6 search hits
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Control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda
(2010)
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Digali Lwalaba
- This thesis investigates control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda. 1. Control of enzyme release in the cricket G. bimaculatus. Using flat-sheet preparations of the caecum, digestive enzyme release was investigated. More trypsin, aminopeptidase and amylase are secreted in the caecum of fed crickets than in unfed crickets, but basal levels of certain enzymes are released continuously even in unfed animals. A variable ratio of nutrients in ingested food leads to a different ratio of digestive enzyme release, but a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Maltose and glucose elevate amylase release from the tissues into the incubation medium, but starch does not. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase, and only low concentrations of peptone increase trypsin release. In crickets, the continuous release of proteases is sufficient to meet the needs for growth, and only moderate stimulation of trypsin results from feeding. Carbohydrates are used for energy, and the release of amylase is adjusted to the amount of food ingested. The neuropeptide allatostatin Grybi-AS 5 elevates the release of amylase in fed females, but not of trypsin or aminopeptidase, however, both amylase and trypsin release are stimulated by AS 5 in unfed crickets. Fed crickets have sufficient trypsin to obtain needed amino acids, but unfed do not, therefore the AS stimulation of trypsin release in unfed crickets makes sense. 2. Control of enzyme release in the larvae of S. frugiperda. A flat-sheet preparation of the ventriculus was used to test the release of amylase, trypsin and aminopeptidase in response to specific nutrients in the food and to specific neuropeptides. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. Dietary carbohydrates are used for energy, and larvae adjust amylase activity to carbohydrate ingestion. Amylase activity is 5-times higher in fed compared to unfed larvae, and sugars in the incubation medium induces more than a 300 % increase in amylase release. Plants contain a low level of protein, but larvae need proteins for growth, thus the larvae can not afford to lose proteins by egestion. Therefore, trypsin activity remains high even in unfed larvae. As a result, proteins and amino acids have little or no effect on trypsin or aminopeptidase release in incubated tissues. The control of enzymes release in response to food is most likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin release, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse- AT stimulates myoactivity. 3. Inhibition of enzyme release in the larvae of S. frugiperda. Exogenous inhibitors are produced by plants, and are ingested by the insect. Endogenous inhibitors are produced by the gut epithelial cells themselves. A dose-dependent inhibition of endogenous enzymes occur in the lumen after feeding L6 larvae with the exogenous serine protease inhibitor from soybean (SBTI), the specific trypsin inhibitor TLCK, an aminopeptidase inhibitor (bestatin), and an amylase inhibitor from wheat. Inhibition in tissue extracts is seen only with higher doses of SBTI and TLCK. Inhibition of enzyme release into the incubation medium is apparent only with very high doses of SBTI. Inhibition in the tissues and inhibition of release indicate a direct cellular response to an inhibitor present in the lumen. The elevation of aminopeptidase activity in response to ingested trypsin inhibitors indicates a cellular synthesis in response to the product of a digestive enzyme. The enzymes investigated are irreversibly inactivated by 10 min at 90°C, but the corresponding inhibitors are not, therefore endogenous inhibitors could be identified. Endogenous inhibitors are present in the ventricular cells and in the lumen. We suggest that the endogenous protease inhibitors may protect the epithelium by inactivation of the trypsin in underfed larvae. This is the first explanation of how insects are able to secrete an active trypsin.
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Hormonal regulation and environmental influences in the reproduction of the butterfly Bicyclus anynana
(2008)
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Thorin Lukas Geister
- Regarding hormonal control of reproduction, the butterfly Bicyclus anynana belongs to a group of the Lepidoptera, in which egg maturation starts after eclosion, and thus vitellogenesis and choriogenesis seem to depend exclusively on juvenile hormones (JH). Using a JH mimic (pyriproxyfen) and an antagonist of JH biosynthesis (MK-801), reproduction in B. anynana could be successfully manipulated towards either a higher or lower fecundity. Especially early fecundity responded to manipulations. Furthermore, both JH III and JH II were found in the hemolymph throughout lifetime. Nevertheless, fecundity and vitellogenin titres were not clearly related to JH titres, as both decreased continuously with age, although JH III titres stayed constant and JH II titres increased. Thus, reproduction in B. anynana is at least to some extent under hormonal control, with JH being probably an important signal especially for the initiation of reproduction. Oviposition temperature induces a plastic response in egg size and number in B. anynana. While more but smaller eggs are laid at high temperatures representing wet season conditions, larger but fewer eggs are oviposited at lower temperatures, which are experienced in the dry season. This plasticity is thought to be adaptive in this species inhabiting seasonal environments. Vitellogenins are representing a major part of eggs and consequently, JH dynamics represents an obvious target for changes in egg size, as vitellogenin synthesis and/or incorporation into developing eggs might increase or decrease through changes in JH titres. Female B. anynana butterflies exposed to different oviposition temperatures showed the expected response to adult temperature, producing fewer but larger eggs at the colder temperature, but more and smaller eggs at the warmer temperature. However, no evidence was found that this striking example of phenotypic plasticity is under hormonal control, as JH III, JH II, vitellogenin titres and egg proteins showed no significant variations across temperatures. Based on these similar patterns across temperatures, the results in this thesis suggest that the temperature-mediated reproductive plasticity is not mediated through JH in B. anynana. The known fitness advantage of the larger eggs produced at lower temperatures in B. anynana may be related to size per se, to a larger absolute amount of nutrients or to relative changes in egg composition. Therefore, this thesis explored the consequences of temperature variation on egg and hatchling composition, and the associated use and turnover of energy and egg compounds in relation to temperature. Overall, larger eggs produced at the lower temperature were achieved by providing these eggs with larger quantities of all compounds investigated and thus more energy, whilst relative egg composition was rather similar to that of smaller eggs laid at the higher temperature. Turnover rates during embryonic development differed across developmental temperatures, suggesting an emphasis on hatchling quality (i.e. protein content) at the more stressful lower temperature, but on storage reserves (i.e. lipids) at the higher temperature. These observed differences may consequently represent adaptive maternal effects. The availability of adequate adult nutrition is essential for successful reproduction in B. anynana, as without access to carbohydrates in the adult stage no eggs will be produced. A commonly used method for estimating the fitness effects of diets is determining the number and sometimes sizes of eggs produced and often not including offspring viability. Five different nutritional treatments were used for female B. anynana butterflies ranging from moist banana, plain sucrose solution, to sucrose solution enriched with lipids, yeast and finally minerals and vitamins. These treatments were analyzed with regard to their effects on egg composition as well as egg hatchling success. Adult diet was demonstrated to have pronounced effects on fecundity, egg composition and egg hatching success, with butterflies feeding on the complex nutrition of banana fruit performing best. Vitamins and minerals in a sucrose-based diet increased fecundity, but did not affect offspring hatching success. Effects of adult diet on egg composition were not straightforward, indicating complex interactions among specific compounds. Total egg energy and water content seemed to be related to hatching success of progeny. The results of this thesis demonstrate that egg composition should be taken into account in such studies, as egg size and number does not necessarily represent a reliable proxy for reproductive energetic investment.
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Evolutionary and proximate constraints on egg size in butterflies
(2007)
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Stephanie Sandra Bauerfeind
- Arthropod egg and thus progeny size is an evolutionary and ecologically significant trait, showing tremendous variation within and across species. The high variation found is caused by a complex set of interacting proximate and evolutionary factors, but despite increasing effort this network is still only partially resolved. Using butterflies as model organisms, this study focuses on two main factors that are assumed to strongly shape variation in offspring size and number: maternal size and maternal nutrition. Both are assumed to affect reproductive traits and thereby maternal and offspring fitness. Phenotypic correlations between maternal size and egg size within various butterfly species as well as a two-trait selection experiment on simultaneous changes in both traits in the butterfly Bicyclus anynana clearly demonstrated that the importance of maternal size in shaping variation in egg size is limited, both in sense of a morphological as well as an evolutionary constraint. These results strongly contrast to the general assumption of a positive scaling relationship between both traits. This is one of the few studies addressing the issue of evolutionary constraints directly, employing artificial two-trait selection which has been proven to be a powerful tool to unravel genetic variances and covariances that underlie the evolution of traits. While the importance of maternal size in shaping variation in egg size is limited, proximate factors including larval and adult crowding as well as the quantity and quality of available food during the larval and adult stage affect variation in reproductive traits to a high degree. In the butterfly B. anynana, larval and adult densities had surprisingly little effects on female reproduction, whereas dietary limitations yielded strong responses in female reproductive output. Larval food stress reduced fecundity and reproductive investment (mediated through a reduction in body size), but effects on egg size were overall marginal. Additional negative effects of adult food stress on fecundity were largely confined to females being fed as larvae ad libitum, while those being previously starved showed reduced performance regardless of adult income. When abundantly fed during the larval stage, a limitation of adult resources reduced reproductive output, proving the need for adult feeding in B. anynana for egg production. Thus, restricted food access in different developmental stages of B. anynana sets different limits to reproduction, either posed by shortage of larval-derived storage reserves (i.e. nitrogenous compounds) or adult income (i.e. carbohydrates). Consequently, restrictions in both, larval- and adult-derived resources, limit reproduction in B. anynana. Further, this study deals with questions regarding effects of different adult dietary compounds for a fruit-feeding butterfly being novel in its reductionist approach and in the breadth of different nutrient classes considered. This study demonstrates that B. anynana relies to a large extent on adult feeding in order to realise full reproductive output. Female B. anynana require adult-derived carbohydrates for egg production and exhibit a tremendous gain in reproductive output when fed on fruit as compared to sucrose solutions. Contrary to initial expectations, I could not pinpoint a single pivotal substance (in addition to sucrose) that was able to elicit a comparably high reproductive performance as banana, although I tested the micronutrients being most abundantly available in banana (minerals: potassium and magnesium chloride; a mixture of vitamins and a combination of both) and all major substances known to be involved in insect egg production (amino acids, cholesterol, polyunsaturated fatty acids). Further, it is also excluded that the growth of microorganisms and fungi (associated with the production of fermentative products like organic acids and alcohols, thereby providing access to additional resources) explains the found results. In conclusion, reproduction does not only depend on a small number of adult-derived nutrients, but on a larger number having relatively small effects each. Thus, resource congruence (the use of nutrient types in a specified ratio) rather than any specific component may be the key to answer the question.
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Development of broadly applicable transgenic tools for the transposon mutagenesis of the red flour beetle, Tribolium castaneum
(2005)
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Ivana Viktorinová
- The discovery of non-species-specific, broad-range transposable elements and the establishment of a universal 3xP3 promoter revolutionized insect transgenesis. It overcame the limitations of the germ-line transformation to be restricted to the model organism Drosophila melanogaster. In combination with discernable fluorescent markers, multi-component systems, such as transposon-based insertional mutagenesis, can now be introduced to various insect species. To drive the transposase gene for transposable element remobilisation, suitable promoters are needed. The broadly conserved thermotolerance factor, Hsp70, is well-characterised in D. melanogaster and its promoter, which is inducible by high temperatures, provides a genetic tool for transient gene activation. In this thesis, I could prove that the D. melanogaster hsp70 promoter is functional in Tribolium castaneum as well. Its observed basal level activity, however, must be considered and limits its use for experiments, where no strict transient gene expression is required. Nevertheless, the D. melanogaster hsp70 promoter will suffice to provide an efficient transposase source in transposon-based mutagenesis screens in T. castaneum. The remobilization of non-autonomous transposable elements in such screens results in novel mutations and tagging of potentially interesting cis-regulatory elements. To further investigate gene functions, misexpression studies are necessary. In D. melanogaster, this can be done by directed binary expression systems. Here I could show, that the combination of Gal4delta/UAST works best in D. melanogaster somatic tissue, whereas the LexA/(LL)4 and the tetracycline-controlled systems seem to function only poorly. All constructs are based on broad range transposons as well as universal markers and promoters, so that they can be used in other insect species to determine the best system. Preliminary tests in T. castaneum, however, showed that there are a number of additional problems that need to be addressed, before a suitable binary expression system can be established for this species. The full genome sequence of T. castaneum is now available. Therefore, interesting mutations, cis-regulatory elements and their biological functions can be directly linked to the sequence level. When target sites of site-specific recombination systems are included in insertional mutagenesis screens, their insertion sites can be precisely identified and designed chromosomal rearrangements (inversions, duplications and deletions) created. Here I could present a universal system, which can be introduced into non-drosophilid species and enables such chromosomal rearrangements, which I could successfully demonstrate in D. melanogaster. Defined inversions suppressed meiotic recombination between inverted and non-inverted regions on homologous chromosomes and can thus serve as defined balancer chromosomes. Also defined deletions/duplications were generated in D. melanogaster. Such aberrations will be crucial in other insect species, like T. castaneum, to safely keep mutation stocks and identify gene functions. Moreover, the separation of terminal inverted repeats by inverting the chromosomal region between two transposable elements resulted in immobilization. This is of a particular interest for applied transgenesis approaches in insect pest management, when transgenic insects will be released into the nature and transposable elements must be efficiently protected from potential cross mobilization in host species.
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The signal transduction of the adipokinetic hormone and regulation of energy metabolism in the cricket, Gryllus bimaculatus de Geer (Ensifera: Gryllidae)
(2004)
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Anurag N. Anand
- The fat body from the cricket, Gryllus bimaculatus incorporates acetate, glycerol or palmitate into lipid in vitro. Adipokinetic hormone (AKH) inhibits acetate incorporation into lipid by the fat body from adult crickets in vitro. AKH inhibits palmitate incorporation into lipid to a small extent, however, it does not influence the incorporation of palmitate into different lipid classes. In the presence of AKH, glycerol incorporation into lipid by the adult cricket fat body increases by several fold. AKH does not influence the incorporation of glycerol into different lipid classes. The fat body incorporates glycerol mainly into triacylglycerol (TAG) and almost exclusively into the backbone. AKH inhibits fatty acid (FA) synthesis but not the coupling of FAs with the glycerol backbone. Triacylglycerol lipase (TGL) from the fat body, in last larval instar and adult crickets, shows an age-dependent pattern of activity. AKH activates TGL, but does not regulate ACCase, ACL and FAS upon treatment of the fat body for a short period, under the assay conditions. The FAS and acetate incorporation activities are highly correlated in an age-dependent manner and are highest on day 2 of the adult stage. The activation of the formation of sn-1,2-DAG from TAG, in the fat body by AKH seems to be via the removal of FAs at positions 1 and 3, followed by reacylation of 2-MAG. The presence of calcium in the incubation medium is crucial for the inhibition of acetate incorporation by AKH; possibly it is essential for the binding of AKH to the receptor. The influx of acetate and calcium into and efflux of calcium from the cytosol are not affected by AKH. The release of calcium from intracellular calcium stores in the fat body, caused by thapsigargin, inhibits acetate incorporation irreversibly and shows a tendency for the activation of TGL. The calcium ionophore ionomycin inhibits acetate incorporation by the fat body reversibly and shows a tendency for TGL activation. Another ionophore, A23187 also inhibits acetate incorporation. Caffeine and theophylline inhibit acetate incorporation by the fat body in a reversible manner and tend to activate TGL. Caffeine seems to act via the release of calcium from intracellular stores and not via increase in the cellular cAMP concentrations. cAMP analogues and agonists do not influence acetate incorporation, however, the agonists, IBMX and forskolin cause a multifold increase in the cAMP concentrations in the fat body. AKH does not affect the cAMP concentrations in the fat body suggesting that cAMP is not involved in the signal transduction. As the activation of protein kinase C by PMA (a phorbol ester) does not affect acetate incorporation, diacylglycerol does not seem to be involved in the AKH signal transduction. The activation and/or translocation of TGL and inhibition of fatty acid synthesis by AKH seems to be via the release of calcium from intracellular calcium stores. Lipid and protein form a major part of the fat body in the penultimate larval instar crickets, while glycogen forms a minor part. However, in comparison with the adult (glycogen content of fat body about 1%) and last larval instar crickets (glycogen content about 3%), the penultimate larval instar crickets contain higher amounts of glycogen (about 9%). AKH inhibits acetate incorporation into lipid by the fat body from penultimate larval instar crickets. The patterns of acetate incorporation and inhibition by AKH are similar in both, males and females. The inhibition is dose-dependent with an EC50 of 79 pM AKH. AKH seems to play an important role in the development and reproduction of insects, in addition to its role during flight metabolism.
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Isolation and characterization of the B-type allatostatin gene of Gryllus bimaculatus de Geer (Ensifera, Gryllidae)
(2004)
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Junling Wang
- 1. Cricket B type allatostatins, which belong to a neuropeptide family sharing the conserved W(X)6Wamide structure, exhibited inhibitory functions on the biosynthesis of juvenile hormones (JH) in vitro in the corpora allata (CA) as well as on ecdysteroid biosynthesis in the ovary of adult crickets (Gryllus bimaculatus). To understand the mechanisms of function of the pleiotropic cricket B type allatostatins, it is necessary to characterize their gene (preprohormone) and study the spatial and temporal expression patterns of the gene. 2. By PCR screening of a random primer cDNA library and by RACE (Rapid Amplification of cDNA Ends), a 535 bp 3´cDNA sequence of the cricket B type allatostatin gene was yielded. This 3´cDNA fragment encodes a putative translation product of 85 amino acids with potential dibasic endoproteolytic cleavage sites, which may allow processing into six peptides including three copies of Grybi-AS B1 (GWQDLNGGWGa) and single copies of Grybi-AS B2 (GWRDLNGGWGa), Grybi-AS B3 (AWRDLSGGWGa), and Grybi-AS B6 (AWNNLGSAWGa), respectively. Three of these deduced peptides were previously isolated from cricket brain extracts by conventional chromatographic techniques and were designated as Grybi-AS B1, Grybi-AS B2, and Grybi-AS B3. The Grybi-AS B6 neuropeptide represents a novel member of the B type allatostatins. 3. The nucleotide sequences encoding the type B allatostatins are high in GC-content and show strong homology. The highest GC-content was found for Grybi-AS B3 with 83.3%. The similarity of the nucleotide sequences encoding Grybi-AS B2 and Grybi-AS B1 is 93.3%, whereas Grybi-AS B2 and B3 share 90% nucleotide identity. 4. By Southern blot analyses, it was proven that the Grybi-AS type B gene is present as a single copy per haploid genome of G. bimaculatus. 5. By RT in situ PCR technique, it could be demonstrated that the Grybi-AS B gene is expressed in various tissues of 1 day old female adult crickets: In the central nervous system the Grybi-AS B gene expression was detected in the brain. In the protocerebrum, strong positive signals were found in the median neurosecretory cells, and to a lesser extent in lateral neurosecretory cells and in neurons. Gene expression was also found in the neurosecretory cells of the deuterocerebrum and the tritocerebrum. Furthermore, Grybi - AS B gene expression was localized in neurosecretory cells of the suboesophageal ganglion (SOG), the thoracic ganglia, and the abdominal ganglia. In the germarium and in primary oocytes of the ovary, Grybi-AS B gene expression was detected as condensed signals in the nuclei, but not in the prefollicular cells or the cytoplasm. With ongoing development of the oocytes, the signals in the nuclei (germinal vesicles) appeared as separated granules with weaker intensity, which finally disappeared, whereas in the follicular cells strong signals became apparent. Grybi-AS B gene expression was also detected in the epithelial cells of the accessory reproductive glands of female crickets. In the caecum and midgut, Grybi-AS B gene expression was found in endocrine secretory and epithelial cells, whereas in the hindgut, positive RT in situ PCR signals were detected in both longitudinal and circular muscles and in the gut epithelial cells. Grybi-AS B gene expression was also found in cells of the fat body and in thoracic (flight) muscles. 6. The results on Grybi-AS B gene expression as obtained by RT in situ PCR were confirmed by RT-PCR and RNA dot blot analyses. The expression of the Grybi-AS B gene in various tissues of adult females varied in an age-dependent manner. In brains of virgin females gene expression increased from the day of emergence until day 8 of adult life. In the ovary of virgin females gene expression showed a maximum at day 4 after ecdysis, whereas in mated females gene expression was high during the first two days and at days 6 to 7, but low inbetween. In caecum and midgut of virgin females gene expression was low during the first 5 days after ecdysis, but peaked at days 6 and 7, whereas in the hindgut gene expression was highest at day 3 of adult life. In the fat body, gene expression showed highest values on day 1 and days 6 to 7 after ecdysis. 7. Gene expression in brain, testes, and accessory reproductive glands of 0 to 3 days old male crickets was also demonstrated by RT-PCR and RNA dot blot analyses.
