Low rates of iridoid glycoside hydrolysis in two Longitarsus leaf beetles with different feeding specialization confer tolerance to iridoid glycoside containing host plants

Iridoid glycosides are plant defence compounds that are deterrent and/or toxic for unadapted herbivores but are readily sequestered by dietary specialists of different insect orders. Hydrolysis of iridoid glycosides by β‐glucosidase leads to protein denaturation. Insect digestive β‐glucosidases thus have the potential to mediate plant–insect interactions. In the present study, mechanisms associated with iridoid glycoside tolerance are investigated in two closely‐related leaf beetle species (Coleoptera: Chrysomelidae) that feed on iridoid glycoside containing host plants. The polyphagous Longitarsus luridus Scopoli does not sequester iridoid glycosides, whereas the specialist Longitarsus tabidus Fabricius sequesters these compounds from its host plants. To study whether the biochemical properties of their β‐glucosidases correspond to the differences in feeding specialization, the number of β‐glucosidase isoforms and their kinetic properties are compared between the two beetle species. To examine the impact of iridoid glycosides on the β‐glucosidase activity of the generalist, L. luridus beetles are kept on host plants with or without iridoid glycosides. Furthermore, β‐glucosidase activities of both species are examined using an artificial β‐glucosidase substrate and the iridoid glycoside aucubin present in their host plants. Both species have one or two β‐glucosidases with different substrate affinities. Interestingly, host plant use does not influence the specific β‐glucosidase activities of the generalist. Both species hydrolyse aucubin with a much lower affinity than the standard substrate. The neutral pH reduces the β‐glucosidase activity of the specialist beetles by approximately 60% relative to its pH optimum. These low rates of aucubin hydrolysis suggest that the ability to sequester iridoid glycosides has evolved as a key to potentially preventing iridoid glycoside hydrolysis by plant‐derived β‐glucosidases.     

Knockdown of Na,K-ATPase β-subunits in Oncopeltus fasciatus induces molting problems and alterations in tracheal morphology

The ubiquitously expressed transmembrane enzyme Na,K-ATPase (NKA) is vital in maintaining functionality of cells. The association of α- and β-subunits is believed to be essential for forming a functional enzyme. In the large milkweed bug Oncopeltus fasciatus four α1-paralogs and four β-subunits exist that can associate into NKA complexes. This diversity raises the question of possible tissue-specific distribution and function. While the α1-subunits are known to modulate cardenolide-resistance and ion-transport efficiency, the functional importance of the β-subunits needed further investigation. We here characterize all four different β-subunits at the cellular, tissue, and whole organismal scales. A knockdown of different β-subunits heavily interferes with molting success resulting in strongly hampered phenotypes. The failure of ecdysis might be related to disrupted septate junction (SJ) formation, also reflected in β2-suppression-induced alteration in tracheal morphology. Our data further suggest the existence of isolated β-subunits forming homomeric or β-heteromeric complexes. This possible standalone and structure-specific distribution of the β-subunits predicts further, yet unknown pump-independent functions. The different effects caused by β knockdowns highlight the importance of the various β-subunits to fulfill tissue-specific requirements.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

F?nge von Habichten (Accipiter gentilis) im Wurzacher Ried: Kritische Fragen zu einem beh?rdlich genehmigten Wiedereinbürgerungsprojekt

Zusammenfassung Während des unter der Trägerschaft des Landesjagdverbandes Baden-Württemberg e. V. seit 1978 laufenden Projektes zur Wiedereinbürgerung des Birkhuhnes im Wurzacher Ried wurde behördlicherseits der Wegfang von Habichten unter bestimmten Auflagen genehmigt. Nach Beringung mußten die Vögel wieder freigelassen werden. Eine vergleichende Untersuchung der von den Projektbetreibern veröffentlichten Berichte und der Beringungsdokumente ergab erhebliche Ungereimtheiten. Die aus den Berichten der Projektbetreiber zu entnehmende Zahl von 168 gefangenen, angeblich beringten und angeblich wieder freigelassenen Habichten steht der Zahl von 98 in den Beringungslisten dokumentierten Vögel gegenüber. Bereits ein Vergleich zwischen der Wiederfundrate der dokumentierten Vögel (1 %) mit der Fänglingfundrate der Vogelwarte Radolfzell (13 %) ergab einen hochsignifikanten Unterschied. Auch der Vergleich mit der Fundrate (15 %) einer anderen, parallel laufenden Verfrachtungsstudie zeigte, daß die Wiederfundrate der Wurzacher Habichte hochsignifikant geringer ist. Die Darstellung der Projektbetreiber, wonach 1978–1981 insgesamt 90 Habichte durch die Landesanstalt für Umweltschutz verfrachtet worden sein sollen, wurde durch die Landesanstalt nicht bestätigt. Die Landesanstalt bestätigte nur fünf verfrachtete Habichte aus dem Wurzacher Projekt. Obwohl der Habichtfang im März nicht erlaubt war, haben die Projektbetreiber insgesamt vier Habichte im März gefangen und zumindest einen (nach eigenen Angaben jedoch alle!) auch noch verfrachtet. Vermutlich im Auftrag von Geflügelzüchtern wurden sieben Habichte, darunter vier Altvögel, innerhalb eines kleinen Gebietes in einer Entfernung von 13–18,5 km vom Wurzacher Ried gefangen. So besteht begründeter Verdacht, daß ein beträchtlicher Teil der im oder um das Wurzacher Ried gefangenen Habichte nicht wieder freigelassen, sondern getötet wurde. Eine Reihe der in den Projektberichten enthaltenen Angaben lassen zudem ganz erhebliche Zweifel an der Fachkompetenz der Projektbetreiber aufkommen.

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Goshawk (Accipiter gentilis) trapping in the nature reserve Wurzacher Ried: critical questions on a Black Grouse (Tetrao tetrix) reintroduction project

Summary A Black Grouse reintroduction project was established in 1978 in the nature reserve Wurzacher Ried in southwestern Germany. Since the beginning of this project the staff had a licence to catch, ring and to release but not to kill goshawks. A comparison of the very low Euring-recovery-rate of 1 percent from this project with the Euring-recovery-rate of the Vogelwarte Radolfzell (13 %) showed a significant difference (p=0.00017). In contrast, we found no difference between the recovery-rate (15 %) of a study with almost similar design conducted by the University of Tübingen (Schmidt-Koenig 1982) only 100 km apart from the Wurzacher Ried and the Euring-recovery-rate of the Vogelwarte Radolfzell (p=0.624). The recovery-rate of the reintroduction project differed, however, significantly from the recovery-rate ofSchmidt-Koenig (p=0.006). Furthermore, we found serious inconsistencies between the project reports and the ringing documents concerning this project. Our results indicate, that most of the goshawks caught in the project Wurzacher Ried may not have been released but illegally killed by the project staff.

     

Heritability,evolvability, phenotypic plasticity and temporal variation in sperm‐competition success of Drosophila melanogaster

Sperm‐competition success (SCS) is seen as centrally important for evolutionary change: superior fathers sire superior sons and thereby inherit the traits that make them superior. Additional hypotheses, that phenotypic plasticity in SCS and sperm ageing explain variation in paternity, are less considered. Even though various alleles have individually been shown to be correlated with variation in SCS, few studies have addressed the heritability, or evolvability, of overall SCS. Those studies that have addressed found low or no heritability and have not examined evolvability. They have further not excluded phenotypic plasticity, and temporal effects on SCS, despite their known dramatic effects on sperm function. In Drosophila melanogaster, we found that both standard components of sperm competition, sperm defence and sperm offence, showed nonsignificant heritability across several offspring cohorts. Instead, our analysis revealed, for the first time, the existence of phenotypic plasticity in SCS across an extreme environment (5% CO₂), and an influence of sperm ageing. Evolvability of SCS was substantial for sperm defence but weak for sperm offence. Our results suggest that the paradigm of explaining evolution by sperm competition is more complex and will benefit from further experimental work on the heritability or evolvability of SCS, measuring phenotypic plasticity, and separating the effects of sperm competition and sperm ageing.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Biased predation could promote convergence yet maintain diversity within Müllerian mimicry rings of Oreina leaf beetles

Müllerian mimicry is a classic example of adaptation, yet Müller's original theory does not account for the diversity often observed in mimicry rings. Here, we aimed to assess how well classical Müllerian mimicry can account for the colour polymorphism found in chemically defended Oreina leaf beetles by using field data and laboratory assays of predator behaviour. We also evaluated the hypothesis that thermoregulation can explain diversity between Oreina mimicry rings. We found that frequencies of each colour morph were positively correlated among species, a critical prediction of Müllerian mimicry. Predators learned to associate colour with chemical defences. Learned avoidance of the green morph of one species protected green morphs of another species. Avoidance of blue morphs was completely generalized to green morphs, but surprisingly, avoidance of green morphs was less generalized to blue morphs. This asymmetrical generalization should favour green morphs: indeed, green morphs persist in blue communities, whereas blue morphs are entirely excluded from green communities. We did not find a correlation between elevation and coloration, rejecting thermoregulation as an explanation for diversity between mimicry rings. Biased predation could explain within‐community diversity in warning coloration, providing a solution to a long‐standing puzzle. We propose testable hypotheses for why asymmetric generalization occurs, and how predators maintain the predominance of blue morphs in a community, despite asymmetric generalization.     

Genbank accession #: AF 124983

Plant Molecular Biology -     

Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.