Browsing by Subject "Insects"

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    Classification and characterization of Ethiopian honey bees (Apis mellifera) based on morphometric, genetic and socio-economic analyses
    (2022) Hailu, Teweldemedhn Gebretinsae; Hasselmann, Martin
    Ethiopia is a major beekeeping country located in northeast Africa where several evolutionary lineages of Apis mellifera contact. A unique practice of honey bee colony marketing which involves broad agro-ecological zones (AEZs) is a developing trend in the northern part of the country such as Tigray region in association with apicultural development. Several studies based on classical morphometry on the Ethiopian honey bee subspecies classification debated from the unique Apis mellifera simensis to five others. Moreover, the genetic diversity, adaptation, gene flow and inter-relationships of the honey bees between AEZs were not disentangled – a challenge for planning sustainable apicultural development and conservation. Therefore, this study was conducted to elucidate the honey bees of Ethiopia in a context of apicultural transformation using integrated methods: morphometrics, genetics, colony market survey and metadata analyses on beekeeping development. The results of geometric morphometric analyses confirmed that Ethiopian honey bees represented by Apis mellifera simensis references belong to a separate lineage (Y) compared to A, O, M and C, and the present sample belonged to Y. This supported the hypothesis of five major honey bee lineages of the honey bee Apis mellifera. Similarly, a maximum likelihood phylogenetic tree analysis based on the mitochondrial COI-COII showed that most of the Ethiopian honey bees belong to lineage Y. However, a substantial proportion of the samples from the northern part of the country clustered with lineage O, which support the hypothesis that there is close contact between Y and O. Both geometric morphometry and classical morphometry differentiated the Ethiopian honey bees from all references including A. m. monticola, A. m. scutellata, A. m. jementica, A. m. adansonii but grouped with A. m. simensis. Genetically, five DraI haplotypes (COI-COII) were found to be randomly distributed across AEZs, indicating a substantial gene flow. Consequently, the level of genetic differentiation among the Ethiopian honey bee subpopulations defined by local areas and AEZs was generally low based on r7-frag nuclear marker, which is identified to be associated with adaptation to habitat elevation in East African honey bees. Similarly, nucleotide diversity consistently decreased with increasing elevation – indicating a reduced effective population size in the highlands. Results obtained from colony market survey showed that the honey bee swarms are reproduced in a few highlands and re-distributed throughout the region. Colony buyers have preferences of color and AEZ of origin of the honey bees, which led to a one-way flow and eroded the overall level of genetic differentiation. However, a marked differentiation was detected between the highland and lowland honey bees in relic communities where an allelic length polymorphism was observed as a signature of local adaptation. Altogether, Ethiopian honey bees belong to the lineage Y and subspecies A. m. simensis, and are characterized by a high level of gene flow enhanced by colony marketing; but a conserved signature of local adaptation to higher elevations was identified in less disturbed communities. Further studies based on genome-wide analyses and field experiments, focusing on undisturbed communities, can provide more insights into adaptation, admixture and management implications. Sustainable bee breeding and extension services that enable local beekeeping without colony trade and transportation will help to promote apiculture and genetic conservation.
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    Lokalisation von Pheromon-Rezeptoren und -Bindeproteinen in antennalen Sensillen von Insekten
    (2007) Gohl, Thomas; Breer, Heinz
    The remarkable reactivity of moth to specific pheromones is based on the extreme selectivity and sensitivity of sensory cells in the male antennae. This feature is supposed to be based on cells equipped with specific receptors. Only the sequencing of the genomes of Bombyx mori and Heliothis virescens provided the possibility to identify candidate genes of olfactory receptors in moths. Upon detailed inspection of candidate receptors it turned out that within the generally very heterogeneous group of receptor-genes a subfamily exists containing members of both species showing striking sequence homology. A conservation of the primary structure of receptors for pheromones has been postulated. For a continuing characterization in these studies different approaches were used to verify that these receptors are indeed expressed in cells of pheromone-sensitive sensilla (sensilla trichodea). By means of ?whole mount? in situ hybridization experiments the RNA of the receptor types BmOR1 and BmOR3 could be visualized in directly neighboring cells reflecting the topology of trichoid sensilla. Also some of the Heliothis receptor types (HR13, HR14, HR16) could be assigned to sensilla trichodea. In addition to the specific receptors, the pheromone binding proteins (PBPs) are expected to play an important role in the detection of hydrophobic pheromone molecules. PBPs are produced by glia-like cells surrounding the sensory neurons. In double in situ hybridization experiments it could be shown that HR13-cells are indeed surrounded by cells expressing HvirPBP1 and HvirPBP2. Analysis comparing the topology of different receptor-types showed that cells expressing HR13 can be assigned to sensilla trichodea type A, whereas HR14 and HR16 are expressed in cells of sensilla trichodea type C. This characteristic expression pattern is considered as a further indication that these candidate-receptors are indeed pheromone-receptors. The assignment of individual receptor-types to distinct sensilla-types provides the basis for investigating the functional implications of receptor-types for the registration of main or minor components of complex pheromone-blends. Further it turned out that HR13 shows coexpression with SNMP1 (sensory neuron membrane protein 1) which is considered as a ?marker?-protein for antennal sensory neurons. This is however not the case for receptor types HR14 and HR16. In search of further SNMP-types screening-experiments were carried out which led to the identification of a novel SNMP-type (SNMP2) of Heliothis virescens. Subsequent studies concerning the expression of SNMP2 showed that the topologic distribution of SNMP2-cells is comparable to SNMP1-cells, but they show a different morphology. Further experiments revealed that SNMP2 is in fact expressed in PBP-producing cells. These findings imply that the proposed putative function of SNMPs has to be reconsidered. One major goal of this study was the attempt, to identify receptor-relevant cells by visualization of mRNA via in situ hybridization but to visualize the localization of the receptor-protein via immunohistochemical approaches. Although the generation of antibodies for olfactory receptors is very difficult, it was possible to raise antibodies specific for receptor type HR13. Using these antibodies in immunohistochemical approaches allowed to also visualize HR13-receptor-protein. By means of double-staining experiments using HR13-specific antisense RNA-probes and anti-HR13 antibodies mRNA and protein were visualized in the same specific cells. Using confocal laserscanning microscopy, it was possible to document that receptor-protein was indeed located in the sensory dendrites. Further, the receptor-protein was also visualized in the axonal processes of sensory cells and the receptor-specific staining revealed that within the antennal nerve HR13-axons appear to be organized in fascicles. These HR13-immunolabeled fascicles were visible until they reach the ?sorting zone? of the antennal lobe; in contrast to mouse olfactory bulb, no receptor specific staining was visible in the antennal lobe.
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    Sequestration of plant toxins in milkweed bugs (Heteroptera: Lygaeinae) : physiological implications and mechanisms
    (2023) Espinosa del Alba, Laura; Petschenka, Georg
    Insect herbivores and plants together are a crucial component of terrestrial macro-biodiversity. Within the realm of plant-insect interactions, phytophagy by insects triggered an “arms-race” dynamic resulting in escalatory adaptation and counter-adaptation over time. This coevolution led to complex phenomena such as sequestration of plant toxins by specialized insects, with the main aim to deter predators. Although sequestration is an extensively reported phenomenon, many physiological aspects and underlying mechanisms remain largely unexplored. Milkweed bugs (Heteroptera: Lygaeinae) constitute a versatile model ideally suited for studying both areas due to their particular evolutionary history. They are primarily associated with plant species in the Apocynaceae which commonly produce cardenolides, but remarkably some milkweed bug species secondarily evolved novel associations with phylogenetically disparate plant families supplying new sources of chemically related or unrelated toxins. Using as model milkweed bugs the cardenolide specialist Oncopeltus fasciatus and Spilostethus saxatilis, a species that shifted to sequestration of the chemically unrelated colchicoids, the present thesis first aimed to develop a new artificial diet that allowed the incorporation of the desired types and amounts of toxins without impairing insect performance. Taking a simplified approach, an artificial diet presented in a pill form and made of 100% organic sunflower meal was established. Despite the fact that the new diet has remarkable energy and nutrient differences with sunflower seeds (the laboratory diet), no differences in terms of insect performance were found between the two diets in O. fasciatus and S. saxatilis. Moreover, the new diet presented an acceptable concentration accuracy and shelf-life for short-term toxin feeding assays. Once established, the new diet was used to investigate the effects of cardenolides and colchicoids on several life-history traits of S. saxatilis, and to compare them with the effect of cardenolides in O. fasciatus. Although both classes of toxins have different molecular targets (cardenolides: Na+/K+-ATPase; colchicoids: tubulin), S. saxatilis was able to sequester them at a cost-free level. In fact, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis after dietary exposure to cardenolides and colchicoids, respectively. Among cardenolides, labriformin is especially toxic for milkweed-specialist Na+/K+-ATPases in vitro. Nevertheless, it was shown to have no costs in terms of growth and fertility at the whole organism level for O. fasciatus. This finding might be an example of reciprocal evolution between milkweed plants and its herbivores, where highly toxic cardenolides specifically targeted to insect specialists are counteracted by tolerance, detoxification, and sequestration strategies. To assess the role of sequestration beyond normal physiological conditions, O. fasciatus and S. saxatilis were orally infected with the bacterium Pseudomonas entomophila. Neither cardenolides nor colchicoids provided a higher resistance or tolerance. Regarding mechanisms of sequestration, the other overarching research topic of the present thesis, both in vivo (whole animal) and in vitro (isolated digestive tracts) approaches showed no reciprocal competition for the same transport mechanism between chemically related and unrelated toxins. Furthermore, the digestive tract of milkweed bugs did not seem to be a critical mediator as it is for other non-sequestering and sequestering species. The time course of sequestration for the model species was resolved from three days to one hour, and the higher levels of colchicoids detected in S. saxatilis compared to the level of cardenolides in O. fasciatus might indicate an early acquisition of defenses with the shift from cardenolide to colchicoid-containing plants. Finally, a hint to preadaptation mechanisms to resist novel toxins was documented in Spilostethus pandurus, a species that belongs to the same genus as S. saxatilis, thereby providing a basis for future investigations.