Predicted novel hypertrehalosaemic peptides of cockroaches are verified by mass spectrometry.

Small neuropeptides from the corpora cardiaca are responsible in cockroaches for the mobilisation of trehalose from the fat body into the haemolymph. Such hypertrehalosaemic hormones (HrTHs) belong to the large family of insect adipokinetic hormones (AKHs); a few HrTHs were previously sequenced from cockroaches, and from genomic and/or transcriptomic information one may predict the genes encoding HrTHs from more species. Definite elucidation of the primary structure of the mature peptide with putative modifications needs analytical chemical methods. In the current study, we use high-resolution mass spectrometry coupled with liquid chromatography to identify unequivocally the HrTHs of 13 cockroach species. Either genomic/transcriptomic information was available for most of the species examined, or from related species. We confirm predicted novel sequences and find hydroxyproline modification for the majority of the peptides. The novel decapeptides are structurally close to Bladi-HrTH, which is found in all seven of the investigated blaberid subfamilies. Bladi-HrTH and all the novel peptides elicit a hypertrehalosaemic response in Periplaneta americana, a blattid cockroach.

Mass Spectrometric Proof of Predicted Peptides: Novel Adipokinetic Hormones in Insects.

The importance of insects in our ecosystems is undeniable. The indiscriminate use of broad-spectrum insecticides is a factor in the decline in insect biomass. We identify and sequence a prominent neuropeptide hormone in insects with an overarching goal to elucidate relatedness and create a database of bioactive peptides that could inform possible cross-activity in biological assays for the identification of a biorational lead compound. The major task of an adipokinetic hormone (AKH) in an insect is the regulation of metabolic events, such as carbohydrate and lipid breakdown in storage tissue during intense muscular work. From genomic and/or transcriptomic information one may predict the genes encoding neuropeptides such as the AKHs of insects. Definite elucidation of the primary structure of the mature peptide with putative post-translational modifications needs analytical chemical methods. Here we use high-resolution mass spectrometry coupled with liquid chromatography to identify unequivocally the AKHs of five insect species (one cockroach, two moths, and two flies) of which either genomic/transcriptomic information was available or sequences from related species. We confirm predicted sequences and discover novel AKH sequences, including one with a post-translational hydroxyproline modification. The additional sequences affirm an evolutionary pattern of dipteran AKHs and a conserved pattern in crambid moths.

Solid-Phase Synthesis of an Insect Pyrokinin Analog Incorporating an Imidazoline Ring as Isosteric Replacement of a trans Peptide Bond.

A facile solid-phase synthetic method for incorporating the imidazoline ring motif, a surrogate for a trans peptide bond, into bioactive peptides is reported. The example described is the synthesis of an imidazoline peptidomimetic analog of an insect pyrokinin neuropeptide via a cyclization reaction of an iminium salt generated from the preceding amino acid and 2,4-diaminopropanoic acid (Dap).

The Adipokinetic Peptides in Diptera: Structure, Function, and Evolutionary Trends.

Nineteen species of various families of the order Diptera and one species from the order Mecoptera are investigated with mass spectrometry for the presence and primary structure of putative adipokinetic hormones (AKHs). Additionally, the peptide structure of putative AKHs in other Diptera are deduced from data mining of publicly available genomic or transcriptomic data. The study aims to demonstrate the structural biodiversity of AKHs in this insect order and also possible evolutionary trends. Sequence analysis of AKHs is achieved by liquid chromatography coupled to mass spectrometry. The corpora cardiaca of almost all dipteran species contain AKH octapeptides, a decapeptide is an exception found only in one species. In general, the dipteran AKHs are order-specific- they are not found in any other insect order with two exceptions only. Four novel AKHs are revealed by mass spectrometry: two in the basal infraorder of Tipulomorpha and two in the brachyceran family Syrphidae. Data mining revealed another four novel AKHs: one in various species of the infraorder Culicumorpha, one in the brachyceran superfamily Asiloidea, one in the family Diopsidae and in a Drosophilidae species, and the last of the novel AKHs is found in yet another Drosophila. In general, there is quite a biodiversity in the lower Diptera, whereas the majority of the cyclorraphan Brachycera produce the octapeptide Phote-HrTH. A hypothetical molecular peptide evolution of dipteran AKHs is suggested to start with an ancestral AKH, such as Glomo-AKH, from which all other AKHs in Diptera to date can evolve via point mutation of one of the base triplets, with one exception.

Structural diversity of adipokinetic hormones in the hyperdiverse coleopteran Cucujiformia.

Seventeen species of the coleopteran series Cucujiformia are investigated for the presence and sequence of putative adipokinetic hormones (AKHs). Cucujiformia includes species from the major superfamilies, that is, Chrysomeloidea, Curculionoidea, Cucujoidea, and Tenebrionoidea. The clade Phytophaga in which the Chrysomeloidea and Curculionoidea reside, harbor very detrimental species for agriculture and forestry. Thus, this study aims not only to demonstrate the structural biodiversity of AKHs in these beetle species and possible evolutionary trends but also to determine whether the AKHs from harmful pest species can be used as lead substances for a future putative insecticide that is harmless to beneficial insects. Sequence analysis of AKHs is achieved by liquid chromatography coupled to mass spectrometry. Most of the investigated species contain AKH octapeptides in their corpora cardiaca, although previously published work also found a few decapeptides, which we comment on. The signature and sole AKH in cerambycidae Chrysomeloidea and Curculionoidea is Peram-CAH-I (pEVNFSPNW amide), which is also found in the majority of chrysomelidae Chrysomeloidea and in the one investigated species of Cucujoidea albeit in a few cases associated with a second AKH which can be either Peram-CAH-II (pELTFTPNW amide), Emppe-AKH (pEVNFTPNW amide), or Micvi-CC (pEINFTPNW amide). The most often encountered AKH in Tenebrionoidea, family Meloidae as well as family Tenebrionidae, is Tenmo-HrTH (pELNFSPNW amide) followed by Pyrap-AKH (pELNFTPNW amide) and a Tenmo-HrTH extended decapeptide (in Meloidae). Finally, we examine AKH sequences from 43 species of cucujiform beetles, including the superfamily Coccinelloidea for a possible lead compound for producing a cucujiform-specific pesticide.

The hypertrehalosaemic neuropeptide conformational twins of cicadas consist of only L-amino acids: are they cis-trans isomers?

It is known for almost 25 years that the corpora cardiaca (neurosecretory glands) of cicadas synthesize two isobaric peptides with hypertrehalosaemic activity denominated Placa-HrTH-I and II. Both decapeptides have the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn amide) and mass, but differ in their chromatographic retention time. The slightly more hydrophobic peptide, Placa-HrTH-II, co-elutes with the synthetic peptide of the same sequence and is less active in biological assays than Placa-HrTH-I. Ion mobility separation in conjunction with high-resolution mass spectrometry detected the differing structural feature between both peptides in the region Pro6-Ser7-Trp8. Here, it was shown that Placa-HrTH-I co-eluted with a synthetic peptide containing D-Pro in position 6, while dextrorotatory amino acid residues in positions 7 and 8 could be excluded in this way. Amino acid hydrolysis followed by chiral analysis using a relative of Marfey's reagent was then used to validate the presence of D-Pro in Placa-HrTH-I. Interestingly, this experiment unambiguously proved both the absence of D-Pro and the presence of L-Pro in Placa-HrTH-I. Racemization as a reason for the structural differences of the twin adipokinetic hormones was hence ruled out and cis-trans isomerism as the likely alternative came into focus. It remains to be investigated if Pro6 in cis-conformation is indeed present and responsible for the increased bioactivity of Placa-HrTH-I.

Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C-terminal amidated octapeptide Manse-AT (6-13). We identified three residues essential for bioactivity (Thr4, Arg6 and Phe8) by assaying alanine-replacement analogs of Manse-AT (6-13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10-13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10-13) validated our hypothesis. The IC50 value of antagonist Manse-AT (10-13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10-13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

Molecular characterization of an adipokinetic hormone-related neuropeptide (AKH) from a mollusk.

Adipokinetic hormones (AKH) are key regulators of energy mobilization in insects. With the growing number of genome sequence available, the existence of genes encoding AKH related peptides has now been established in protostomes. Here we investigated the occurrence of a mature AKH-like neuropeptide (Cg-AKH) in the oyster Crassostrea gigas. We unambiguously elucidated the primary structure of this neuropeptide by mass spectrometry from peptidic extracts of oyster visceral ganglia. Cg-AKH mature peptide (pQVSFSTNWGS-amide) represents an additional member of the AKH family of peptides. The organization of Cg-AKH encoding gene and its corresponding transcript is also described. Cg-AKH gene was found to be expressed in the nervous system though at extremely low levels compared to other neuropeptide encoding genes such as the oyster GnRH gene. Although both reproduction and feeding are known to affect the energy balance in oysters, no significant differential expression of Cg-AKH gene could be evidenced in relation with the nutritional status or along the reproductive cycle. The possible involvement of Cg-AKH in the regulation of energy balance in oyster remains an open question.

Novel members of the adipokinetic hormone family in beetles of the superfamily Scarabaeoidea.

Eight beetle species of the superfamily Scarabaeoidea were investigated with respect to peptides belonging to the adipokinetic hormone (AKH) family in their neurohemal organs, the corpora cardiaca (CC). The following beetle families are represented: Scarabaeidae, Lucanidae, and Geotrupidae. AKH peptides were identified through a heterospecific trehalose-mobilizing bioassay and by sequence analyses, using liquid chromatography coupled to positive electrospray mass spectrometry (LC-ESI-MS) and analysis of the tandem MS2 spectra obtained by collision-induced dissociation. All the beetle species have octapeptide AKHs; some have two AKHs, while others have only one. Novel AKH members were found in Euoniticellus intermedius and Circellium bacchus (family Scarabaeidae), as well as in Dorcus parallelipipedus (family Lucanidae). Two species of the family Geotrupidae and two species of the Scarabaeidae subfamily Cetoniinae contain one known AKH peptide, Melme-CC, while E. intermedius produces a novel peptide code named Euoin-AKH: pEINFTTGWamide. Two AKH peptides were each identified in CC of C. bacchus and D. parallelipipedus: the novel Cirba-AKH: pEFNFSAGWamide and the known peptide, Scade-CC-I in the former, and the novel Dorpa-AKH: pEVNYSPVW amide and the known peptide, Melme-CC in the latter. Kheper bonelli (subfamily Scarabaeinae) also has two AKHs, the known Scade-CC-I and Scade-CC-II. All the novel peptides were synthesized and the amino acid sequence assignments were unequivocally confirmed by co-elution of the synthetic peptides with their natural equivalent, and identical MS parameters of the two forms. The novel synthetic peptides are all active in inducing hypertrehalosemia in cockroaches.

The discovery of a novel antagonist - Manduca sexta allatotropin analogue - as an insect midgut active ion transport inhibitor.

BACKGROUND: The midgut is an important site for both nutrient absorption and ionic regulation in lepidopteran larvae, major pests in agriculture. The larval lepidopteran midgut has become a potent insecticide target over the past few decades. Recent studies have shown that an insect neuropeptide, Manduca sexta allatotropin (Manse-AT), exhibits inhibition of active ion transport (AIT) across the larval midgut epithelium. The full characteristic of the AIT inhibition capacity of Manse-AT is essential to assay. In this study, AIT inhibition across the M. sexta midgut by Manse-AT and its analogues in a range of concentrations was assayed. The structure-activity relationship of Manse-AT was also studied by truncated and alanine-replacement strategies. RESULTS: Our results identified three residues, Thr4, Arg6 and Phe8, as the most important components for activity on the midgut. Replacement of Glu1, Met2 and Met3 reduced the potency of the analogues. The conservative substitution of Gly7 with alanine had little effect on the potency of the analogues. We demonstrated for the first time that Manse-AT (10-13) behaves as a potent antagonist in vitro on active ion transport across the epithelium of the posterior midgut in M. sexta. CONCLUSION: Structure-activity studies of Manse-AT are useful in developing lead compounds for the design and testing of synthetic antagonists, ultimately to develop potent and specific pest control strategies. Manse-AT (10-13) has been discovered as the first Manse-AT antagonist, with a significant effect and a short sequence compared with other insect neuropeptides. It may be a new potential pest control agent in the future. © 2016 Society of Chemical Industry.

Two novel tyrosine-containing peptides (Tyr(4)) of the adipokinetic hormone family in beetles of the families Coccinellidae and Silphidae.

Novel members of the adipokinetic hormone family of peptides have been identified from the corpora cardiaca (CC) of two species of beetles representing two families, the Silphidae and the Coccinellidae. A crude CC extract (0.3 gland equivalents) of the burying beetle, Nicrophorus vespilloides, was active in mobilizing trehalose in a heterologous assay using the cockroach Periplaneta americana, whereas the CC extract (0.5 gland equivalents) of the ladybird beetle, Harmonia axyridis, exhibited no hypertrehalosemic activity. Primary sequences of one adipokinetic hormone from each species were elucidated by liquid chromatography coupled to electrospray mass spectrometry (LC-MS). The multiple MS(N) electrospray mass data revealed an octapeptide with an unusual tyrosine residue at position 4 for each species: pGlu-Leu-Thr-Tyr-Ser-Thr-Gly-Trp amide for N. vespilloides (code-named Nicve-AKH) and pGlu-Ile-Asn-Tyr-Ser-Thr-Gly-Trp amide for H. axyridis (code-named Harax-AKH). Assignment of the correct sequences was confirmed by synthesis of the peptides and co-elution in reversed-phase high-performance liquid chromatography with fluorescence detection or by LC-MS. Moreover, synthetic peptides were shown to be active in the heterologous cockroach assay system, but Harax-AKH only at a dose of 30 pmol, which explains the negative result with the crude CC extract. It appears that the tyrosine residue at position 4 can be used as a diagnostic feature for certain beetle adipokinetic peptides, because this feature has not been found in another order other than Coleoptera.

Isolation and identification of two novel attractant compounds from Chinese cockroach (Eupolyphaga sinensis Walker) by combination of HSCCC, NMR and CD techniques.

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hextane-ethyl acetate-methanol-water (1.5:1:1.5:1, v/v/v/v) was applied to the isolation and purification of attractants from Chinese cockroach, Eupolyphaga sinensis Walker. Two new attractants with attractant activity towards the male insects were obtained from the extract sample in a one-step separation. Their purities were determined by HPLC. Subsequent MS, NMR and CD analyses have led to the characterization of (R)-3-ethyl-6,8-dihydroxy-7-methyl-3,4-dihydroisochromen-1-one (1) and (R)-6,8-dihydroxy-3,7-dimethyl-3,4-dihydroisochromen-1-one (2), two novel isocumarin type attractants. Based on these results, it is concluded that HSCCC is a viable separation method option for purifying insect attractants, while effectively maintaining the attracting activity of the isolates. This is the first attempt to apply counter-current chromatography technique to separate attractants from Chinese cockroach.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

Functional characterization of the adipokinetic hormone in the pea aphid, Acyrthosiphon pisum.

Aphids are important plant phloem-sucking pests and detailed knowledge about the hormonal control of their metabolism can potentially contribute to the development of methods for their management. The insect metabolism is predominantly controlled by neuropeptides belonging to the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH). The main goal of this study was to obtain the sequence of AKH transcripts and analyze its expression in all polyphenic female forms of the pea aphid, Acyrthosiphon pisum. The neuropeptide is expressed in the brain of all female forms and in the ovaries of the both (wingless and winged) parthenogenetic forms. The form of active Acypi-AKH decapeptide was confirmed by the LC/MS and +ESI tandem mass spectrometry. The highest relative amount of Acypi-AKH was recorded in winged virginoparae. Furthermore, a potential role of this hormone when directly applied to the aphid was studied as well. Interestingly, no significant increase of trehalose in the wingless virginoparae after application of synthetic Acypi-AKH was detected. Yet this treatment did affect the level of protective polyol (mannitol) and furthermore led to increased activity of the detoxification enzyme glutathione S-transferase. The possible physiological function of AKH in A. pisum under the stress conditions is discussed.

Adipokinetic hormones (AKHs) of sphingid Lepidoptera, including the identification of a second M. sexta AKH.

The adipokinetic hormones (AKHs) from the corpora cardiaca (CC) of representative species from all three subfamilies of the Sphingidae (hawkmoths) were investigated using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) and liquid chromatography electrospray ion trap mass spectrometry (LC-ESI MS), including a re-examination of the AKH complement of the tobacco hawkmoth, Manduca sexta. In addition to larvae and adults of M. sexta (subfamily: Sphinginae), adults from the following subfamilies were examined: Macroglossinae (large elephant hawkmoth, Deilephila elpenor), Smerinthinae (poplar hawkmoth, Laothoe populi and eyed hawkmoth, Smerinthus ocellata), and Sphinginae (death's head hawkmoth, Acherontia atropos). All moths are shown to have the nonapeptide Manse-AKH (pELTFTSSWGamide) [corrected] in their CC, together with a second AKH, which, on the basis of mass ions ([M+Na](+), [M+K](+)) and partial sequence analysis is identical in all species examined. The structure of this AKH was extracted from the CC [corrected] of adult M. sexta and shown, by ESI-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), to be a novel decapeptide AKH with a sequence of pELTFSSWGQamide. [corrected]. The new peptide has been code named Manse-AKH-II. Sequence confirmation was obtained from identical MS studies with synthetic Manse-AKH-II and with the native peptide. Manse-AKH-II has significant lipid-mobilizing activity when injected at low dose (5pmol) into newly emerged adult M. sexta. The potential implications of a second AKH, in M. sexta in particular, are discussed in relation to putative receptor(s).

The effects of linear and cyclic analogs of Locmi-DH, Dippu-DH(46) and Dippu-DH(31) on appetitive behavior in Locusta migratoria.

The effects of analogs of the diuretic peptides Locmi-DH, Dippu-DH(46) and Dippu-DH(31) on two aspects of appetitive behavior are investigated in previously food-deprived nymphs of Locusta migratoria. The analogs tested are the C-terminal 15-mer and nonapeptides and their corresponding cyclic analogs. At a nominal dose of 1pmol injected per nymph, the linear fragments and their cyclic analogs of Dippu-DH(46) display no significant effects on the latency to feed or on the length of the first meal in nymphs. However, at the same dose, the linear fragments of Dippu-DH(31) and their cyclic analogs, and analogs of Locmi-DH modulate appetitive behavior: they are anorexigenic in reducing the duration of the first meal, and generally increasing the latency to feed. The cyclic analogs of Dippu-DH(31) are at least as effective as their linear counterparts in influencing these aspects of appetitive behavior in locust nymphs.

Peptidomics and peptide hormone processing in the Drosophila midgut.

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH(31), CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism.

Isolation and characterization of the cDNA encoding DH(31) in the kissing bug, Rhodnius prolixus.

Rhodnius prolixus undergoes a period of rapid diuresis after ingesting large blood meals. Neurohormones with either diuretic or anti-diuretic activity control diuresis by acting on several tissues including the Malpighian tubules. One of the neurohormones that potentially plays a role in diuresis is diuretic hormone 31 (DH(31)) which belongs to the insect calcitonin-like family of diuretic hormones. Here we determine the complete cDNA sequences of three Rhopr-DH(31) splice variants (Rhopr-DH(31)-A, Rhopr-DH(31)-B and Rhopr-DH(31)-C) and characterize their expression in unfed fifth-instar R. prolixus. Reverse transcriptase-PCR demonstrates that Rhopr-DH(31) is predominantly expressed in the central nervous system (CNS) of unfed fifth-instars. However, the expression of the three splice variants differs with Rhopr-DH(31)-B expression being the highest followed by Rhopr-DH(31)-A and Rhopr-DH(31)-C, as determined using semi-quantitative Southern blot analysis. Fluorescent in situ hybridization reveals that Rhopr-DH(31) is expressed in a variety of cells in the CNS, including some neurosecretory cells.

Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding.

We cloned the gene that encodes prothoracicotropic hormone (PTTH) in the northern house mosquito, Culex pipiens, and investigated its expression profile in short-day (diapause-destined) and long-day (nondiapause-destined) individuals from the fourth-instar larval stage to 2 months of adulthood, as well as after a blood meal. The deduced C. pipiens PTTH (Cupip-PTTH) amino acid sequence contains seven cysteines with a specific spacing pattern. Sequence alignment suggests that Cupip-PTTH is 23% identical to Drosophila melanogaster PTTH, but is ≥59% identical to the PTTHs of other mosquitoes. Cupip-PTTH has structural characteristics similar to those of Bombyx mori PTTH and some vertebrate nerve growth factors with cysteine-knot motifs. PTTH transcripts exhibit a daily cycling profile during the final (fourth) larval instar, with peak abundance occurring late in the scotophase. The fourth-larval instar stage is one day longer in short-day larvae than in long-day larvae, resulting in larger larvae and adults. This additional day of larval development is associated with one extra PTTH cycle. No cycling was observed in pupae, but PTTH transcripts were slightly higher in short-day pupae than in long-day pupae throughout much of the pupal stage. PTTH expression persisted at a nearly constant level in diapausing adult females for the first month but then dropped by ∼50%, while expression decreased at the beginning of adulthood in nondiapausing females and then remained at a low level as long as the females were denied a blood meal. However, when nondiapausing females were offered a blood meal, PTTH transcripts rose approximately 7 fold in 2 h and remained elevated for 24 h. A few diapausing females (∼10%) will take a blood meal when placed in close proximity to a host, but much of the blood is ejected and such meals do not result in mature eggs. Yet, elevated PTTH mRNA expression was also observed in diapausing females that were force fed. Our results thus point to several distinctions in PTTH expression between short-day and long-day mosquitoes, but both types of females responded to a blood meal by elevating levels of PTTH mRNA.

Adipokinetic hormones provide inference for the phylogeny of odonata.

Adipokinetic neuropeptides from the corpora cardiaca of 17 species of Odonata encompassing mainly the families Corduliidae and Libellulidae were isolated and structurally elucidated using liquid chromatography coupled with ion trap electrospray ionization mass spectrometry. It became evident that all species of the family Corduliidae studied express the peptide code-named Libau-AKH (pGlu-Val-Asn-Phe-Thr-Pro-Ser-Trp amide), which is also present in all but one libellulid species, Erythemis simplicicollis which expresses Erysi-AKH (pGlu-Leu-Asn-Phe-Thr-Pro-Ser-Trp amide). This divergence from all other Libellulids is due to a nonsynonymous missense single nucleotide polymorphism (SNP) in the nucleotide coding sequence (CDS) of prepro-AKH CDS and supports the polyphyletic nature of Sympetrinae and other subfamilies of libellulids. Despite this exception, these findings then support the hypothesis that Corduliidae and Libellulidae are closely related as stated in most phylogenies. The presence of Anaim-AKH (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide) in Macromiidae likely distinguishes species in this family from Corduliidae. Current molecular genetic phylogenies and our AKH findings suggest that Syncordulia gracilis, which expresses Anaim-AKH, does not belong in Corduliidae. Evolution of AKHs in anisopteran Odonata are likely due to nucleotide substitution involving nonsynonymous missense SNPs in the CDS of prepro-AKH.