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.

Oxidation Products of Tryptophan and Proline in Adipokinetic Hormones-Artifacts or Post-Translational Modifications?

BACKGROUND: Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are short peptides with blocked termini and Trp in position 8. Often, proline occupies position 6. Few post-translational modifications have been found, including hydroxyproline ([Hyp6]) and kynurenine. Our recent data suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought and we here investigate if they are natural or artifactual. METHODS: From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS). Synthetic [Hyp6]-AKHs were tested in an in vivo metabolic assay. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were analyzed. B. atropos CC were placed into a depolarizing saline and the released AKHs were measured. RESULTS: Hyp was detected in several decapeptides from cockroaches. The modified form accompanied the AKH at concentrations below 7%. The [Hyp6]-AKHs of B. atropos were present in fresh CC preparations and were shown to be releasable from the CC ex vivo. Synthetic [Hyp6]-containing peptides tested positively in a hypertrehalosemic bioassay. Hydroxyprolination was also detected for Manto-CC from the termite Kalotermes flavicollis and for Tetsu-AKH of the grasshopper, Tetrix subulata. Oxidized Trp-containing forms of Nicve-AKH were found in species of the burying beetle genus Nicrophorus. CONCLUSIONS: Trp oxidation is known to occur easily during sample handling and is likely the reason for the present findings. For hydroxyprolination, however, the experimental evidence suggests endogenous processes.

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 proline effect and the tryptophan immonium ion assist in de novo sequencing of adipokinetic hormones.

Adipokinetic hormones (AKHs) in Arthopoda are characterized by special sequence features including limited choices of amino acid residues in certain positions, such as Trp in position 8. Over 100 different AKHs have been described, but de novo sequencing of novel peptide hormones can be a challenge. In a project of analyzing corpora cardiaca extracts from two fly species, two different moths, a termite and a beetle for their AKHs, we noted specific patterns in the fragmentation spectra of octapeptides in electrospray Q-TOF experiments resulting from the presence of Pro in position 6. The preference for cleavage N-terminal to Pro residues created an abundant y3″-ion, which, in conjunction with the two b-ions resulting from the fragmentation before and after Pro, provided a marker pattern. As Pro6 occurs in about 61% of known AKHs, this information is highly relevant for sequence elucidation. Moreover, the default presence of Trp8 allowed the use of its immonium ion for AKH candidate identification. In addition, we assembled the known AKH sequences from the literature and sequences of AKH-type format found in the Uniprot database in a single online resource. These efforts assisted in the analysis workflow and led to the assignment of two novel AKHs and evidence for the presence of Melme-CC and Dorpa-AKH in the corpus cardiacum of the scarab beetle Sinodendron cylindricum.

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.

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).

Antifungal Compounds from Microbial Symbionts Associated with Aquatic Animals and Cellular Targets: A Review.

Fungal infections continue to be a serious public health problem, leading to an estimated 1.6 million deaths annually. It remains a major cause of mortality for people with a weak or affected immune system, such as those suffering from cancer under aggressive chemotherapies. On the other hand, pathogenic fungi are counted among the most destructive factors affecting crops, causing a third of all food crop losses annually and critically affecting the worldwide economy and food security. However, the limited number currently available and the cytotoxicity of the conventional antifungal drugs, which are not yet properly diversified in terms of mode of action, in addition to resistance phenomena, make the search for new antifungals imperative to improve both human health and food protection. Symbiosis has been a crucial alternative for drug discovery, through which many antimicrobials have been discovered. This review highlights some antifungal models of a defensive symbiosis of microbial symbiont natural products derived from interacting with aquatic animals as one of the best opportunities. Some recorded compounds with supposed novel cell targets such as apoptosis could lead to the development of a multitherapy involving the mutual treatment of fungal infections and other metabolic diseases involving apoptosis in their pathogenesis pathways.

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.

In Silico Screening for Pesticide Candidates against the Desert Locust Schistocerca gregaria.

Adipokinetic hormone (AKH) is one of the most important metabolic neuropeptides in insects, with actions similar to glucagon in vertebrates. AKH regulates carbohydrate and fat metabolism by mobilizing trehalose and diacylglycerol into circulation from glycogen and triacylglycerol stores, respectively, in the fat body. The short peptide (8 to 10 amino acids long) exerts its function by binding to a rhodopsin-like G protein-coupled receptor located in the cell membrane of the fat body. The AKH receptor (AKHR) is, thus, a potential target for the development of novel specific (peptide) mimetics to control pest insects, such as locusts, which are feared for their prolific breeding, swarm-forming behavior and voracious appetite. Previously, we proposed a model of the interaction between the three endogenous AKHs of the desert locust, Schistocerca gregaria, and the cognate AKHR (Jackson et al., Peer J. 7, e7514, 2019). In the current study we have performed in silico screening of two databases (NCI Open 2012 library and Zinc20) to identify compounds which may fit the endogenous Schgr-AKH-II binding site on the AKHR of S. gregaria. In all, 354 compounds were found to fit the binding site with glide scores < −8. Using the glide scores and binding energies, 7 docked compounds were selected for molecular dynamic simulation in a phosphatidylcholine membrane. Of these 7 compounds, 4 had binding energies which would allow them to compete with Schgr-AKH-II for the receptor binding site and so are proposed as agonistic ligand candidates. One of the ligands, ZINC000257251537, was tested in a homospecific in vivo biological assay and found to have significant antagonistic activity.

The Adipokinetic Peptides of Hemiptera: Structure, Function, and Evolutionary Trends.

The Hemiptera comprise the most species-rich order of the hemimetabolous insects. Members of a number of superfamilies, most notably especially the more basal ones such as white flies, psyllids and aphids, belong to the most destructive agricultural insects known worldwide. At the other end of the phylogenetic tree are hemipterans that are notorious medical pests (e.g. kissing bugs). Most of the hemipteran species are good flyers, and lipid oxidation plays a pivotal role to power the contraction of flight muscles and, in aquatic water bugs, also deliver the ATP for the extensive swimming action of the leg muscles. Mobilization of stored lipids (mostly triacylglycerols in the fat body) to circulating diacylglycerols in the hemolymph is regulated by a set of small neuropeptides, the adipokinetic hormones (AKHs). We searched the literature and publicly available databases of transcriptomes and genomes to present here AKH sequences from 191 hemipteran species. Only few of these peptides were sequenced via Edman degradation or mass spectrometry, and even fewer were characterized with molecular biology methods; thus, the majority of the AKHs we have identified by bioinformatics are merely predicted sequences at this stage. Nonetheless, a total of 42 AKH primary sequences are assigned to Hemiptera. About 50% of these structures occur also in other insect orders, while the remaining 50% are currently unique for Hemiptera. We find 9 novel AKHs not shown to be synthesized before in any insect. Most of the hemipteran AKHs are octapeptides (28) but there is an impressive number of decapeptides (12) compared to other speciose orders such as Diptera and Lepidoptera. We attempt to construct a hypothetical molecular peptide evolution of hemipteran AKHs and find quite a bit of overlapping with current phylogenetic ideas of the Hemiptera. Lastly, we discuss the possibility to use the sequence of the aphid AKH as lead peptide for the research into a peptide mimetic fulfilling criteria of a green insecticide.

The effect of pyriproxyfen on the concentration of circulating metabolic fuel molecules and chemical elements in the hemolymph of Acraea horta L. (Lepidoptera: Nymphalidae): A quantitative analysis.

Many pollinating insects expand their niche to adjacent agricultural areas and are, therefore, exposed to chemical insecticides. Acraea horta L. (Lepidoptera: Nymphalidae) is a pollinator butterfly widely distributed in the Southern African region. The objectives of this work were to evaluate carbohydrate, lipid and chemical elements in the hemolymph of A. horta exposed to pyriproxyfen, a juvenile hormone analog (JHA). Last instar larvae (L6: day 1 or day 2) were topically exposed to an aqueous solution of pyriproxyfen (100 µg of the active ingredient per insect) or to diluent (control group). Hemolymph was collected after adult eclosion to determine total carbohydrate and lipid concentrations: in the control group lipids were present in lower concentrations than carbohydrates and there was no significant difference in metabolite levels between sexes; a similar pattern with similar levels were measured in the treated group, except that lipid concentrations in treated males were lower, and carbohydrate concentrations in treated females were lower than the control values. Morphologically intact adult males from treated larvae were subjected to free flight; their hemolymph carbohydrate levels were significantly reduced and did not recover to starting levels in a 30 min rest period following the exhaustive flight episode. To assess the effect of pyriproxyfen on a different stage of development, 48 h old butterflies were treated in the same way as described for the L6 larvae above; hemolymph samples were taken 48 h later for metabolite measurements and for quantification of chemical elements: carbohydrate levels decreased significantly after pyriproxyfen exposure, while lipid levels increased; inorganic elements measured in untreated adults were more abundant in females, with a general decrease in concentration following pyriproxyfen exposure, except for an increase in Fe levels in males and Cl levels in females. The quantitative changes measured in A. horta hemolymph via biochemical and chemical element analyses may indicate distinct physiological interferences beyond the main mode of action of pyriproxyfen on JH activity. In conclusion, the use and quantification of pyriproxyfen should be carefully evaluated prior to application in areas where A. horta and other pollinator species occur.

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.

Structure-Activity Studies on the Hypertrehalosemic Hormone II of the Stick Insect Carausius morosus (Phasmatodea): Carbohydrate-Mobilization and Cardio-Stimulatory Activities.

The corpora cardiaca of the Indian stick insect, Carausius morosus, synthesize two decapeptide neuropeptides of the adipokinetic hormone (AKH) family, both of which can increase the trehalose levels in the hemolymph when the stick insect is ligated between the head and the thorax. Here, we use two biological assays to assess the potencies of 19 AKH analogs in ligated C. morosus: the carbohydrate-mobilizing assay measures the change in the levels of circulating carbohydrates following injection of a substance, while the semi-exposed heart assay measures a change in heart beat rate after the peptide is applied onto the heart. With the endogenous AKH (Carmo-HrTH-II) as lead peptide, we report here on seven naturally-occurring AKH peptides (bioanalogs) selected for testing because of a single or double amino acid replacement, or for being octapeptides. Single amino acid substitutions by an alanine residue at all positions of Carmo-HrTH-II, as well as analogs modified at the termini were also investigated to give a comprehensive view of ligand-receptor interaction at the physiological level in a hemimetabolous insect that practices thanatosis (feigning death). Only small changes are elicited in the bioassays, but the results from the two tests are comparable bar one or two anomalies. Results show that analogs modified at the termini have no or reduced activity. Regarding structural requirements of a ligand, the C. morosus AKH receptor appears to be strict: octapeptides are not preferred and many of the decapeptide analogs failed to reach 50% activity relative to Carmo-HrTH-II. The data implies that the AKH receptor in C. morosus mostly does not tolerate shorter peptides and single amino acid replacements in most places of the native AKH peptide. This information is important if environmentally friendly insect-specific pesticides are made based on an insect AKH as lead peptide: stick insects that are normally not viewed as pest insects may not be easily targeted by cross-reactive AKH mimetics directed at harmful insects, due to the very specific amino acid requirements to activate the C. morosus AKH receptor.

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.

Unique Members of the Adipokinetic Hormone Family in Butterflies and Moths (Insecta, Lepidoptera).

Lepidoptera is amongst one of the four most speciose insect orders and ecologically very successful because of their ability to fly. Insect flight is always aerobic and exacts a high metabolic demand on the animal. A family of structurally related neuropeptides, generically referred to as adipokinetic hormones (AKHs), play a key role in triggering the release of readily utilizable fuel metabolites into the hemolymph from the storage forms in the fat body. We used mass spectrometry to elucidate AKH sequences from 34 species of Lepidoptera and searched the literature and publicly available databases to compile (in a phylogenetic context) a comprehensive list of all Lepidoptera sequences published/predicted from a total of 76 species. We then used the resulting set of 15 biochemically characterized AKHs in a physiological assay that measures lipid or carbohydrate mobilization in three different lepidopteran species to learn about the functional cross-activity (receptor-ligand interactions) amongst the different butterfly/moth families. Our results include novel peptide structures, demonstrate structural diversity, phylogenetic trends in peptide distribution and order-specificity of Lepidoptera AKHs. There is almost an equal occurrence of octa-, nona-, and decapeptides, with an unparalleled emphasis on nonapeptides than in any insect order. Primitive species make Peram-CAH-II, an octapeptide found also in other orders; the lepidopteran signature peptide is Manse-AKH. Not all of the 15 tested AKHs are active in Pieris brassicae; this provides insight into structure-activity specificity and could be useful for further investigations into possible biorational insecticide development.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

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.

Five Neuropeptide Ligands Meet One Receptor: How Does This Tally? A Structure-Activity Relationship Study Using Adipokinetic Bioassays With the Sphingid Moth, Hippotion eson.

Adipokinetic hormones (AKHs) play a major role in mobilizing stored energy metabolites during energetic demand in insects. We showed previously (i) the sphingid moth Hippotion eson synthesizes the highest number of AKHs ever recorded, viz. five, in its corpus cardiacum: two octa- (Hipes-AKH-I and II), two nona- (Hipes-AKH-III and Manse-AKH), and one decapeptide (Manse-AKH-II), which are all active in lipid mobilization (1). (ii) Lacol-AKH from a noctuid moth showed maximal AKH activity in H. eson despite sequence differences and analogs based on Lacol-AKH with modifications at positions 2, 3, 8, or at the termini, as well as C-terminally shortened analogs had reduced or no activity (2). Here we report on N-terminally shortened and modified analogs of the lead peptide, as well as single amino acid substitutions at positions 1, 4, 5, 6, and 7 by an alanine residue. Ala1 and Glu1 instead of pGlu are not tolerated well to bind to the H. eson AKH receptor, whereas Gln1 has high activity, suggesting it is endogenously cyclized. Replacing residue 5 or 7 with Ala did not alter activity much, in contrast with changes at position 4 or 6. Similarly, eliminating pGlu1, Leu2, or Thr3 from Lacol-AKH severely interfered with biological activity. This indicates that there is no core peptide sequence that can elicit the adipokinetic effect and that the overall conformation of the active peptide is required for a physiological response. AKHs achieve a biological action through binding to a receptor located on fat body cells. To date, one AKH receptor has been identified in any given insect species; we infer the same for H. eson. We aligned lepidopteran AKH receptor sequences and note that these are very similar. The results of our study is, therefore, also applicable to ligand-receptor interaction of other lepidopteran species. This information is important for the consideration of peptide mimetics to combat lepidopteran pest insects.