Processes In Invertebrates Research Articles

Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress

Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0–61.2% and 45.0–47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 105 cfu shrimp−1 revealed significant increase of LvTH mRNA expression in haemocytes within 30–120 min and in brain within 15–30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30–60 and 15–60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30–60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation.

Politics for the Polyps

Daniel Harris, “Politics for the Polyps: The Compound Organism as a ‘Peculiar Form of Communism’ in Charles Kingsley’s Alton Locke and The Water-Babies” (pp. 64–88) Charles Kingsley’s novels and political writings are saturated with references to physiological processes in marine invertebrates. In particular, the forms of his novels take their inspiration from functional arrangements in colonial organisms such as corals, in which “individual” polyps are physiologically linked to their neighbors. Alton Locke (1851) and The Water-Babies (1863) attempt to explain the benefits of cooperative economic practices (e.g., the associative workshop) and the dangers of cooperative political practices (e.g., the Chartist mass meeting) by jettisoning British Enlightenment assumptions about personal identity. Instead, Kingsley’s novels use discontinuous and communal physiological processes in invertebrates, such as corals and jellyfish, as frameworks for representing psychological and political development. Ultimately, Kingsley seeks to intervene in mid-century debates about how to individuate members of the working class by suggesting that reformist measures must be grounded in a physiological understanding of individuation that contravenes psychological definitions of individuality.

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light.

Light-regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha-melanocyte-stimulating hormone (α-MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α-MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye-dependent, while functional data in vivo point to GABAA receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α-MSH secretion, which is used normally during background adaptation.

Ferritin has an important immune function in the ark shell Scapharca broughtonii

Ferritin, the principle cytosolic iron storage protein in the majority of living organisms, has important roles during immune process in invertebrates. Detailed information about ferritin in the ark shell Scapharca broughtonii, however, has been very limited. In this study, full-length ferritin (termed SbFer) was cloned by the rapid amplication of cDNA ends (RACE) method based upon the sequence from the transcriptome library. The cDNA contained a 182 bp 5′-untranslated region, a 519 bp open reading frame encoding a polypeptide of 172 amino acids, a 229 bp 3′-untranslated region, and three introns (902, 373 and 402 bp) embedded in four exons. There was an iron response element (IRE) in the 5′-untranslated region. The deduced amino acid sequence of SbFer possessed many characteristics of vertebrate H type ferritin, shared 63%–91% identity with mollusks and greater identity with vertebrate H type ferritin compared to the L type. The SbFer gene expression pattern examined by quantitative real-time PCR showed ferritin mRNA was expressed in all ark shell tissues examined. The highest levels of expression were found in hemocytes with decreasing levels of expression in foot, mantle, gill, adductor muscle and hepatopancreas. A challenge with Vibrio anguillarum resulted in time-dependent significant upregulation of SbFer mRNA, indicating SbFer participated actively in the bacterial defense process. Further analysis of the antibacterial activity indicated recombinant SbFer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria. Taken together, these results suggested strongly that ferritin of the ark shell is involved in immune defense against microbial infection and it is a constitutive and inducible acute-phase protein.

Comparative Diagenesis and Rare Earth Element Variation in Miocene Invertebrate and Vertebrate Fossils from Panama

AbstractThe incorporation of rare earth elements (REEs) into the mineral lattices of skeletonized fossils has been used, particularly in vertebrates, to understand diagenesis and other postmortem paleoenvironmental parameters. Little is known about similar processes in invertebrates. Invertebrate and vertebrate fossils were analyzed for REE concentrations and patterns from five Miocene formations in central Panama (∼20–6 m.yr. ago). These formations (Culebra, Cucaracha, Gatun, Chucunaque, and Chagres) contain sediments interpreted to range from freshwater to shallow marine environments (depth of <300 m). We also compared different compositional types of fossils, including hydroxylapatite (bone) and aragonite (otoliths) in vertebrates and calcite and aragonite in invertebrates. REEs from modern invertebrates indicate that, as previously reported for vertebrates, invertebrates do not incorporate significant amounts of REEs into their mineral lattices during life; therefore, uptake must primarily occur during diagenesis. Comparisons of REEN concentrations (REE concentrations normalized to Post-Archean Australian Shale) of the fossils from Panama indicate differences in diagenesis. Within our largest sample from the Gatun Formation, there are significance differences in REEN concentrations and patterns within different compositional types. For example, shark teeth have the highest REEN concentrations and otoliths the lowest, with these differences likely a result of physical, chemical, and mineralogical factors. Calcitic and aragonitic fossils from the Gatun Formation show REEN patterns indicating differential uptake of individual elements during diagenesis. The cerium anomaly, previously used to indicate relative paleobathymetry, did not discriminate water depth in the marine formations. Comparative analysis of REEs in vertebrates and invertebrates provides a powerful tool to understand diagenetic processes, as demonstrated in the five formations and different compositional types studied from the Miocene of Panama.

Dual effect of nitric oxide on phenoloxidase-mediated melanization

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO2 at the active site of PO followed by oxidation of DOPA by NO2. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.

The NOTCH signaling pathway in normal and malignant blood cell production.

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.

Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles.

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic musculature, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.

Heparan sulfate: a key regulator of embryonic stem cell fate

Heparan sulfate (HS) belongs to a class of glycosaminoglycans and is a highly sulfated, linear polysaccharide. HS biosynthesis and modification involves numerous enzymes. HS exists as part of glycoproteins named HS proteoglycans, which are expressed abundantly on the cell surface and in the extracellular matrix. HS interacts with numerous proteins, including growth factors, morphogens, and adhesion molecules, and thereby regulates important developmental processes in invertebrates and vertebrates. Embryonic stem cells (ESCs) are distinguished by their characteristics of self-renewal and pluripotency. Self-renewal allows ESCs to proliferate indefinitely in their undifferentiated state, whereas pluripotency implies their capacity to differentiate into the three germ layers and ultimately all cell types of the adult body. Both traits are tightly regulated by numerous cell signaling pathways. Recent studies have highlighted the importance of HS in the modulation of ESC functions, specifically their lineage fate. Here, we review the current advances that have been made in understanding the structural changes of HS during ESC differentiation and in deciphering the molecular mechanisms by which HS modulates cell fate. Finally, we discuss the applications of heparinoids and chemical inhibitors of HS biosynthesis for the manipulation of ESC culture and directed differentiation.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: Cloning, characterization and expression analysis

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

The cloning, phylogenetic relationship and distribution pattern of two new putative GPCR-type octopamine receptors in the desert locust ( Schistocerca gregaria)

The biogenic amine octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems. It plays a prominent role in modulating multiple physiological and behavioural processes in invertebrates. Octopamine exerts its effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. We found two partial sequences of putative octopamine receptors in the desert locust Schistocerca gregaria (SgOctαR and SgOctβR) and investigated their transcript levels in males and females of both phases and during the transition between long-term solitarious and gregarious locusts. The transcript levels of SgOctαR are the highest in the central nervous system, whereas those of SgOctβR are the highest in the flight muscles, followed by the central nervous system. Both SgOctαR and SgOctβR show higher transcript levels in long-term gregarious locusts as compared to solitarious ones. The rise of SgOctβR transcript levels already appears during the first 4 h of gregarisation, during which also the behavioural changes take place.

Food intake, digestive enzyme activity and contraction of the gut is regulated by sulfakinins from the cricket Gryllus bimaculatus

Event Abstract Back to Event Food intake, digestive enzyme activity and contraction of the gut is regulated by sulfakinins from the cricket Gryllus bimaculatus Martina Meyering-Vos1*, Sandra Diersch1, Reinhard Predel2 and Joseph Woodring1 1 University of Bayreuth, Animal Ecology I, Germany 2 University of Jena, Institute of Zoology, Germany In vertebrates, a variety of mechanisms are involved in the regulation of food intake and energy balance, and recent developments in insect research indicate that the endocrine regulation of these processes in invertebrates is at least as complex. Sulfakinins (SK) are neuropeptides structurally displaying sequence homology with the hormonally active portion of the vertebrate gastrin/CCK peptide family. In Gryllus bimaculatus two sulfakinins (I, II) are encoded on the gene and the peptides could be identified in brain extracts from adult females by mass spectrometry analysis. In Gryllus bimaculatus after in vivo degradation of the specific sulfakinin mRNAs by the RNA interference method, the expression is reduced and this leads to a drastic increase in the food-intake indicating the inhibitory bioactivity of the peptides. Synthetic SK I peptide has a stimulatory effect on the release of α-amylase activity in the ventriculus, whereas SK II stimulates the release of this enzyme in the caecum of unfed crickets. Additionally, the sulfakinins stimulate the release of proteases into the caecum. SK I stimulates trypsin release and in a similar manner as gastrin stimulates biosynthesis in vertebrates. SK II increases most specifically the released aminopeptidase activity. Finally, SK I stimulates the myoactivity of the crop, wheras SK II has no effect. Acknowledgements This work was supported by the DFG Me 2034/1-3. Keywords: amylase, food intake regulation, Gastrointestinal System, gut myoactivity, Neuropeptide, Protease, satiety, Sulfakinin Conference: 25th Conference of the European Comparative Endocrinologists, Pécs, Hungary, 31 Aug - 4 Sep, 2010. Presentation Type: Conference Presentation Topic: Comparative endocrinology Citation: Meyering-Vos M, Diersch S, Predel R and Woodring J (2010). Food intake, digestive enzyme activity and contraction of the gut is regulated by sulfakinins from the cricket Gryllus bimaculatus. Front. Endocrinol. Conference Abstract: 25th Conference of the European Comparative Endocrinologists. doi: 10.3389/conf.fendo.2010.01.00081 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Sep 2010; Published Online: 13 Sep 2010. * Correspondence: Dr. Martina Meyering-Vos, University of Bayreuth, Animal Ecology I, Bayreuth, Germany, martina.meyering-vos@uni-bayreuth.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Martina Meyering-Vos Sandra Diersch Reinhard Predel Joseph Woodring Google Martina Meyering-Vos Sandra Diersch Reinhard Predel Joseph Woodring Google Scholar Martina Meyering-Vos Sandra Diersch Reinhard Predel Joseph Woodring PubMed Martina Meyering-Vos Sandra Diersch Reinhard Predel Joseph Woodring Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Weak influences of physical and chemical factors on the morphogenetic process (in Invertebrates)

Analysis is presented of long-term data obtained in investigation of the effects of weak influences on morphogenetic processes in invertebrates (regeneration of planarians Dugesia (Girardia) tigrina and postembryonic development of insects, the grain beetle Tenebrio molitor). Weak physical and chemical factors were used: electromagnetic radiation, constant, alternating, and combined magnetic fields, and low concentrations of solutions of neuropeptides. It is shown that these influences are characterized by instability and opposite directions (from stimulatory to inhibitory). The dependence of the effects on external factors and the course of internal processes was established.

The possible evolutionary role of tumors in the origin of new cell types

The ability of tumor cells to differentiate in combination with their ability to express genes that are not expressed in normal tissues, may result in the emergence of new cell types in evolution. Tumors may play an evolutionary role by providing conditions (space and resources) for the expression of newly evolving genes. Genetically or epigenetically predetermined tumors at the early stages of progression, benign tumors, and some tumor-like processes in invertebrates and plants, all of which are modes of excess cell growth which provide evolving multicellular organisms with extra cell masses, are considered as potentially evolutionarily meaningful. Malignant tumors at the late stages of progression, however, are not. The preexisting cell types of multicellular organisms had restricted potential for the expression of newly evolving genes. Because of regulation and gene competition, some of the newly evolving genes may stay silent. Multicellular organisms would need excess cell masses for the expression of newly evolving genes. The preexisting cell types cannot provide such excess cell masses because of limitations imposed on the number of possible cell divisions. Tumors could provide the evolving multicellular organisms with the excess cell masses for the expression of newly evolving genes. We suggest that tumors could be a sort of proving ground (or reservoir) for the expression of newly evolving genes that originate in the course of genome evolution in the DNA of germ cells (i.e., not in tumor cells themselves). The case in which the expression of a newly evolving gene in tumors results in the origin of a new function would be associated with the origin of new feedback and regulatory circuits, as in root nodules in legumes and macromelanophores in Xiphophorus fishes. Tumor cells would differentiate, resulting in a new cell type for the given multicellular species. This cell type would be inherited because of epigenomic mechanisms similar to those in preexisting cell types. Populations of tumor-bearing organisms with genetically or epigenetically programmed tumors could represent the transition between established species of organisms at different stages of progressive evolution. Experimental confirmation of the prediction of the hypothesis of evolution by tumor cells differentiation concerning the expression of evolutionarily new genes and/or silent (neutrally evolving) sequences in tumor cells is presented.

Roaches, apoptosis and the ovarian clock: use it or lose it

F or many female animals, including humans, aging is accompanied by declines in reproductive success. Fecundity declines are often attributed to a loss of ovulatory capacity, either due to depletion of finite stores of ovarian follicles or loss of ability to generate viable eggs. In the view of evolutionary senescence and life-history theory, reproductive aging is an expected result of genetic and physiological trade-offs between investment in reproduction and long-term somatic maintenance, and of weakened natural selection on older organisms (Kirkwood and Austad, 2000). Nutritional interventions, such as food restriction, have been shown to affect aging and reproductive lifespan in a wide variety of invertebrate and vertebrate species, and experimental manipulation of the timing and rate of reproduction often have a robust influence on aging and longevity (Partridge et al., 2005). But relatively little research has focused on how altered nutrition and reproduction early in the life span affect specific physiological or cellular changes correlated with ovarian senescence and female reproductive failure. In an intriguing new study, Edvardsson et al. (2009) estimated quantitative genetic variation in the regulation of aging-related rates of ovarian apoptosis (a measure of ovarian senescence) under both severe food restriction and ad lib feeding in the African cockroach, Nauphoeta cinerea (Figure 1). It is important to note that they found rates of ovarian apoptosis to be almost fourfold higher in food-deprived than ad lib-fed females. In addition, they report reductions in female reproductive lifespan and increased rates of ovarian apoptosis after food restriction (but not food excess) when combined with experimentally delayed first reproduction. Their results suggest that, at least under certain environmental circumstances, this cellular correlate of infertility is under additive genetic control, and hence is subject to natural selection. Previous work by members of this group reported that loss of fertility in female cockroaches forced them to delay their first mating after sexual maturity, which is associated with higher rates of oocyte loss through apoptosis throughout the reproductive life span, effectively forcing females to adopt a ‘use it or lose it’ strategy (Moore and Sharma, 2005). In sexually reproducing animals, the balance between the rates of mitosis and apoptosis (programmed cell death) is continuously shifting during the healthy lifespan of an organism, including the developmental period (Hussein, 2005). During ovarian development and sexual maturation, coordinated waves of apoptosis set the stage for healthy oogenesis and staging of preovulatory follicles. As the ovary ages, higher rates of apoptosis are a benchmark of impending infertility. But why study ovarian aging in cockroaches in the first place? Nauphoeta arguably is a much less popular laboratory animal than its distant insect relative, the fruit fly Drosophila melanogaster, the classical model for genetic studies and endocrinological basis of trade-offs between fecundity and longevity. Moreover, our molecular toolkit for understanding basic aging processes in invertebrates is far better developed in both Drosophila and the nematode, Caenorhabditis elegans. But cockroaches have established—if relatively unsung—utility for studies of basic neuroendocrinology and reproduction. Even more important from a comparative aging standpoint, insects exhibit immense diversity in their reproductive biology that may or may not be reflected in differences in the proximate mechanisms underlying reproductive trade-offs. While Drosophila is a marvelous genetic model for studies of reproductive trade-offs, unlike vertebrates and many other animals, fruit flies are oviparous, with ovaries that continuously generate new oogonia and viable new eggs over the course of the normal reproductive life span. Nauphoeta, in contrast, is ovoviviparous, and bears live offspring. ‘Pregnant’ female roaches carry embryos in a brood pouch until they reach the firstinstar stage. Rather than reproducing continuously, they exhibit cycles of oocyte maturation followed by this specialized form of parental care. In this and previous studies by Moore and Moore (2001), delays in first reproduction combined with severe food deprivation reduced female roaches’ lifetime reproductive success and induced early infertility. In a general sense, these findings parallel the results of a plethora of food restriction studies from the aging literature. In the lexicon of biogerontology, however, ‘food restriction’ usually is not equated with complete food deprivation, as in the cockroach study. Instead, it usually entails a significant caloric restriction of 20–30% of normal ad lib food intake without actual starvation or malnourishment, and with adjustments in essential micronutrients to compensate for reduced food intake. In Drosophila and many other animal species, less

Molecular characterization and localization of the first tyramine receptor of the American cockroach (Periplaneta americana)

The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 ∼350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

Retinol esterification in the hepatopancreas of the crayfish (Procambarus clarkii)

Vitamin A (retinol) is a necessary nutrient for all higher animals, including many invertebrates which use it as their visual chromophore. All of these animals must get vitamin A in their diet and must be able not only to transport this hydrophobic vitamin to target cells but must also convert it into active forms of the vitamin, such as retinal and retinoic acid. In mammals, retinol is converted into retinyl esters for export from the small intestine, for storage in the liver, and as part of the visual cycle. Little is known about these processes in invertebrates. Mammals have two enzymes that can catalyze the conversion of retinol to retinyl‐esters: lecithin:retinol acyltransferase (LRAT) and acyl‐coA:retinol acyltransferase (ARAT). LRAT is the physiologically important one in mammals. We have identified an ARAT activity in microsomes from the hepatopancrease of the freshwater crayfish, Procambarus clarkii. We were unable to detect any LRAT activity, suggesting crayfish only have one enzyme that can catalyze this reaction. This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax's for both retinol and the acyl‐CoA.

Catecholaminergic responses to environmental stress in the hemolymph of zhikong scallop Chlamys farreri

Catecholamines have long been known to play essential roles in several physiological processes in invertebrates. In this study, enzyme immunoassays were used to examine the levels of catecholamines (including adrenaline (AD), noradrenaline (NA) and dopamine (DA)) in the hemolymph of the scallop, Chlamys farreri, in responses to selected environmental stress. These stressors include high temperature (acute transfer from 17 to 28 degrees C), low salinity (acute transfer from salinity 31 to 20 psu) and air exposure, which are commonly encountered in aquaculture practices or in their natural habitat. The results showed that both AD and NA concentrations increased significantly in response to all the stressors. The levels of these catecholamines peaked 72 hr after acute temperature or salinity stresses, and then decreased gradually. For scallops subjected to air exposure, the duration of AD concentration elevation depended on the exposure temperature with a higher air temperature resulting in a shorter time to return to control level. In contrast, the NA responses were long lasting and no obvious decreases were observed after reaching the peak value. Interestingly, the DA concentration showed an inverse trend in response to stresses compared with AD or NA. These results suggested that the catecholaminergic responses in bivalve hemolymph will provide useful references in predicting the success of aquaculture practices and monitoring the environmental stresses.

Isolation and characterization of Wnt pathway‐related genes from Porifera

The Wnt signal acts by binding to Frizzled receptors, with the subsequent activation of two different signal transduction cascades, the canonical and the non-canonical Wnt pathways, involved in cell growth, differentiation, migration and fate. The canonical pathway functions through the translocation of beta-catenin to the nucleus and the activation of TCF/LEF transcription factors; it plays an important role in developmental patterning and cell fate decisions during embryogenesis. The non-canonical Wnt pathway is responsible for the planar cell polarity process in invertebrates, and for the convergent-extension movements during vertebrate gastrulation. The final effect of the non-canonical Wnt pathway is the rearrangement of the cell cytoskeleton, through the activation of the subfamily of Ras-like small GTPases. In a recent report we described for the first time the isolation of a Wnt-related gene, Sd-Frizzled, from the most basal animal phylum, the Porifera. In the present study we report the isolation and phylogenetic characterization of several Wnt pathway-related genes from the sponge Suberites domuncula: Sd-TCF/LEF, Sd-GSK3, a recently discovered molecule with a putative function as a Wnt regulator (Sd-LZIC), the small Rho GTPases Sd-RhoA, Sd-Cdc42, and their effector Sd-mrlc. Also the isolation of a secreted frizzled related protein sFRP from another sponge species (Lubomirskia baicalensis) is reported.

Chironomids: suitable test organisms for risk assessment investigations on the potential endocrine disrupting properties of pesticides

Selecting an appropriate invertebrate assay has been a primary goal of national and international testing programs for endocrine disrupting chemicals. The available information on the endocrine system, its hormones and their modes of action in controlling physiological processes in invertebrates is limited and the selection of appropriate test species still presents a challenge. This paper outlines the development of a higher-tier full life cycle (FLC) test for pesticides with the non-biting midge Chironomus riparius (Insecta, Diptera, Chironomidae). As an insect, C. riparius represents the species' richest and ecologically one of the most important groups of invertebrates. In addition, the endocrine system of insects is one of the best studied among the invertebrates. Acute and chronic tests with Chironomus spp. are commonly used for testing and risk assessment of agrochemicals. A chironomid FLC test protocol has been developed and its suitability investigated in an inter-laboratory comparison. The protocol used is based on existing OECD and US-EPA test methods. To verify the suitability of the test to generate endpoints that encompass adverse effects on the arthropod endocrine system, a juvenile hormone analog was selected as positive control substance. Results have demonstrated that the proposed chironomid FLC can be performed in separate laboratories and that the selected arthropod juvenile hormone mimic causes effects. However, the observed toxicity is not proof of an endocrine disruptive mechanism and could equally be evoked by other compounds. Contrary to a screening assay, which aims at revealing a substance's mode-of-action, the FLC test generates robust, population-relevant endpoints that can be used in the risk assessment of agrochemicals. Since the initial results presented in this paper are encouraging we propose to complete the validation of this assay under OECD with high priority.