Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes

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Fysiologiske egenskaper ved aldring i honningbier (Apis mellifera)

Aging and a decline in physiological resilience occur in most living organisms, including honey bee workers. Aging research in different organisms—from yeast, worms, insects, and mice to humans—has investigated several causes. Aging is explained by different theories, often separated into programmed aging and stochastic-damage-caused aging. Programmed aging suggests that aging is controlled by a biological timetable, gene regulation, or a program plan. Stochastic-damage-caused theories explain aging by an accumulation of random damages in organs or cells. In addition, immunity and disease defense are reduced by increased age, together with learning ability and other cognitive functions. In aging studies, it can be advantageous to use model organisms to identify evolutionarily conserved mechanisms. Model organisms offer the opportunity to perform controlled experiments, wholelifespan studies, and experimental intervention with mortality as a direct readout. In this thesis, honey bee workers are used as the model organism. They have an interesting division of labor, where a young worker bee usually starts as nurse bee, which cleans and takes care of the brood within the hive. After a few weeks, she usually transfers to foraging activities to collect nectar, pollen, and water in the field. In honey bee workers, their physiological age and resilience are related to worker caste, where nurses are the physiologically youngest and foragers the physiologically oldest phenotype. A third caste of worker is the winter bees (diutinus), which are the most stress resilient phenotype and the longest-lived. Through my thesis project, I have studied the three worker castes of the honey bee to in more depth. My research goal was to help build a better understanding of their phenotypic differences. One interesting finding in this thesis is that very old forager bees can be more resilient than younger forager bees. On the other hand, my work also demonstrates that nurses have better cellular immunity than foragers and winter bees, thereby supporting the idea that worker bees’ aging is more dependent on their tasks than on chronological age, and that aging is most pronounced in foragers and less so in nurses and winter bees. The findings illustrate that aging patterns can be difficult to explain by a single theory or framework, since I can conclude that the most aged phenotype, the foragers, can be both resilient and fragile.Aldring og nedgang i fysiologisk motstandsdyktighet inntreffer i de aller fleste levende organismer, inkludert honningbienes arbeidere. Aldringsforskning har undersøkt flere årsaker i ulike organismer, fra gjærsopp, ormer, insekter, mus til mennesker. Aldring kan forklares av flere teorier hvor de ofte blir separert i programmert aldring og stokastisk-skade forårsaket aldring. Programmert aldring antyder at aldring styres av en biologisk klokke, genregulering eller en programplan. Stokastisk-skade forårsakede teorier forklarer aldring ved en akkumulering av tilfeldige skader i organer eller celler. Ved økende alder reduseres immunitet og motstandsdyktighet mot sykdommer, sammen med evnen til å lære og andre kognitive funksjoner. I aldringsstudier kan det være fordelaktig å bruke modellorganismer for å identifisere evolusjonært konserverte mekanismer. Modellorganismer gir oss muligheten til å utføre kontrollerte eksperimenter, studere livsløps og utføre eksperimentelle inngrep med dødelighet som en direkte avlesning. I denne avhandlingen brukes honningbier som modellorganisme. De har en interessant arbeidsfordeling, hvor en ung arbeiderbie vanligvis begynner som en ammebie som rengjør og tar vare på larvene i bikuben. Etter noen uker går hun over til å være en trekkbie, dette innebærer å samle nektar, pollen og vann. En arbeiderbies fysiologiske alder og motstandsdyktighet har en sammenheng med arbeiderkasten den tilhører, hvor ammebiene er en fysiologisk yngre fenotype og trekkbiene er fysiologisk eldre. En tredje arbeidertype er vinterbier (diutinus), denne er den mest motstandsdyktige fenotypen mot stress og er den lengst-levende. I denne avhandlingen har jeg studert de tre arbeiderkastene til honningbier i dybden. Mitt forskningsmål var å bygge en bedre forståelse av de fenotypiske forskjellene. Et interessant funn i denne avhandlingen er at svært gamle trekkbier kan være mer motstandsdyktige enn yngre trekkbier. På en annen side viser arbeidet at ammebier har bedre cellulær immunitet enn trekk og vinterbier og derfor støtter ideen om at arbeiderbienes aldring er mer avhengig av deres oppgave enn kronologisk alder, og at aldring er mest uttalt i trekkbier og mindre i amme og vinterbier. Funnene illustrerer at aldringsmønstre kan være vanskelig å forklare ved en enkelt teori eller rammeverk, ettersom at jeg kan konkludere med at den eldste fenotypen, trekkbiene, kan være både motstandsdyktige og sårbare.Research Council of Norwa

Physiological features of aging in honey bees (Apis mellifera)

Aging and a decline in physiological resilience occur in most living organisms, including honey bee workers. Aging research in different organisms—from yeast, worms, insects, and mice to humans—has investigated several causes. Aging is explained by different theories, often separated into programmed aging and stochastic-damage-caused aging. Programmed aging suggests that aging is controlled by a biological timetable, gene regulation, or a program plan. Stochastic-damage-caused theories explain aging by an accumulation of random damages in organs or cells. In addition, immunity and disease defense are reduced by increased age, together with learning ability and other cognitive functions. In aging studies, it can be advantageous to use model organisms to identify evolutionarily conserved mechanisms. Model organisms offer the opportunity to perform controlled experiments, wholelifespan studies, and experimental intervention with mortality as a direct readout. In this thesis, honey bee workers are used as the model organism. They have an interesting division of labor, where a young worker bee usually starts as nurse bee, which cleans and takes care of the brood within the hive. After a few weeks, she usually transfers to foraging activities to collect nectar, pollen, and water in the field. In honey bee workers, their physiological age and resilience are related to worker caste, where nurses are the physiologically youngest and foragers the physiologically oldest phenotype. A third caste of worker is the winter bees (diutinus), which are the most stress resilient phenotype and the longest-lived. Through my thesis project, I have studied the three worker castes of the honey bee to in more depth. My research goal was to help build a better understanding of their phenotypic differences. One interesting finding in this thesis is that very old forager bees can be more resilient than younger forager bees. On the other hand, my work also demonstrates that nurses have better cellular immunity than foragers and winter bees, thereby supporting the idea that worker bees’ aging is more dependent on their tasks than on chronological age, and that aging is most pronounced in foragers and less so in nurses and winter bees. The findings illustrate that aging patterns can be difficult to explain by a single theory or framework, since I can conclude that the most aged phenotype, the foragers, can be both resilient and fragile

Measurements of phagocytic events do not differ between phagocytic markers and are not affected by a bacterial challenge.

<p><b>A</b> Quantitative analyses of the latex bead and CM-Dil uptake with high-resolution microscopy yield similar results (T-test: <i>P</i>>0.05). The relative ratio of marker positive hemocytes in mature nurse bees is shown as mean ± SE. <b>B</b> Activation with <i>E</i>.<i>coli</i> or Grace’s insect media as control does not change the relative ratio of the phagocytic cells in mature nurse bees compared to the individuals with no activation, shown as mean ± SE.</p

Correlation analysis, relation of mitosis and phagocytic hemocytes or vitellogenin positive hemocytes.

<p><b>A</b> Phagocytic activity correlated to mitotic activity in nurses shows a significant negative correlation (r² = 0.35, <i>P</i><0.05). This indicates that an individual has either a high phagocytic activity or a high mitotic activity. In forager and winter bees, no correlation was detectable. <b>B</b> Winter bees show a negative correlation between Vg levels and mitotic activity (r² = 0.63, <i>P</i><0.001). This indicates that that the winter bees with a low number of vitellogenin positive hemocytes have more hemocytes that undergo mitosis. A significant correlation was not detected in nurse or forager bees.</p

Quantification of Vg positive cells from hemolymph in nurses, foragers and winter bees.

<p><b>A</b> A dot plot separated with FSC and SSC shows the gating for removing small particles and the background noise. <b>B</b> Immunostaining of hemocytes with and without the Vg primary antibody. The histogram shows hemocytes incubated with the secondary antibody only (control: black) and hemocytes incubated with both the Vg primary and secondary antibodies (grey). <b>C</b> Dot plot from a Vg positive sample, where the gating approach (the horizontal and vertical lines) are adapted from the controls of DAPI and Vg antibody. The two regions to the right are counted as hemocytes with the upper right region as Vg positive hemocytes. <b>D</b> The mean percentage of the Vg positive hemocytes is higher in winter bees compared to nurses and forager bees (One-way ANOVA: <i>P</i><0.01). The data are presented as means ± SEM.</p