDRFRTVAELNKYMADLTEYYTVLGRPRFa QMHPPKRPEHFRNMDELNVYLDKLRQYYTILGRPRFa AVEPPRRPEHFRNIEELNKYLAELRQYYTILGRPRFa

Mollusca

Cephalopoda Gastropoda

Sepiolida Opisthobranchia Pulmonata

Idiosepius paradoxus NPF Aplysia californica NPF Lymnaea stagnalis NPY Helix aspersa NPF Lottia gigantea NPF

MFAPPNRPAEFKSPEELRQYMKALNEYYAIVGRPRFa DNSEMLAPPPRPEEFTSAQQLRQYLAALNEYYSIMGRPRFa TEAMLTPPERPEEFKNPNELRKYLKALNEYYAIVGRPRFa STQMLSPPERPREFRHPNELRQYLKELNEYYAIMGRTRFa MLAPPDRPSEFRSPDELRRYLKALNEYYAIVGRPRFa

Docoglassa Platyhelminthes

Cestoda

Cyclophyllidea

Trematoda

Strigeidida

Turbellaria

Macrostomida Seriata Tricladida

Rotifera a b c

Monogononta

Plioma

Moniezia expansa NPF Taenia solium NPF Schistosoma japonicum NPF Schistosoma mansoni NPF Macrostomum lignano NPF Schmidtea mediterranea NPF1 Schmidtea mediterranea NPF2 Arthurdendyus triangulatus NPF Brachionus plicatilis NPF1 Brachionus plicatilis NPF2

NPF-like sequences determined by bioinformatics. NPF-like sequences determined by Edman degradation NPF-like sequences determined by cloning.

Sequence

PDQDSIVNPSDLVLDNKAALRDYLRQINEYFAIIGRPRFa GALTESRKQIFRNVKEFRRYLQRLDEWLAITGRPRFa AQALAKLMTLFYTSDAFNKYMENLDAYYMLRGRPRFa AQALAKLMSLFYTSDAFNKYMENLDAYYMLRGRPRFa QIQPPAKPDRFTTVEQFDTYMKQLEAYLMLIGRQRFa SGLTKQKYSLFSGPEDLRNYLRQLNEYIALSSRPRYa KVVYLKSRNHFRSDEDYVSYLRKVQKYIQLYGRPRFa KVVHLRPRSSFSSEDEYQIYLRNVSKYIQLYGRPRFa YPTPPPVPSQFLTPNDVQKYLNQLRNYYMVVGRPRFa LPPPPAKPERFTSKQQLKEYLVKLHEYYAIIGRPRFa

Reference/ accession number CO048390a EY648133a GO145290a DB912252a [37]b,c; M98854 [47]b; AJ238276 [26]b FC808552a [31]b; AJ242779 EL746900a [20]c,a; AY533028 [20]c; AY662954 EG951019a DN296735a DN302040a [9]b; [11]c FM906081a FM923948a

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17 cm  12 cm  6.5 cm plastic box [Tri-State Plastics, Inc.] with moistened sand/vermiculite mixture and pine slats measuring 4 cm  10 cm  1 cm). Tissue-specific cDNA was produced as above (DNase treated) from specific regions of the nervous system and gut dissected from workers (5 tissues pooled for processing). Transcript expression was assessed by PCR with the primers UPNPF 1 and NPF Rev, and tissue cDNA as template or total RNA to identify possible gDNA contamination (40 cycles; 94 8C, 20 s; 58.0 8C, 20 s; 72 8C, 1.5 min). Primers for b-actin (Tables 2a and 2b: F1 Bact and R1 Bact) similarly were used to verify the integrity of the tissue cDNA. PCR products as visualized in gels were photographed with GeneSnap 6.03 (SynGene). The quantities of NPF-like peptide in worker hemolymph (1.5 termite equivalents), heads (0.3 termite equivalents) and midguts (0.5 termite equivalents) were quantified with the heterologous RIA as above. Viability of workers was assessed by placing them in a Petri dish with blue-dyed cellulose for 72 h, and only those with blue gut tracts were used. Hemolymph was collected by opening three abdomens in 200 ml ice-cold 0.2% acetic acid with Complete Mini protease inhibitor cocktail (Roche Diagnostics) in 0.1 M phosphate buffered saline (PBS) for 2.5 min, then the solution was transferred to a tube on ice. Heads and midguts were dissected from termites in the same solution and transferred to tubes with fresh solution (three tissues/200 ml) for homogenization with a plastic pestle and sonication (2 s), while iced. Processed tissue samples were centrifuged (5 min, 11,300  g, 4 8C), and 150 ml of supernatant was lyophilized. 2.8. Ref NPF synthesis Peptides were synthesized with Fmoc chemistry in an Applied Biosystems 431A Peptide Synthesizer and, after precipitation with ether, purified with HPLC on a ProsphereTM C18 column (gradient program: 20% B, 5 min, 20–40% B, 5 min, 40–55% B, 30 min, 55– 100% B, 5 min; 4 ml/min; monitored at 275 nm). MALDI-TOF mass spectrometry confirmed the mass of the synthetic peptides (Mass Spectrometry Facility, University of Georgia). Peptide sequences synthesized were: Ref NPF1–30: KPSDPEQLADTLKYLEELDRFYSQVARPRF-NH2. Ref NPF1–36: VPSVWAKPSDPEQLADTLKYLEELDRFYSQVARPRF-NH2. Drm NPF: SNSRPPRKNDVNTMADAYKFLQDLDTYYGDRARVRF-NH2.

Ref NPF synthetic peptides were not soluble in water alone, so a small volume of dimethylformamide (DMF) was used to solubilize prior to dilution with saline for assays (final DMF concentration was 0.01% in assay solutions). 2.9. Gut motility assays 2.9.1. Impedance monitor Termites were transported to University of Toronto at Mississauga (Mississauga, ON) where assays were performed in the laboratory of Ian Orchard. The alimentary tract of workers (3rd to 5th instar) was pulled from of the abdomen while held in 100 ml of desert locust, Schistocerca gregaria, saline [48]. The foregut was cut free from the midgut posterior to the stomodeal valve and transferred to 100 ml fresh saline in a Sylgard (Paisley Products, Scarborough, ON, Canada) coated dish where the esophagus and anterior midgut were secured with minuten pins. Electrodes of the impedance converter were placed within 300 mm of the foregut at the crop-proventriculus junction. Electrodes were attached to an impedance converter (Model 2991, UFI, Morro Bay, CA) set in alternating current mode, and output signals sent to a linear chart recorder (0.1 V and 2 cm/ min; Linear 1200, Barnstead International). Foreguts were

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equilibrated for 15 min, followed by a 5 min background activity period, and 100 ml saline was exchanged every 5 min for aeration. Foreguts were exposed to different concentrations and combinations of leucokinin (LK I – Bachem Torrance, CA, USA), SchistoFLRFamide (Peninsula Laboratories, Belmont, CA, USA), Ref NPF1–36, Drm NPF, or serotonin (Sigma-Aldrich Canada) for 5 min, after which the tissue was rinsed four times and reequilibrated in fresh saline for 5 min. Up to four incubations with peptide or serotonin were performed per foregut preparation. 2.9.2. Force transducer Gut tracts dissected from workers as above were cut anterior to the pyloric valve, and a hair was tied around the junction of the midgut and hindgut. The hindgut was then transferred to 100 ml fresh saline in a Sylgard coated dish, and cuticle surrounding the rectum was secured to the bottom of the dish with minuten pins. The other end of the hair was tied to a miniature force transducer (Aksjeselapet Mikro-Elektronikk, Norway). After equilibration, gut movements in the presence of peptides or serotonin were recorded with AcqKnowledge software (Version 3.5.3, ß1998, BIOPAC Systems Inc.). Frequency and amplitude of force transducer readings were quantified with Clampfit software (ß2005, Molecular Devices Corporation). Readings were calculated over a 4.5 min time span within each 5 min incubation period. Means of percent increase of frequency and amplitude by serotonin and LK I were tested to see if they were significantly different from zero using a one-tailed t-test (a = 0.05) (Microsoft1 Office Excel, 2003). Means of percent inhibition of frequency and amplitude by Drm NPF were also compared to see if they were significantly different from zero using a one-tailed t-test (a = 0.05) (Microsoft1 Office Excel, 2003). 3. Results 3.1. Characterization of R. flavipes NPF The 80% CH3CN eluate from the solid phase extraction of the termite extract solution contained the most immunoreactive peptide (3.76 nmol Hez MP-I equivalents total) as estimated by heterologous RIA. This enriched extract was subjected to eight HPLC steps (Fig. 1A). In the final step, a single peak of immunoreactive peptide ‘B’ (8.4 pmol Hez MP-I equivalents by heterologous RIA) was resolved to apparent homogeneity (Fig. 1B). The mass of peptide ‘B’ was 3667.2 Da and N-terminal sequencing revealed a partial sequence: XPSDPEQLADTLXYLQEL. A series of PCR amplification steps starting with degenerate primers to this sequence and cDNA from head and midgut led to the identification of a PCR product (541 bp) encoding an open reading frame of a prepropeptide (Fig. 2A). The prepropeptide of 88 amino acids has a signal peptide, mature NPF region, and Cterminal peptide (Fig. 2A). The predicted signal peptide is cleaved between Ala27 and Lys28 (SignalP analysis [3]). The following 31 amino acids comprise a homolog of invertebrate NPFs (see below) with the last residue Gly58, a putative amide donor. This residue is followed by a dibasic Lys–Arg site, at which the C-peptide is presumably cleaved and the terminal Phe57 amidated, typifying an NPF. The mass of peptide ‘B’ corresponds to the 31 amino acid long sequence after cleavage of the C-peptide but prior to processing of Gly58. The predicted and processed sequence is that of an amidated peptide comprised of 30 amino acids (see also Section 4) with conserved NPF features, thus indicating that this cDNA encodes an authentic NPF in R. flavipes. A larger PCR product was detected in each sample, in addition to the 438 bp PCR product. The sequence of the larger product was

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Fig. 2. (A) Nucleotide sequence (GenBank GQ480479) and amino acid sequence for the prepropeptide of Reticulitermes flavipes NPF (Ref NPF). Putative signal peptide is in bold, and the putative mature 30 amino acid long peptide is underlined. The sequence is presumably processed at Gly58 forming an amide at the C-terminus. The C-peptide is in italics. (B) Additional 111 bp sequence (GenBank GQ480480) and in-frame amino acid translation in the larger form of the NPF transcript. Translation of the putative insert region is highlighted in gray. The 111 bp sequence occurs between bps 287 and 288 of the shorter transcript (arrowhead).

identical to the Ref NPF cDNA sequence except for a 111 bp insert (Fig. 2B). 3.2. Alignments and cladograms Sequences of 31 unique NPFs were determined in Arthropoda by peptide sequencing, cloning, or bioinformatics including 24 species of insects and 3 species of crustaceans (Table 2a). Another 18 sequences occur among other invertebrates, including annelids, mollusks, platyhelminths, and rotifers (Table 2b). Phylogenetic analysis of the collected sequences revealed two major groupings of NPFs (Fig. 3). Ref NPF occurs in a group comprised exclusively of arthropods, herein termed the arthropod clade (Fig. 3). The other group contains NPFs of annelids, mollusks, platyhelminths and rotifers, as well as NPFs from 7 insect species, herein termed the invertebrate clade (Fig. 3). A distinctive consensus NPF sequence is exhibited by each clade (Fig. 4; note that Drosophila and Schistosoma species are represented only once). The consensus sequence within the arthropod clade contains 24 amino acids (Fig. 4). Ref NPF strongly resembles this consensus, with identity for 16 amino acids and conservation of 4 additional amino acids (Fig. 4). The respective consensus sequence for the invertebrate clade contains 22 amino acids (Fig. 4); however, Ref NPF exhibits identity for only 9 amino acids and conservation of 3 additional ones (Fig. 4). These patterns support the placement of Ref NPF within the arthropod clade. Characteristic sequences for both clades exhibit strong conservation in the C-terminal region and little conservation in the N-terminal region. 3.3. Synthetic Ref NPFs After synthesis, Ref NPF1–30 and Ref NPF1–36 were purified by HPLC, and their masses (3610.7 and 4250.1 Da, respectively) compared to the predicted ones of 3609.8 and 4249.2 Da. RIAs with antisera to Hez MP-I and Drm NPF were compared to determine the relative affinity of each for the synthetic Ref NPFs. Ref NPF1–30 and

Ref NPF1–36 were diluted in RIA buffer to final concentrations from 0.1 fmol to 10 pmol. Hez MP-I and Drm NPF were used for the corresponding standard curves. Hez MP-I antiserum exhibited a similar affinity for both Ref NPFs (Fig. 5) but bound Hez MP-I with 10-fold greater affinity. The Drm NPF antiserum did not recognize either Ref NPF even at 10 pmol (data not shown). 3.4. Tissue specific Ref NPF-like content and Ref NPF transcripts The binding efficiency of Hez MP-I antiserum to synthetic Ref NPF1–30 and Hez MP-I by RIA facilitated an estimation of Ref NPF1– 30 in R. flavipes tissues. Regression lines were calculated from the linear region of the standard curves for Hez MP-I (1–25 fmol; slope = 0.59) and Ref NPF1–30 (5–100 fmol; slope = 0.55) and compared. Fed workers given dyed cellulose for 72 h (visible dye in the gut) had 394  33 fmol/head and 455  27 fmol/midgut (average  SE; extract of 7 tissue preparations each) of Ref NPF1–30 equivalents. NPF-like material was not detected in pooled hemolymph samples. Ref NPF transcripts, as determined by RT-PCR, were present in foregut, midgut, and all nervous system regions of workers (Fig. 6). 3.5. Gut motility assays 3.5.1. Impedance monitor Contraction patterns by different foregut regions were distinguished by careful observation and recorded with the impedance monitor. Specific placement of the electrodes discriminated contractions made by the crop-proventriculus junction from background noise (Fig. 7). Serotonin generally increased the frequency of contractions by all regions of the foregut and the duration of contractions increased with concentration. Serotonin at 107 M induced a burst of contractions lasting 45 s (Fig. 7B and D), followed by inactivity for 1 min, and then contractions lasting 45 s at the crop-proventriculus junction. Higher concentrations of serotonin (5  107 and 106 M) sustained contractions for the

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Fig. 5. Cross-reactivity of Hez MP-I antiserum to synthetic peptides as determined by RIA. Reactivity is expressed as a bound/free (B/F) ratio of the tested peptide (SEM) versus the log dose of the peptides.

Fig. 6. RT-PCR detection of Ref NPF transcripts in tissues of Reticulitermes flavipes workers. UPNPF 1 and NPF Rev primers (Table 1) were used on cDNA from brains (Br), subesophageal ganglia (SG), thoracic ganglia (T), abdominal ganglia (A), foregut (FG), midgut (MG), anterior hindgut (HG) and rectum (R). The presence of cDNA was confirmed with R. flavipes-specific b-actin forward and reverse primers (Table 1). RNA controls were run with Ref NPF primers to confirm the absence of genomic DNA.

Fig. 3. Cladogram of invertebrate NPF sequences. NPF sequences were aligned with Clustal X and trimmed. The dendrogram was constructed by Jukes-Cantor distance analysis of the sequences within GCG. *Denotes arthropod NPF sequences.

5 min monitoring period (Fig. 7A and C). Saline washes abolished serotonin-stimulated contractions. Ref NPF1–36, Drm NPF, and LK I alone over a range of concentrations did not affect contractions of this junction. SchistoFLRFamide (106 M), as a control, inhibited contractions induced with serotonin (106 and 107 M) (Fig. 7D) but in similar experiments with serotonin the NPFs had no inhibitory effect (Fig. 7A–C). 3.5.2. Force transducer The activity of dissected hindguts varied after attachment to the force transducer. Most exhibited spontaneous contractions during the equilibration period, but in the majority activity ceased before a baseline reading on gut activity was made. Both serotonin and LK I stimulated hindgut contractions. Serotonin significantly increased the frequency and amplitude of contractions at 106 and 107 M but not at 108 M (Table 3, Fig. 8). LK I induced contractions at a lower concentration (109 M, Table 3) than serotonin, and these contractions had greater amplitude and more

Table 3 Increase of frequency and amplitude of contractions in hindguts from Reticulitermes flavipes workers over background and saline washes when incubated for 5 min with serotonin or LK I (x-fold increase SEM). Each hindgut was washed 4 in saline between incubations. Treatment 108 M 107 M 106 M 109 M 108 M 107 M * **

serotonin serotonin serotonin LK I LK I LK I

Average frequency increase

Average amplitude increase

n

8.96  5.60 14.75  4.31 * 5.90  2.25 * 5.05  1.96 ** 17.24  4.93 ** 6.15  1.90

0.42  0.68 0.52  0.21 0.22  0.05 * 0.34  0.14 ** 0.78  0.22 ** 1.53  0.47

5 20 5 5 5 5

**

** **

Means significantly greater than zero: p < 0.05. Means significantly greater than zero: p < 0.02.

regular spacing than those induced by serotonin. Ref NPF1–30 and Ref NPF1–36 alone did not activate isolated hindgut contractions or inhibit those induced spontaneously or with serotonin. In contrast, Drm NPF (106 and 107 M) significantly inhibited the frequency of hindgut contractions induced by serotonin (Fig. 9A). Drm NPF at 106 M significantly reduced the frequency of hindgut contractions induced by a range of LK I concentrations (109, 108 and

Fig. 4. Manual alignment of Ref NPF with ‘‘arthropod’’ and ‘‘invertebrate’’ NPF consensus sequences. A single NPF sequence per genus from the representative clade was used to assemble each consensus to avoid bias from overrepresented groups. ( ) Majority identical. ( ) Majority conserved. ($) All residues identical. (*) All residues conserved.

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Fig. 9. Average percentage frequency inhibition (SEM) of serotonin-induced or spontaneous R. flavipes hindgut contractions by Drm NPF. Five hindguts were incubated twice for each data point. (A) 107 M serotonin with Drm NPF. (B) Spontaneous contractions with Drm NPF. Means significantly greater than zero: *p  0.02; **p  0.001.

Fig. 7. Impedance monitor readings of Reticulitermes flavipes crop-proventricular junction contractions (arrows) in response to serotonin and (A) Ref NPF1–36, (B and C) Drm NPF, and (D) SchistoFLRFamide.

107 M). Drm NPF (106 M) also reduced the frequency of spontaneously induced hindgut contractions (Fig. 9B). Drm NPF was most effective at reducing contraction frequency at 106 M, the highest concentration tested. The amplitude of hindgut contractions was not significantly affected by Drm NPF. 4. Discussion

Fig. 8. Force transducer readings of R. flavipes hindgut contractions in response to (A) 107 M serotonin and 106 M Drm NPF and (B) 109 M LK I and 106 M Drm NPF. Peptide and biogenic amine solutions were added 30 s before the traces shown.

Molecular characterization of Ref NPF adds another member to the NPY/NPF family and in particular, a second NPF for the Exopterygota/Hemimetabola (Table 2a). The sequence of Ref NPF provides a basis for phylogenetic analysis, detailed examination of tissue expression, and exploration of physiological actions. Cladograms from phylogenetic analysis of invertebrate NPF sequences reveal two evident archetypes (Fig. 3). One group consists entirely of arthropod sequences and contains Ref NPF (Fig. 3). The other group contains not only sequences from nonarthropod invertebrates, but also NPFs from seven insects (Fig. 3). Intriguingly, the insects, Nasonia vitripennis, Pediculus humanus, Ostrinia nubilalis, and Bombyx mori (see also [39]) each have two NPF sequences, one in the ‘‘arthropod clade’’ and one in the ‘‘invertebrate clade’’ (Fig. 3), the latter also containing insect NPFs for Acyrthosiphon pisum, Apis mellifera and R. prolixus. Collectively these observations suggest duplication of an ancestral NPF gene, so that two NPFs existed in an early arthropod ancestor. One NPF gene apparently predominated among arthropods, while the more ancestral form still exists in some insects and in other invertebrates. Availability of sequence information for NPFs is sparse for invertebrates. Nonetheless, associations among related species are generally evident within the larger clades. The relatedness of termite Ref NPF to migratory locust Lom NPF is expected, as both are Exopterygote insects. The association of the Kuruma shrimp Maj NPF with Ref NPF is less clear because of a limited number of

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crustacean sequences. Distinctive groupings are evident for Drosophila species, mosquitoes, mollusks, and annelids (Fig. 3). Similarity between Ref NPF and the deduced consensus sequence of arthropod NPFs is evident by inspection (Fig. 4). These sequences exhibit substantial identity of consensus amino acids and lesser identity with the consensus sequence of invertebrate NPFs. The need for more crustacean sequences is highlighted by the small peptides from giant tiger prawn, Penaus monodon [44], which also resemble Ref NPF. The high conservation of insect NPF sequences favored the identification and isolation of an immunoreactive peptide from termite tissues and its subsequent quantification in tissues (see below) with an antiserum against Hez MP-I, as it had previously for isolation of Drm NPF [6]. However, NPF in the hemolymph was either absent, undetectable by the Hez MP-I antiserum, or circulating amounts were below the detection limit of our heterologous RIA. Newly described lepidopteran NPFs (cf. Table 2a) reveal the sequence of Hez MP-I, QAARPRFa, to be a Cterminal fragment of the presumptive Hez NPF. Interestingly, an antiserum specific for Drm NPF that recognized Aea NPF [46] did not detect significant immunoreactive material in termite extracts or synthetic Ref NPF (data not shown). The Drm NPF sequence is somewhat atypical in comparison to other insect NPFs (Table 2a), particularly in the C-terminus, whereas the Hez MP-I sequence proved a more functional antigen to detect NPF sequences. The issue of the length of Ref NPF is somewhat perplexing. An inconclusive residue corresponding to Lys from the Ref NPF cDNA sequence was the first residue revealed by N-terminus sequencing of the purified peptide. Lys28 at the N-terminus would correspond to a mature amidated peptide of 30 amino acids. Indeed, SignalP analysis [3] of Ref NPF cDNA predicted signal peptide cleavage between residues Ala27 and Lys28 (Fig. 2). Such a product is shorter than the prototypical 36 amino acid peptide characteristic of most other invertebrate NPFs [8]. The expected 36 residue NPF resulting from a putative signal peptide cleavage between residues Ile20 and Val21 was not isolated. Only one of the three groups of fractions in the termite extract containing NPF-immunoreactive material was purified to homogeneity (Fig. 1). The remaining fractions could have additional forms of NPF or sNPFs, since the Hez MP-I antiserum also detected an Ae. aegypti sNPF [34]. An alternate explanation is that the Ref NPF isolated and sequenced was a larger peptide partially degraded at the susceptible basic site (Lys28) during extraction in a hot acid solution. Such a circumstance has precedence in the isolation of Drm NPF, for which a 29 amino acid peptide was initially sequenced and the full-length 36 residue form was only found subsequently by mass spectrometry [6]. A similar predicament was found in the identification of Lom NPF [8], for which a mass corresponding to the prediction of the signal sequence cleavage was not found. Drm NPF8–36 and Drm NPF1–36 were found to bind to the Anopheles gambiae NPF receptor with equal affinity [14]. This circumstance is consistent with the absence of conserved amino acids in the N-terminal regions of NPFs (cf. Tables 2a and 2b). Among NPF genes, a prototypical intron provides additional evidence for NPY-NPF homology [31]. Each vertebrate NPY, PPY and PP gene, contains an intron of variable size in the region that corresponds to the penultimate Arg residue in the coding sequence [31]. Such an intron exists in NPF genes from An. gambiae [14], B. mori [39], Ae. aegypti, and A. mellifera, but not in D. melanogaster [6]. An additional 111 bp sequence in the longer Ref NPF product amplified from cDNA (Fig. 2) is similarly positioned. The two transcripts are identical except for the 111 bp region suggesting they are not transcripts from two different genes. Whether this product comprises an incompletely processed intron or an alternatively spliced transcript was not resolved in our study, but it was not a result of genomic contamination as no products

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were amplified from DNase treated total RNA (Fig. 6). A comparable incomplete splice product also occurred in An. gambiae NPF PCR products [14], similar to those for An. gambiae adipokinetic hormone I, for which introns are not removed in transcripts from the abdominal tissue [22]. Curiously, the translation of the longer termite NPF transcript would result in a peptide with an internal region resembling an NPF-like sequence (cf. Fig. 2B). A similar occurrence has been reported in B. mori where an alternatively spliced transcript codes for a possible NPF (B. mori NPF 1b) although detection of this peptide has not been confirmed [39]. Further exploration with mass spectrometry and in situ hybridization may resolve the nature of the longer transcript in R. flavipes. Characterization of Ref NPF expression in the brain–gut axis of this termite provides an impetus to explore potential biological activities, as well as comparisons of the effects of Ref NPF1–30 and Ref NPF1–36 to shed light on the processing of the pro-peptide. The presence of NPF-like material in axons on the foregut and midgut of workers suggested that the endogenous NPF may be involved in the regulation of gut motility [34]. In this study, the effect of serotonin, LK I, and SchistoFLRFamide on motility by gut regions isolated from R. flavipes workers was similar to that reported for other insects [1,35,40]. Ref and Drm NPFs had no evident effect on crop-proventriculus contractions of R. flavipes foreguts, but they were stimulated by serotonin and inhibited by SchistoFLRFamide, thus indicating that foregut preparations used in this experiment were viable and receptive to biogenic amines and other peptides. Hindgut contractions of R. flavipes were stimulated by both LK I and serotonin. LK I is a stimulator of hindgut tissue for inhibition assays [40]. Drm NPF inhibited spontaneous and induced hindgut contractions (Fig. 9), suggesting the presence of NPF receptors in this region, although no NPF-containing axons were detected on the anterior hindgut of R. flavipes [34]. A network of NPF-like axons covers the hindgut of other insects such as H. zea [18] and R. prolixus [15], and both Drm NPF and Ang NPF inhibited R. prolixus hindgut contractions [16]. Furthermore, neither synthetic Ref NPF1–30 nor NPF1–36 influenced hindgut activity in our assays suggesting that other peptides in termites may more closely resemble Drm NPF and interact with receptors on the hindgut. However, results from bioassays using heterologous neuropeptides must be viewed with caution. Unfortunately there was insufficient native Ref NPF peptide remaining after the purification and sequencing to examine its activity directly on alimentary preparations. Although Drm NPF and Ref NPF exhibit only limited similarity (cf. Table 2a), the actions of the fly peptide on the termite tissue are intriguing. The function of NPF is not resolved for any insect other than D. melanogaster. Phenotypes resulting from altered genetic expression of NPF or its receptor show that NPF promotes feeding-related behaviors that prevent starvation among other behavior processes [30,50,51]. There are even fewer reports of NPF effects on tissues isolated from insects [16,27,35]. Despite the different results obtained with Ref NPF and Drm NPF, this study showed that serotonin and two neuropeptides known to affect gut contraction in other insects are similarly potent in termite gut assays. Actions related to feeding and digestion are the bane of termite activities for humans and understanding their regulation, which likely includes NPF, may lead to the discovery of alternative strategies to current management tactics [13]. Acknowledgements This research project was supported in part by the Microchemical and Proteomics Core of the Emory University School of Medicine, Atlanta, GA. We thank Stephen Garczynski for assistance with the RIA, Kevin Clark for synthesis of Ref NPFs and Drm NPF,

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