Chiral secondary amino acids, their importance, and methods of analysis.

Naturally occurring secondary amino acids, with proline as the main representative, contain an alpha-imino group in a cycle that is typically four-, five-, and six-membered. The unique ring structure exhibits exceptional properties-conformational rigidity, chemical stability, and specific roles in protein structure and folding. Many proline analogues have been used as valuable compounds for the study of metabolism of both prokaryotic and eukaryotic cells and for the synthesis of compounds with desired biological, pharmaceutical, or industrial properties. The D-forms of secondary amino acids play different roles in living organisms than the L-forms. They have different metabolic pathways, biological, physiological, and pharmacological effects, they can be indicators of changes and also serve as biomarkers of diseases. In the scientific literature, the number of articles examining D-amino acids in biological samples is increasing. The review summarises information on the occurrence and importance of D- and L-secondary amino acids-azetidic acid, proline, hydroxyprolines, pipecolic, nipecotic, hydroxypipecolic acids and related peptides containing these D-AAs, as well as the main analytical methods (mostly chromatographic) used for their enantiomeric determination in different matrices (biological samples, plants, food, water, and soil).

A chiral GC-MS method for analysis of secondary amino acids after heptafluorobutyl chloroformate & methylamine derivatization.

L-amino acids (L-AAs) play different important roles in the physiology of all living organisms. Their chiral counterparts, D-amino acids (D-AAs) are increasingly being recognized as essential molecules in many biological systems. Secondary amino acids with cyclic structures, such as prolines, exhibit conformational rigidity and thus unique properties in the structural and protein folding. Despite their widespread occurrence, much less attention was paid to their chiral analysis, particularly when the minor, typically D-enantiomer, is present in low amounts in a complex biological matrix. In this paper, a cost-effective, chiral GC-MS method is described for capillary Chirasil-L-Val separation of nine cyclic secondary amino acid enantiomers with four-, five-, and six-membered rings, involving azetidine-2-carboxylic acid, pipecolic acid, nipecotic acid, proline, isomeric cis/trans 3-hydroxy, 4-hydroxyproline, and cis/trans-5-hydroxy-L-pipecolic acid in the excess of its enantiomeric antipode. The sample preparation involves in-situ derivatization with heptafluorobutyl chloroformate, simultaneous liquid-liquid micro-extraction into isooctane followed by amidation of the arising low-polar derivatives with methylamine, an evaporation step, re-dissolution, and final GC-MS analysis. The developed method was used for analyses of human biofluids, biologically active peptides containing chiral proline constituents, and collagen.

Heptafluorobutyl Chloroformate-Based Sample Preparation Protocol for Nonchiral and Chiral Amino Acid Analysis by Gas Chromatography-Mass Spectrometry.

Gas chromatography (GC) is a commonly used technique in amino acid analysis (AAA). However, one of the requirements of the application of GC for AAA is a need for the polar analytes to be converted into their volatile, thermally stable derivatives. In the last two decades, alkyl chloroformates (RCFs) have become attractive derivatization reagents. The reagents react immediately with most amino acid functional groups in aqueous matrices, and the process can easily be coupled with liquid-liquid extraction of the resulting less polar derivatives into immiscible organic phase. Here we describe a simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate (HFBCF) followed by subsequent chiral as well as nonchiral GC/MS (mass spectrometric) analysis on a respective nonpolar fused silica and an enantioselective Chirasil-Val capillary column.

Tasty rewards for ants: differences in elaiosome and seed metabolite profiles are consistent across species and reflect taxonomic relatedness.

Diaspores of myrmecochorous plants consist of a seed (or fruit) and an attached appendage (elaiosome) which attracts ants. The elaiosome is a food resource for ants, whereas the seed is an energy source for subsequent germination and plant establishment. Although myrmecochory occurs in many phylogenetically unrelated lineages, multiple phylogenetic lineages display similar variation in elaiosome and seed metabolite composition due to convergent evolution. We focused on four families (Amaryllidaceae, Boraginaceae, Papaveraceae and Poaceae) each represented by two species from different genera. Diaspores of three populations per species were sampled and concentrations of 60 metabolites from five groups (amino acids, fatty acids, organic acids, polyols and sugars) were determined for both elaiosomes and seeds. Variability in metabolite composition was decomposed by hierarchical ANOVA and variation partitioning using redundancy analysis (reflecting both species nested within families, crossed with seed vs. elaiosome). Differences in the metabolite composition of elaiosomes and seeds were consistent across multiple phylogenetic origins (with more pronounced differences at the level of individual metabolites than at the level of metabolite groups) and supported the idea of convergent evolution under strong selection pressure. Elaiosomes contained higher amounts of easily digestible metabolites (especially amino acids) than seeds. Fatty acids were not more concentrated in elaiosomes, which contradicts the literal translation of "elaiosome" (= oil body). The differentiation of metabolite composition closely reflected taxonomic relatedness, particularly at the family level. Differences among populations within species were small, so the metabolite composition can thus be considered as a trait with relatively low intraspecific variability.

GC-MS Metabolomic Profiling of Protic Metabolites Following Heptafluorobutyl Chloroformate Mediated Dispersive Liquid Microextraction Sample Preparation Protocol.

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.

Physiological basis for low-temperature survival and storage of quiescent larvae of the fruit fly Drosophila melanogaster.

The cryopreservation techniques proposed for embryos of the fruit fly Drosophila melanogaster are not yet ready for practical use. Alternative methods for long-term storage of D. melanogaster strains, although urgently needed, do not exist. Herein, we describe a narrow interval of low temperatures under which the larvae of D. melanogaster can be stored in quiescence for up to two months. The development of larvae was arrested at the pre-wandering stage under fluctuating thermal regime (FTR), which simultaneously resulted in diminishing the accumulation of indirect chill injuries. Our physiological, metabolomic, and transcriptomic analyses revealed that compared to larvae stored at constant low temperatures, the larvae stored under FTR conditions were able to decrease the rates of depletion of energy substrates, exploited brief warm episodes of FTR for homeostatic control of metabolite levels, and more efficiently exerted protection against oxidative damage.

Profiling of urinary amino-carboxylic metabolites by in-situ heptafluorobutyl chloroformate mediated sample preparation and gas chromatography-mass spectrometry.

A novel 1,1,1,2,2,3,3-heptafluorobutyl chloroformate reagent (HFBCF) was examined for in-situ derivatization of amino-carboxylic metabolites in human urine. The arising reaction products exhibit greatly reduced polarity which facilitates combining the derivatization and liquid-liquid microextraction (LLME) from an aqueous urine into an isooctane phase and immediate gas chromatographic-mas spectrometric analysis (GC-MS). The sample preparation protocol is simple, proceeds without an alcohol excess and provides cleaner extracts than other urinary GC-MS based methods. Moreover, thiol metabolites bound in disulfide bonds can be released by reduction with tris(3-hydroxypropyl)phosphine (THP) prior to the developed derivatization and LLME step. In order to evaluate potential of the novel method for GC-MS metabolomics, reaction products of 153 urinary metabolites with HFBCF, particularly those possessing amino and carboxyl groups (56 amino acids and their conjugates, 84 organic acids, 9 biogenic amines, 4 other polar analytes) and two internal standards were investigated in detail by GC-MS and liquid chromatography-mass spectrometry (LC-MS). One hundred and twenty metabolites (78%) yielded a single product, 25 (16%) and 2 metabolites (2-methylcitrate, citrate) generated two and more derivatives. From the examined set, analytically applicable products of 5 metabolites were not detected; the derivatives of 3 metabolites were only suitable for LC-MS analysis. Electron ionization (EI) of the examined analytes contained characteristic, diagnostic ions enabling to distinguish related and isomeric structures. The new method was validated for 132 metabolites using two internal standards in artificial urine and with special attention to potential disease biomarker candidates. The developed sample preparation protocol was finally evaluated by means of a certified organic acid standard mixture in urine and by GC-MS analysis of 100 morning urines obtained from healthy patients (50 males and 50 females), where 112 physiological metabolites were quantified in a 25 µL sample aliquot. The quantification data for the set were satisfactory, most metabolites were found within the range reported in the reference human metabolome (HMDB) database and literature. The reported results suggest that the described method has been a novel promising tool for targeted GC-MS based metabolomic analysis in urine.

Reprint of: Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth, Cydia pomonella.

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (Tm) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.

Comparative ecophysiology of cold-tolerance-related traits: assessing range expansion potential for an invasive insect at high latitude.

Survival at high latitude requires the capability to cope with seasonally imposed stress, such as low winter temperatures or large temperature fluctuations. The Colorado potato beetle, Leptinotarsa decemlineata, is an invasive pest of potato that has rapidly spread from low latitudes to higher latitudes. During the last 30 years, a decrease in range expansion speed is apparent in Europe. We use a comparative approach to assess whether this could be due to an inability of L. decemlineata to cope with the harsher winters encountered at high latitude, when compared to two native northern chrysomelid beetles with similar overwintering ecology. We investigated several cold-tolerance-related physiological traits at different time points during winter. Cold tolerance followed a latitudinal pattern; the northern species were more tolerant to short-term subzero temperatures than the invasive L. decemlineata. The other northern species, the knotgrass leaf beetle, Chrysolina polita, was found to tolerate internal freezing. Interestingly, the pattern for overwinter survival at 5°C was the opposite and higher in L. decemlineata than the northern species and could be related to behavioral differences between species in overwintering location selection and a potential physiological trade-off between tolerance to cold shock and to chronic cold exposure. Furthermore, while the northern species accumulated large amounts of different sugars and polyols with probable cryoprotectant functions, none were detected in L. decemlineata at high concentrations. This lack of cryoprotectant accumulation could explain the difference in cold tolerance between the species and also suggests that a lack of physiological capacity to tolerate low temperatures could slow further latitudinal range expansion of L. decemlineata.

Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth, Cydia pomonella.

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (Tm) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.

Cost effective, robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: application to insects.

The quantification of phospholipid classes and the determination of their molecular structures are crucial in physiological and medical studies. This paper's target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation processes of poikilothermic organisms. We introduce a set of simple and cost-effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid-phase extraction and high-performance thin-layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chromatography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high-performance liquid chromatography with mass spectrometry, which gives information about the headgroup moiety and attached fatty acids. The total content of each phospholipids class is assessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high-performance liquid chromatography analysis.

Physiology of cold tolerance in the bark beetle, Pityogenes chalcographus and its overwintering in spruce stands.

The seasonal development of physiological features underlying gradual acquisition of relatively high cold tolerance in overwintering adults of the bark beetles, Pityogenes chalcographus was described. Prior to overwintering, the beetles accumulated carbohydrate reserves in the form of glycogen and trehalose. These reserves were partially converted to glycerol during peaking winter so that glycerol concentration reached 1.4M in average, which corresponds to approximately one quarter of the beetle dry mass. Whole body supercooling points decreased from -12.8°C in average at the beginning of dormancy (August) to -26.3°C in average during peaking winter (January). More than 75% of January-collected beetles survived at -5°C for 30days, at -15°C for 60days and more than 40% of them survived at -26°C for 12h. High resistance against inoculation of body fluids with external ice crystals, and low mortality, was observed when January-collected beetles were encased in an ice block for 14days. Thus, the physiological limits of cold tolerance measured at individual level in laboratory were safely sufficient for survival of P. chalcographus at any conceivable cold spell that may occur in Central Europe. In contrast, the field experiment showed that winter survival fluctuated between 23.8% and 69.2% at a population level depending on microclimatic conditions in different altitudes and overwintering locations (standing tree trunk or ground level). The meaning of laboratory-assessed physiological limits of cold tolerance for predictions of population winter survival in the field is discussed.

Overwintering strategy and mechanisms of cold tolerance in the codling moth (Cydia pomonella).

BACKGROUND: The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations. PRINCIPAL FINDINGS: We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately -15.3 °C during summer to -26.3 °C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to -15 °C, even in partially frozen state. CONCLUSION: Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).

Physiological and biochemical responses to cold and drought in the rock-dwelling pulmonate snail, Chondrina avenacea.

The pulmonate snail Chondrina avenacea lives on exposed rock walls where it experiences drastic daily and seasonal fluctuations of abiotic conditions and food availability. We found that tolerance to dry conditions was maintained at a very high level throughout the year and was mainly based on the snails' ability to promptly enter into estivation (quiescence) whenever they experienced drying out of their environment. Snails rapidly suppressed their metabolism and minimized their water loss using discontinuous gas exchange pattern. The metabolic suppression probably included periods of tissue hypoxia and anaerobism as indicated by accumulation of typical end products of anaerobic metabolism: lactate, alanine and succinate. Though the drought-induced metabolic suppression was sufficient to stimulate moderate increase of supercooling capacity, the seasonally highest levels of supercooling capacity and the highest tolerance to subzero temperatures were tightly linked to hibernation (diapause). Hibernating snails did not survive freezing of their body fluids and instead relied on supercooling strategy which allowed them to survive when air temperatures dropped to as low as -21 °C. No accumulation of low-molecular weight compounds (potential cryoprotectants) was detected in hibernating snails except for small amounts of the end products of anaerobic metabolism.

Myo-inositol as a main metabolite in overwintering flies: seasonal metabolomic profiles and cold stress tolerance in a northern drosophilid fly.

Coping with seasonal changes in temperature is an important factor underlying the ability of insects to survive over the harsh winter conditions in the northern temperate zone, and only a few drosophilids have been able to colonize sub-polar habitats. Information on their winter physiology is needed as it may shed light on the adaptive mechanisms of overwintering when compared with abundant data on the thermal physiology of more southern species, such as Drosophila melanogaster. Here we report the first seasonal metabolite analysis in a Drosophila species. We traced changes in the cold tolerance and metabolomic profiles in adult Drosophila montana flies that were exposed to thermoperiods and photoperiods similar to changes in environmental conditions of their natural habitat in northern Finland. The cold tolerance of diapausing flies increased noticeably towards the onset of winter; their chill coma recovery times showed a seasonal minimum between late autumn and early spring, whereas their survival after cold exposure remained high until late spring. The flies had already moderately accumulated glucose, trehalose and proline in autumn, but the single largest change occurred in myo-inositol concentrations. This increased up to 400-fold during the winter and peaked at 147 nmol mg(-1) fresh mass, which is among the largest reported accumulations of this compound in insects.

Conversion of the chill susceptible fruit fly larva (Drosophila melanogaster) to a freeze tolerant organism.

Among vertebrates, only a few species of amphibians and reptiles tolerate the formation of ice crystals in their body fluids. Freeze tolerance is much more widespread in invertebrates, especially in overwintering insects. Evolutionary adaptations for freeze tolerance are considered to be highly complex. Here we show that surprisingly simple laboratory manipulations can change the chill susceptible insect to the freeze tolerant one. Larvae of Drosophila melanogaster, a fruit fly of tropical origin with a weak innate capacity to tolerate mild chilling, can survive when approximately 50% of their body water freezes. To achieve this goal, synergy of two fundamental prerequisites is required: (i) shutdown of larval development by exposing larvae to low temperatures (dormancy) and (ii) incorporating the free amino acid proline in tissues by feeding larvae a proline-augmented diet (cryopreservation).

Heptafluorobutyl chloroformate-based sample preparation protocol for chiral and nonchiral amino acid analysis by gas chromatography.

Gas chromatography (GC) is a commonly used technique in amino acid analysis (AAA). However, one of the requirements of the application of GC for AAA is a need for the polar analytes to be converted into their volatile, thermally stable derivatives. In the last two decades, alkyl chloroformates have become attractive derivatization reagents. The reagents react immediately with most amino acid functional groups in aqueous matrices and the process can easily be coupled with liquid-liquid extraction of the resulting less-polar derivatives into immiscible organic phase. Here, we describe a simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate followed by subsequent chiral as well as nonchiral GC/mass spectrometric analysis on a respective nonpolar fused silica and an enantioselective Chirasil-Val capillary column.

Long-term cold acclimation extends survival time at 0°C and modifies the metabolomic profiles of the larvae of the fruit fly Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum. PRINCIPAL FINDINGS: We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines. CONCLUSION: Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen.

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

A novel function of red pigment-concentrating hormone in crustaceans: Porcellio scaber (Isopoda) as a model species.

The RP HPLC and LC/MS QTOF analyses of the methanolic CNS extract from isopod crustacean the woodlouse, Porcellio scaber revealed a presence of the red pigment-concentrating hormone (Panbo-RPCH) in this species. It has been shown that this neuropeptide plays a role in mobilization of energy stores: topical treatments of P. scaber individuals by Panbo-RPCH in a concentration 20 pmol/microl increased the level of glucose in haemolymph about 4 times, while the level of trehalose was only doubled. The results demonstrated that glucose was the main carbohydrate mobilized by the Panbo-RPCH treatment: glucose was responsible for about 97% of total carbohydrate increasing. Despite the demonstration of hyperglycaemic activity of Panbo-RPCH, no stimulatory effect of this hormone on the locomotory activity of P. scaber was observed. The present study is the first discovery of an occurrence of Panbo-RPCH and its hyperglycaemic activity in the representative of the isopod crustaceans. The relationship of the function of Panbo-RPCH in P. scaber to the role of this neuropeptide and adipokinetic hormones in insects is discussed.