Uncapped Cells Research Articles

The development of honey bee colonies assessed using a new semi-automated brood counting method: CombCount.

Precise, objective data on brood and honey levels in honey bee colonies can be obtained through the analysis of hive frame photographs. However, accurate analysis of all the frame photographs from medium- to large-scale experiments is time-consuming. This limits the number of hives than can be practically included in honeybee studies. Faster estimation methods exist but they significantly decrease precision and their use requires a larger sample size to maintain statistical power. To resolve this issue, we created ‘CombCount’ a python program that automatically detects uncapped cells to speed up measurements of capped brood and capped honey on photos of frames. CombCount does not require programming skills, it was designed to facilitate colony-level research in honeybees and to provide a fast, free, and accurate alternative to older methods based on visual estimations. Six observers measured the same photos of thirty different frames both with CombCount and by manually outlining the entire capped areas with ImageJ. The results obtained were highly similar between both the observers and the two methods, but measurements with CombCount were 3.2 times faster than with ImageJ (4 and 13 min per side of the frame, respectively) and all observers were faster when using CombCount rather than ImageJ. CombCount was used to measure the proportions of capped brood and capped honey on each frame of 16 hives over a year as they developed from packages to full-size colonies over about 60 days. Our data describe the formation of brood and honey stores during the establishment of a new colony.

Variable temperature PXRD investigation of the phase changes during the dehydration of potassium Tutton salts

The thermal dehydration of the potassium Tutton salts K2M(SO4)2·6H2O (M = Mg, Co, Ni, Cu, Zn) was investigated using thermal gravimetric analysis (TG), differential scanning calorimetry (DSC), FTIR, and variable temperature powder X-ray diffraction. While each Tutton salts lost all six waters of hydration when heated to 500 K, the decomposition pathway depended on the divalent metal cation. K2Ni(SO4)2·6H2O lost all six waters in a single step, and K2Cu(SO4)2·6H2O consistently lost water in two steps in capped and uncapped cells. In contrast, multiple decomposition pathways were observed for the magnesium, cobalt, and zinc Tutton salts when capped and uncapped TG cells were used. K2Zn(SO4)2·6H2O lost the waters of hydration in a single step in an uncapped cell and in two steps in a capped cell. Both K2Mg(SO4)2·6H2O and K2Co(SO4)2·6H2O decomposed in a series of steps where the stability of the intermediates depended on the cell configuration. A greater number of phases were often observed in DSC and capped-cells TG experiments. A quasi-equilibrium model is presented that could explain this observation. These results highlight that experimental conditions play a critical role in the observed thermal decomposition pathway of Tutton salts.

Honey bee hygienic behaviour does not incur a cost via removal of healthy brood

In the honey bee, hygienic behaviour, the removal of dead or diseased brood from capped cells by workers, is a heritable trait that confers colony-level resistance against brood diseases. This behaviour is quite rare. Only c. 10% of unselected colonies show high levels of hygiene. Previous studies suggested that hygiene might be rare because it also results in the removal of healthy brood, thereby imposing an ongoing cost even when brood diseases are absent. We tested this hypothesis by quantifying hygienic behaviour in 10 colonies using a standard technique, the freeze-killed brood (FKB) bioassay. At the same time, we also quantified the removal of untreated brood. The study colonies showed a wide range in hygienic behaviour, removing 19.7-100% of the FKB. The removal of untreated brood ranged from 2% to 44.4%. However, there was no correlation between the two removal rates for any of the four age groups of untreated brood studied (eggs, young larvae, older larvae from uncapped cells and larvae/pupae from capped cells). These results do not support the cost-to-healthy-brood hypothesis for the rarity of hygienic behaviour.

Resistance to Chalkbrood Disease in Apis mellifera L. (Hymenoptera: Apidae) Colonies with Different Hygienic Behaviour

Chalkbrood disease affects the larvae of honeybees Apis mellifera L. and is caused by the fungus Ascosphaera apis. Infected larvae die when they are stretched in the cap cell and suffer a gradual hardening that ends in a very hard structure (mummie). Several studies have demonstrated that colonies that express an efficient hygienic behaviour (uncapping of cell and subsequent removal of dead brood) exhibit a higher resistance to the disease. However, it remains unclear whether the advantage of hygienic colonies over less hygienic ones lies in the ability to remove mummies or in the early detection of infected larvae and its cannibalization before they harden. To elucidate this aspect, the hygienic behaviour of 24 colonies, which were subsequently provided with pollen cakes containig A. apis, was evaluated. The number of mummies and the number of partially cannibalized and whole larvae in uncapped cells were recorded. The most hygienic colonies controlled the disease better. These colonies also had a higher tendency to uncap cells that contained infected larvae and cannibalize them. The presence of A. apis in partially cannibalized and whole larvae in uncapped cells indicate that the advantage of hygienic colonies over less hygienic ones lies in the early detection of infected larvae death and their quick removal from the cell before they become mummies.

“Entombed Pollen”: A new condition in honey bee colonies associated with increased risk of colony mortality

Here we describe a new phenomenon, entombed pollen, which is highly associated with increased colony mortality. Entombed pollen is sunken, capped cells amidst “normal”, uncapped cells of stored pollen, and some of the pollen contained within these cells is brick red in color. There appears to be a lack of microbial agents in the pollen, and larvae and adult bees do not have an increased rate of mortality when they are fed diets supplemented with entombed pollen in vitro, suggesting that the pollen itself is not directly responsible for increased colony mortality. However, the increased incidence of entombed pollen in reused wax comb suggests that there is a transmittable factor common to the phenomenon and colony mortality. In addition, there were elevated pesticide levels, notably of the fungicide chlorothalonil, in entombed pollen. Additional studies are needed to determine if there is a causal relationship between entombed pollen, chemical residues, and colony mortality.

Simplified methods of evaluating colonies for levels of Varroa Sensitive Hygiene (VSH)

SummaryVarroa Sensitive Hygiene (VSH) is a trait of honey bees, Apis mellifera, that supports resistance to Varroa destructor mites. Components of VSH were evaluated to identify simple methods for selection of the trait. Mite population growth was measured in colonies with variable levels of VSH in two field trials using 24 and 16 colonies. Mite population growth was significantly lower in VSH and hybrid colonies than in control (i.e., unselected) colonies. In resident brood with mite infestations below 5%, the percentage of uncapped pupal cells did not differ significantly among VSH, hybrid and control colonies, but the percentage of recapped cells was highest in VSH colonies (P = 0.03). When brood from more highly infested colonies (9–49% of pupae infested) was introduced for forty hours, VSH colonies reduced infestation more than control colonies (P< 0.01) but final mite fertility was similar (P= 0.12). When infested brood was exposed in colonies for one week, VSH colonies reduced both mite fertility (P= 0.05) and mite infestation (P= 0.02). When highly infested brood was exposed to a subset of colonies for two hours, control colonies uncapped no or few cells while uncapping in VSH colonies was variable but on average was much higher. Mite population growth in individual colonies was negatively correlated with reduced infestation after forty hours of brood exposure and with reduced mite fertility after one week. The simpler and shorter-term measures (relative to measuring mite population growth) of uncapping, recapping, and reductions in infestation and mite fertility may facilitate selection of VSH by more bee breeders.

Bees with Varroa Sensitive Hygiene preferentially remove mite infested pupae aged ≤ five days post capping

SummarySuppressed Mite Reproduction (SMR) is a trait of honey bees that provides resistance to Varroa destructor. The mechanism of resistance in SMR bees is the removal of infested pupae from capped brood, so a better name is VSH bees (acronym for Varroa Sensitive Hygiene). This study compared the removal of infested brood by VSH and control bees to determine whether VSH bees removed infested pupae of different ages at similar rates. A pair of infested combs containing all stages of pupae were transferred into each host colony (six VSH and six control colonies) for 40 hours. VSH bees removed significantly more (55%) infested cells (singly and multiply infested), than controls (13%). They removed significantly more (66%) singly infested pupae aged from one to five days post capping (cohort A) than did controls (16%). The two types did not differ in the removal of singly infested pupae aged five to 10 days post capping (cohort B) (5–22%). Many pupae were found in uncapped cells at the end of the test, and most of the uncapped pupae were infested with mites. None of the uncapped cells contained prepupae, the development stage occurring during the first three days post capping. Thus, removal of infested pupae may be triggered by stimuli in cells with pupae aged 3–5 days post capping.

Inefficient task partitioning among nonhygienic honeybees, Apis mellifera L., and implications for disease transmission

Hygienic behaviour in honeybee colonies involves the recognition and removal of diseased brood by worker bees. This task is further partitioned into subtasks of uncapping cells with diseased brood and removing the cell contents. Worker bees that express hygienic behaviour remove diseased brood from the colony before the pathogen reaches the infectious stage. Although all honeybees are capable of uncapping and removing diseased brood, only those colonies with hygienic bees, the genetic specialists, do so rapidly and hence efficiently limit disease transmission. Colonies with nonhygienic bees, however, uncap and remove diseased brood only after it becomes infectious, resulting in the handling and transmission of pathogens. To understand the behavioural repertoire of individual nonhygienic bees and how the performance of the behaviour is influenced by the presence of hygienic bees as nestmates, we observed nonhygienic bees that were maintained in colonies with varying proportions of hygienic and nonhygienic bees. Our results indicated that nonhygienic bees in mixed colonies were not stimulated to perform hygienic behaviour to a higher extent in the presence of hygienic bees. The rate, duration and probability of uncapping and removing dead brood by nonhygienic bees was significantly reduced in the presence of hygienic bees. In mixed colonies, as compared to a colony of hygienic bees, a higher proportion of uncapped cells were subsequently recapped, resulting in delayed removal of dead brood. This inefficient task partitioning would allow the pathogen to reach the infectious stage and increase the probability of disease transmission.

Uncapping of pupal cells by European bees in the United States as responses to Varroa destructor and Galleria mellonella

SummaryWe investigated the uncapping of pupal cells by honey bees in the United States as responses to infestation with V. destructor and G. mellonella. In a group of 15 colonies, we counted the number of uncapped cells with pupae, and estimated the total sealed brood area. At the same time, the infestation by V. destructor and evidence of wax moth activity were measured in uncapped pupal cells, in cells immediately adjacent (neighbours), and in cells along linear transects. The relative amount of uncapped pupal cells (uncapped pupal cells/total sealed cells) increased with infestation (linear regression R2 = 0.64, slope = 0.16). Varroa mite infestation of uncapped cells (30%) significantly exceeded that of neighbours (14%) and also that of transect cells (12%), but infestations of neighbour and transect cells were similar. The relationship between infestation of uncapped cells and that of other cells (neighbour or transect cells) had a slope significantly higher than one, suggesting that discrimination of infested cells increases with overall colony infestation. Frequencies of wax moth activity (presence of larvae, frass, tunnelling and webbing) were highest in uncapped cells (33%), lower in neighbour cells (21%) and extremely low in transect cells (4%). We followed the opening, removing and resealing of pupal cells every 24 h in a colony with one of the highest infestations with varroa mites and with one of the lowest levels of wax moths, and in a second colony with the opposite infestation levels. Many opened or partly removed cells were found in a different condition, suggesting a dynamic process under conditions of high infestation.

Effect of prey quantity and temperature on nest demography of social wasps

Abstract. 1. To determine the effect of prey quantity and temperature on nest demography of social wasps (Polistes fuscatus), field experiments were conducted, in which wasps were provided with a low quantity of caterpillars (approximately nest subsistence level) or high quantity (three times as much). In addition, in the third year, the nest boxes were modified to be relatively cool (white and insulated) or warm (black).2. In 1997 and 1998, high‐food nests had a high proportion of cells containing developing offspring, produced more offspring, and had disproportionately more female offspring compared with low‐food nests.3. In 1999, the cool and warm nest boxes exhibited a daily average difference of 1.3 °C and a maximum difference of 6.9 °C. The warm high‐food nests produced more offspring than any other treatment. The cool high‐food nests produced similar numbers of offspring as the cool low‐food and warm low‐food treatments.4. As indicated by a surplus of uncapped cells, foundresses of low‐food nests, which would have had the egg‐laying potential to produce many offspring, apparently sacrificed eggs to provide a food supplement for the developing offspring. This pattern plus more time observed foraging suggests considerable expenditure of energy by foundresses, in response to the low supply of food.5. Importantly, these results indicate that an interactive effect of prey quantity and temperature on offspring production occurred, which may reflect the different effects on developmental rate and growth rate at cool versus warm nest temperatures.

Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (alpha M beta 2' CD11b/CD18) during neutrophil polarization.

Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.

Capping of the surface OKT3 binding molecule prevents the T-cell proliferative response to antigens: Evidence that this molecule conveys the activation signal

The human T-lymphocyte receptor for antigen appears to have been localized to a cluster of associated surface glycoprotein molecules, among which is the OKT3 binding molecule. We have tested the hypothesis that selective removal of the OKT3 binding molecule eliminates the cellular immune response to antigens by clearing the surface of the molecule that conveys the activation signal. Enriched T cells were obtained from donors immune to purified protein derivative (PPD), streptokinase-streptodornase (SK-SD), or keyhole limpet hemocyanin (KLH). T-3 molecules were cleared from the cell surface by capping with OKT3 and F(ab') 2 goat anti-mouse IgG. Regeneration of surface molecules was prevented by culturing the T-3 capped cells with OKT3 625 ng/ml. The capacity of capped T cells to proliferate in culture with antibody in response to antigens, alloantigens, and the mitogens, PHA and ionophore A23187, was compared to uncapped cells pretreated with media and to capped cells permitted to regenerate the OKT3 binding molecule. T-3 capped cells cultured in the presence of antibody failed to proliferate to antigens or alloantigens. However, T-3 capped cells cocultured with antibody also did not significantly proliferate to PHA, but did respond to A23187. In contrast, both media-treated T cells and cells which had regenerated the OKT3 binding molecule proliferated to mitogens, antigens, or alloantigens. The requirement for the OKT3 binding molecule was determined by utilizing T-1, T-4, and T-8 capped cells. T-1, T-4, or T-8 capped cells cultured in the presence of OKT1, OKT4, or OKT8 proliferated in response to antigens, alloantigens, and mitogens. These results demonstrate that in the absence of the OKT3 binding molecule, antigens, alloantigens, and PHA failed to induce a significant cellular proliferative response. In the absence of this molecule, PHA cannot bind to its carbohydrate moiety, and therefore cannot activate T cells to proliferate. These data support the concept that the molecule binding OKT3 conveys the transmembrane signal resulting in cellular activation.

Mitogenic stimulation and the redistribution of concanavalin A receptors on lymphocytes

When mouse spleen (Ig −) cells undergo maximal mitogenic stimulation by optimal concentrations of concanavalin A (conA), the Ig − cells form caps of conA very slowly, with 50% of maximum cap formation occurring after about 10 h and maximal capping after about 24 h. Anti-conA antibody added after optimal conA accelerates the rate of cap formation and effectively blocks mitogenic stimulation (< 10%) by optimal conA concentrations when the rate of capping is increased more than about 2-fold. The effect of anti-conA antibody in accelerating cap formation by optimal conA is antagonized by cytochalasin D (CD), which substantially restores the mitogenic action of optimal conA. Thus there is an inverse relationship between rate of cap formation and extent of mitogenic stimulation. Further experiments showed that if anti-conA antibody, α-methyl mannoside or EGTA were added at increasing intervals after the addition of conA, these inhibitors block the stimulation of the cells with very similar time courses. Addition of appropriate concentrations of an inhibitor at the same time as optimal conA blocks mitogenic stimulation completely, but has negligible effects after 24 h. The extent of stimulation which occurs after the addition of inhibitor at intermediate times closely follows the extent of cap formation at the same time. The simplest interpretation of these results is that mitogenic action by optimal conA can be blocked by ( i) accelerated capping of uncapped cells; or ( ii) by the removal of either conA or calcium before, but not after, cap formation has occurred. These results suggest that the rate of cap formation by conA, and the presence of external calcium (>10 −4 M) in the medium for some unspecified period before cap formation occurs are both significant factors in generating the primary mitogenic signals which commit the cells to DNA synthesis.

A Simple Method for Extracting Larval Honeybees from Uncapped Cells

SummaryA quick method of extracting several grams of honeybee larvae from combs is described; it permits removal without use of water, forceps or probing instruments, which often injure and contaminate high percentages of larvae. The method is faster than others that have been described.

Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins.

The relationship between microtubules and concanavalin A surface receptors during concanavalin A capping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 4 degree or 37 degree and then incubated at 37 degree for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization.