Comparative analysis of adipokinetic hormones and their receptors in Blattodea reveals novel patterns of gene evolution.

Adipokinetic hormone (AKH) is a neuropeptide produced in the insect corpora cardiaca that plays an essential role in mobilising carbohydrates and lipids from the fat body to the haemolymph. AKH acts by binding to a rhodopsin-like G protein-coupled receptor (GPCR), the adipokinetic hormone receptor (AKHR). In this study, we tackle AKH ligand and receptor gene evolution as well as the evolutionary origins of AKH gene paralogues from the order Blattodea (termites and cockroaches). Phylogenetic analyses of AKH precursor sequences point to an ancient AKH gene duplication event in the common ancestor of Blaberoidea, yielding a new group of putative decapeptides. In total, 16 different AKH peptides from 90 species were obtained. Two octapeptides and seven putatively novel decapeptides are predicted for the first time. AKH receptor sequences from 18 species, spanning solitary cockroaches and subsocial wood roaches as well as lower and higher termites, were subsequently acquired using classical molecular methods and in silico approaches employing transcriptomic data. Aligned AKHR open reading frames revealed 7 highly conserved transmembrane regions, a typical arrangement for GPCRs. Phylogenetic analyses based on AKHR sequences support accepted relationships among termite, subsocial (Cryptocercus spp.) and solitary cockroach lineages to a large extent, while putative post-translational modification sites do not greatly differ between solitary and subsocial roaches and social termites. Our study provides important information not only for AKH and AKHR functional research but also for further analyses interested in their development as potential candidates for biorational pest control agents against invasive termites and cockroaches.

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.

The unique C-mannosylated hypertrehalosemic hormone of Carausius morosus: Identity, release, and biological activity.

Previous studies had shown that the corpora cardiaca (CC) of the Indian stick insect, Carausius morosus, synthesizes two hypertrehalosemic hormones (HrTHs)-decapeptides which differ in the way that the chromatographically less-hydrophobic form, code-named Carmo-HrTH-I, is modified by an unique C-mannosylated tryptophan residue at position 8. The availability of milligram amounts of this modified peptide in synthetic form now makes it possible to study physico-chemical and physiological properties. This study revealed that the synthetic peptide co-elutes with the natural peptide from the CC chromatographically, is heat stable for at least 30 min at 100°C, and causes hyperlipemia in acceptor locusts (a heterologous bioassay) and hypertrehalosemia in ligated stick insects (conspecific bioassay). In vitro incubation of Carmo-HrTH-I together with stick insect hemolymph (a natural source of peptidases) demonstrated clearly via chromatographic separation that the C-mannosylated Trp bond is stable and is not broken down to Carmo-HrTH-II (the more-hydrophobic decapeptide with an unmodified Trp residue). This notwithstanding, breakdown of Carmo-HrTH-I did take place, and the half-life of the compound was calculated as about 5 min. Finally, the natural peptide is releasable when CC are treated in vitro with a depolarizing saline (high potassium concentration) suggesting its role as true HrTHs in the stick insect. In conclusion, the results indicate that Carmo-HrTH-I which is synthesized in the CC is released into the hemolymph, binds to a HrTH receptor in the fat body, activates the carbohydrate metabolism pathway and is quickly inactivated in the hemolymph by (an) as yet unknown peptidase(s).

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.

Synthesis of C-Mannosylated Glycopeptides Enabled by Ni-Catalyzed Photoreductive Cross-Coupling Reactions.

The biological functions of tryptophan C-mannosylation are poorly understood, in part, due to a dearth of methods for preparing pure glycopeptides and glycoproteins with this modification. To address this issue, efficient and scalable methods are required for installing this protein modification. Here, we describe unique Ni-catalyzed cross-coupling conditions that utilize photocatalysis or a Hantzsch ester photoreductant to couple glycosyl halides with (hetero)aryl bromides, thereby enabling the α-C-mannosylation of 2-bromo-tryptophan, peptides thereof, and (hetero)aryl bromides more generally. We also report that 2-(α-d-mannopyranosyl)-L-tryptophan undergoes facile anomerization in the presence of acid: something that must be considered when preparing and handling peptides with this modification. These developments enabled the first automated solid-phase peptide syntheses of C-mannosylated glycopeptides, which we used to map the epitope of an antibody, as well as providing the first verified synthesis of Carmo-HrTH-I, a C-mannosylated insect hormone. To complement this approach, we also performed late-stage tryptophan C-mannosylation on a diverse array of peptides, demonstrating the broad scope and utility of this methodology for preparing glycopeptides.

Biochemically identified neuropeptides in a caddisfly (Trichoptera) and a pygmy mole cricket (Orthoptera: Caelifera: Tridactyloidea).

One representative of the order Trichoptera, namely the caddisfly Chaetopteryx villosa, was investigated along with the pygmy mole cricket Xya capensis which is a representative of the most basal superfamily of the caeliferan Orthoptera, that is, the Tridactyloidea. From both clades neuropeptides have not been biochemically characterized before this study. Here, members of the adipokinetic hormone family (AKHs) are sequenced via liquid chromatography (LC)-ion trap mass spectrometry from methanolic extracts from the corpora cardiaca of respective species. The corpora cardiaca were dissected, methanolic extracts prepared, peptides separated by liquid chromatography (LC), and AKHs detected and sequenced by ion trap mass spectrometry. Both species investigated contain an octapeptide AKH: the trichopteran species has the peptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Ser-Trp amide; the ambiguity of the isobaric amino acids Leu and Ile at position two was solved by comparing retention times on LC and by co-elution with the synthetic Leu2 -form. This peptide is known as Aedae-AKH and found in certain dipteran species and in an alderfly (Megaloptera). The tridactyloid species contains the peptide with the sequence pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide which had first been identified in a member of the order Mantophasmatodea and is called Manto-CC. Comparisons are made between the AKH complements of the sister groups Trichoptera and Lepidoptera and their possible relatedness and, on the other hand, between the AKH of X. capensis with those of closely related caeliferan superfamilies. The biology of the two studied species is used to speculate about a possible function of the elucidated hormones. Lastly, the use of a larval stage as starting material for structural neuropeptide information is discussed.

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.

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.

D-Proline: Comment to "An overview on D-amino acids".

An excellent 2017 review in this journal about D-amino acids by Genchi aims for a comprehensive representation of the current state of knowledge. Unfortunately, information about both D-proline and proline racemase is almost entirely missing. In our investigations into D/L-Pro-containing neuropeptides in cicadas, we have performed literature surveys in this context. Proline racemases in bacteria are known since 1957; their function has been studied mostly in prokaryotes and, more recently, proline racemase was identified in the unicellular eukaryotic parasite Trypanosoma cruzi. Published data on D-proline and/or proline racemase in other species are rare or non-existent.

Influence of aminergic and peptidergic substances on heart beat frequency in the stick insect Carausius morosus (Insecta, Phasmatodea).

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi-exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo-HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo-HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo-HrTH-II, thereby rendering the HrTH peptide "not active." In ligated stick insects, 6.7 × 10-8  M Carmo-HrTH-II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose-dependent manner; inhibition was achieved with 10-4  M of octopamine.

Analysis of Peptide Ligand Specificity of Different Insect Adipokinetic Hormone Receptors.

Adipokinetic hormone (AKH) is a highly researched insect neuropeptide that induces the mobilization of carbohydrates and lipids from the fat body at times of high physical activity, such as flight and locomotion. As a naturally occurring ligand, AKH has undergone quite a number of amino acid changes throughout evolution, and in some insect species multiple AKHs are present. AKH acts by binding to a rhodopsin-like G protein-coupled receptor, which is related to the vertebrate gonadotropin-releasing hormone receptors. In the current study, we have cloned AKH receptors (AKHRs) from seven different species, covering a wide phylogenetic range of insect orders: the fruit fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti (Diptera); the red flour beetle, Tribolium castaneum, and the large pine weevil, Hylobius abietis (Coleoptera); the honeybee, Apis mellifera (Hymenoptera); the pea aphid, Acyrthosiphon pisum (Hemiptera); and the desert locust, Schistocerca gregaria (Orthoptera). The agonistic activity of different insect AKHs, including the respective endogenous AKHs, at these receptors was tested with a bioluminescence-based assay in Chinese hamster ovary cells. All receptors were activated by their endogenous ligand in the nanomolar range. Based on our data, we can refute the previously formulated hypothesis that a functional AKH signaling system is absent in the beneficial species, Apis mellifera. Furthermore, our data also suggest that some of the investigated AKH receptors, such as the mosquito AKHR, are more selective for the endogenous (conspecific) ligand, while others, such as the locust AKHR, are more promiscuous and can be activated by AKHs from many other insects. This information will be of high importance when further analyzing the potential use of AKHRs as targets for developing novel pest control agents.

The African froghopper Ptyelus flavescens (suborder: Cicadomorpha) contains two novel and one known peptides of the adipokinetic hormone (AKH) family: structure, function and comparison with aphid AKH (suborder: Sternorrhyncha).

The rationale of "green pesticides" in food security is to use information about endogenous hormones of pest insects to make peptide mimetics that will act against the pest insects to alter their behaviour or physiology, while taking care not to harm beneficial insects or other organisms in the food chain. Such "green" insecticides are designed thus, on the basis of neuropeptide ligand-receptor interaction and it is of paramount interest to have finally a mimetic at hand that is harmful only to pest insects. For this concept to work, one has to identify the ligands in pest and beneficial insects. In this study we investigate adipokinetic hormones (AKHs) from a hemipteran source. The most harmful hemipterans on an economic scale are aphids (Hemiptera: Sternorrhyncha: Aphidoidea) of which the AKH is known. Here we identify the AKH complement of a member of a related suborder, the raintree bug or froghopper Ptyelus flavescens (Hemiptera: Cicadomorpha: Cercopoidea). Identification and sequence elucidation of the adipokinetic peptides of this species was achieved by a heterospecific and conspecific trehalose-mobilizing bioassay, and by liquid chromatography coupled to positive electrospray mass spectrometry (LC-ESI-MS) including tandem MS2 spectra obtained by collision-induced dissociation. High resolution MS was employed to distinguish between Gln and Lys residues in the peptides. Three AKHs are discovered in the raintree bug: an octapeptide (Peram-CAH-I: pEVNFSPNW amide) previously known from cockroaches, and two novel decapeptides (Ptyfl-AKH-I: pEINFSTGWGQ amide and Ptyfl-AKH-II: pEINFSTAWGQ amide). The novel peptides were synthesized and the sequence assignments were unequivocally confirmed by co-elution of synthetic peptides and the natural equivalent and by identical MS data of the two forms. A conspecific bioassay in the froghopper describe the endogenous peptide Ptyfl-AKH-I as hypertrehalosemic. In heterologous bioassays the two novel AKHs induce an increase of circulating carbohydrates in cockroaches: Ptyfl-AKH-I is much more active than Ptyfl-AKH-II. Moreover, if the Ile2 in Ptyfl-AKH-II is replaced with a Leu2 residue, biological activity is further diminished. The current data show that the raintree AKH decapeptides differ by four amino acids from the aphid AKH (Acypi-AKH: pEVNFTPTWGQ amide). Therefore, it may be permissible to use the aphid AKH ligand-receptor pair to develop a "green" insecticide to target aphid metabolism.

The hypertrehalosemic neuropeptides of cicadas are structural isomers-evidence by ion mobility mass spectrometry.

It has been known for more than 20 years that the neurosecretory glands of the cicadas, the corpora cardiaca, synthesize two isobaric peptides with hypertrehalosemic activity. Both decapeptides have exactly the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH2) and mass but differ in their retention time in reversed-phase liquid chromatography. A synthetic peptide with the same sequence elutes together with the second more hydrophobic peptide peak of the natural cicada extract. It is not clear what modification is causing the described observations. Therefore, in the current study, ion mobility separation in conjunction with high-resolution mass spectrometry was used to investigate this phenomenon as it was sensitive to changes in conformation. It detected different drift times in buffer gas for both the intact peptides and some of their fragment ions. Based on the ion mobility and fragment ion intensity of the corresponding ions, it is concluded that the region Pro6-Ser7-Trp8 contains a structural feature differing from the L-amino acids present in the known peptide. Whether the conformer is the result of racemization or other biochemical processes needs to be further investigated.

The adipokinetic hormone of the coleopteran suborder Adephaga: Structure, function, and comparison of distribution in other insects.

The aim of the current study is to identify the adipokinetic hormone(s) (AKHs) of a basal suborder of the species-rich Coleoptera, the Adephaga, and possibly learn more about the ancestral AKH of beetles. Moreover, we wanted to compare the ancestral AKH with AKHs of more advanced beetles, of which a number are pest insects. This would allow us to assess whether AKH mimetics would be suitable as insecticides, that is, be harmful to the pest species but not to the beneficial species. Nine species of the Adephaga were investigated and all synthesize only one octapeptide in the corpus cardiacum, as revealed by Edman degradation sequencing techniques or by mass spectrometry. The amino acid sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp corresponds to Schgr-AKH-II that was first identified in the desert locust. It is assumed that Schgr-AKH-II-the peptide of a basal beetle clade-is the ancestral AKH for beetles. Some other beetle families, as well as some Hymenoptera (including honey bees) also contain this peptide, whereas most of the pest beetle species have different AKHs. This argues that those peptides and their receptors should be explored for developing mimetics with insecticidal properties. A scenario where Schgr-AKH-II (the only AKH of Adephaga) is used as basic molecular structure to derive almost all other known beetle AKHs via single step mutations is very likely, and supports the interpretation that Schgr-AKH-II is the ancestral AKH of Coleoptera.

The adipokinetic hormone of Mantodea in comparison to other Dictyoptera.

Six species of the order Mantodea (praying mantises) are investigated for the presence and sequence of putative adipokinetic hormones (AKHs). The selected species span a wide evolutionary range of various families and subfamilies of the clade Mantodea. The corpora cardiaca of the different species are dissected, methanolic extracts prepared, peptides separated by liquid chromatography, and AKHs detected and sequenced by ion trap mass spectrometry. All six species investigated contain an octapeptide with the primary structure pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp amide, which is code-named Emppe-AKH and had been found earlier in three other species of Mantodea. Conspecific bioassays with the species Creoboter sp. (family Hymenopodidae) reveal an adipokinetic but not a hypertrehalosemic function of Emppe-AKH. Comparison with other members of the Dictyoptera (cockroaches, termites) show that Emppe-AKH is only found in certain termites, which have been recently placed into the Blattaria (cockroaches) as sister group to the family Cryptocercidae. Termites and cockroaches both show biodiversity in the sequence of AKHs, and some cockroach species even contain two AKHs. In contrast, all praying mantises-irrespective of their phylogenetic position-synthesize uniformly only one and the same octapeptide Emppe-AKH.

Identification and bioactivity evaluation of the first neuropeptide from the lesser-known insect order Embioptera (webspinner).

A species of the poorly studied order Embioptera, the webspinner Oligotoma saundersii, is investigated for its complement of neuropeptides of the adipokinetic hormone (AKH) family. A methanolic extract of its corpora cardiaca (CC) is able to effect carbohydrate mobilization in the cockroach, Periplaneta americana, and liquid chromatography coupled to electrospray ionization mass spectrometry clearly identified one decapeptide as a member of the AKH family in the CC of O. saundersii. The primary structure of this peptide, code-named Olisa-AKH, is elucidated as pEVNFSPNWGG amide. It is a novel member of the AKH family and in its synthetic form it has strong hypertrehalosemic activity in the American cockroach. This effect may be explained by its near-identical structure compared with one of the endogenous cockroach AKH peptides. An analog with the reversed order of the proline and asparagine residues, viz. N(6)P(7)-Olisa-AKH, had negligible activity thus, shedding light on the requirements of the cockroach AKH receptor. From reversed-phase high-performance liquid chromatography experiments, we can conclude that the CC from an individual webspinner contains less than one pmol of Olisa-AKH. Comparison of the AKH sequences from the major orders of the Polyneoptera does not point to a close phylogenetic relationship between webspinners and stick insects.

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.

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.

The decapod red pigment-concentrating hormone (Panbo-RPCH) is the first identified neuropeptide of the order Plecoptera and is interpreted as homoplastic character state.

This paper presents the first neuropeptide structure, identified by mass spectrometry, in two species of Plectoptera (stoneflies) and in one species of the coleopteran family Lycidae. In all three species, the octapeptide Panbo-RPCH (first identified in Pandalus borealis as a red pigment-concentrating hormone: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp amide) is present. A review of the literature available on invertebrate neuropeptides that are identified or predicted from expressed sequence tags, transcriptome shotgun assemblies, and from fully sequenced genomes, show that Panbo-RPCH is found in Malacostraca (Crustacea) and certain hemipteran Heteroptera (Insecta). To date, Panbo-RPCH has not been shown present in non-Malacostracan crustaceans, nor in basal taxa of the Insecta (Archaeognatha, Zygentoma, Ephemeroptera, Odonata). The present data adds to knowledge on the distribution of Panbo-RPCH, and when taking into account the most accepted, current phylogenetics of the Crustacea-Hexapoda relationship, this distribution of Panbo-RPCH in Malacostraca, Plecoptera, some hemipteran Heteroptera and in Coleoptera (Lycidae) can best be explained by homoplasy, implying parallel evolution.

The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding.

Neuropeptides are the main regulators of physiological, developmental, and behavioural processes in insects. Three insect neuropeptide systems, the adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone/corazonin-related peptide (ACP), and their cognate receptors, are related to the vertebrate gonadotropin (GnRH) system and form the GnRH superfamily of peptides. In the current study, the two signalling systems, AKH and ACP, of the yellow fever mosquito, Aedes aegypti, were comparatively investigated with respect to ligand binding to their respective receptors. To achieve this, the solution structure of the hormones was determined by nuclear magnetic resonance distance restraint methodology. Atomic-scale models of the two G protein-coupled receptors were constructed with the help of homology modelling. Thereafter, the binding sites of the receptors were identified by blind docking of the ligands to the receptors, and models were derived for each hormone system showing how the ligands are bound to their receptors. Lastly, the two models were validated by comparing the computational results with experimentally derived data available from the literature. This mostly resulted in an acceptable agreement, proving the models to be largely correct and usable. The identification of an antagonist versus a true agonist may, however, require additional testing. The computational data also explains the exclusivity of the two systems that bind only the cognate ligand. This study forms the basis for further drug discovery studies.