Micro-computed tomography of pupal metamorphosis in the solitary bee Megachile rotundata

Insect metamorphosis involves a complex change in form and function. In this study, we examined the development of the solitary bee, Megachile rotundata, using micro-computed tomography (μCT) and volume analysis. We describe volumetric changes of brain, tracheae, flight muscles, gut, and fat bodies in prepupal, pupal, and adult M. rotundata. We observed that individual organ systems have distinct patterns of developmental progression, which vary in their timing and duration. This has important implications for commercial management of this agriculturally relevant pollinator.     

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at −80 °C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.     

Colony disturbance and solitary nest initiation by workers in the obligately eusocial sweat bee, Halictus ligatus

Sweat bees are one of the most socially polymorphic lineages on the planet. In obligately eusocial species, newly enclosed females may become either queens or workers, depending on the environmental and social circumstances of the nest into which they emerge. In socially polymorphic species, females also have the option of nesting solitarily, founding a nest and raising future reproductives alone, without the help of other adult females. Halictus ligatus is a widespread Nearctic, ground-nesting sweat bee. It has been particularly well studied in Ontario, where detailed studies have described it as obligately eusocial. Here we report evidence that the flexibility of female H. ligatus actually extends to expressing behaviour more typical of socially polymorphic species, those in which some individuals reproduce solitarily. In a population in southern Ontario, black wasps (Astata sp.) emerged from the soil beneath the nesting aggregation and proceeded to excavate their own nesting tunnels, dislocating many H. ligatus nest entrances. Young workers whose natal nests were destroyed by the wasp activity constructed new nests, so under very specific circumstances, it is possible for potential altruists to nest solitarily.     

DNA fingerprinting analysis in the solitary bee Megachile rotundata: variability and nest mate genetic relationships.

The most conventional approach for evaluating genetic variability in an insect population involves assessing the degree of enzyme polymorphism. Hymenoptera display a relatively low level of genetic variability compared with most insect species. DNA probes consisting of tandemly repeated sequences are powerful tools for detecting polymorphisms when employed to develop DNA fingerprinting (DNAfp) profiles in a wide range of organisms. This report describes genetic variability in the solitary bee species Megachile rotundata as assessed by DNAfp using the M13 sequence and a synthetic oligonucleotide sequence homologous to a hypervariable region of the alpha-globin gene. DNAfp comparisons among offspring were used to analyze genealogical structure in M. rotundata nests. The results indicate that polyandry, by a large number of males, is not a common phenomenon in M. rotundata bee species. In the present analysis, it is likely that the broods raised in single nests are mostly the offspring of one singly mated female. However, the data does not preclude that for certain nests two males could have been involved in the mating process.     

Substrate specificity of esterases in a solitary bee, Megachile rotundata (Hymenoptera: Megachilidae): Variability in sex, age and life stage

The esterases of Megachile rotundata (F.) were examined with respect to 36 substrates and comparisons were made between males and females. Differences in substrate disposition with respect to aromatic, aliphatic and thioesters were found between life stages. Overall, thioesters and relatively short aliphatic chain (3–4C) aromatic esters were metabolized most readily. With respect to age, males lose significantly more esterase activity with time than females, which may be a consequence of overall protein Ioss in late life. Based upon evidence from other bee families, possible ecological roles of M. rotundata esterases in nesting are suggested.     

Interaction between the solitary bee Chelostoma florisomne and its nest parasite Sapyga clavicornis − empty cells reduce the impact of parasites

1. Nesting behaviour and interactions between the bee Chelostoma florisomne (L.) (Megachilidae) and its nest parasite Sapyga clavicornis (L.) (Sapygidae) were studied through continual observations of individuals and dissections of bee nests. Protection of bee offspring is based on (1) the bee’s discovery and removal of parasite eggs deposited prior to the construction of a cell closure, (2) minimising the time when fully provisioned cells might be parasitised successfully, and (3) the construction of empty cells in front of brood cells. 2. An empty cell was found in front of 64.4% of all brood cells and, if the outermost brood cell in a nest was excluded, in front of 74.3% of inner brood cells. A vestibule closure is most often constructed in front of the outermost brood cell. 3. Following oviposition, the bee made only five flights, which together lasted 6–13 min, to construct a cell closure. A cell closure does not prevent the nest parasite from oviposition inside the brood cell, however, and parasite eggs deposited through the cell closure are not detected and removed by the bee. Only an additional cell closure, i.e. the formation of an empty cell, may protect a brood cell when the bee is not in the nest. The nest parasite often oviposited through the additional cell closure but its offspring were then trapped in the empty cell and starved to death. 4. Only 5.4% of the inner brood cells that were protected by an empty cell were parasitised, compared with 28.9% of those without an anterior, empty cell; 27.4% of the empty cells contained dead parasite offspring (eggs and larvae). Thus, the empty cells provided significant protection and, combined with additional means of protection of brood cells, led to a low degree of parasitism. More than 77% of the wasp offspring died at an early stage due to intraspecific interference competition within brood cells and as result of the wasps’ oviposition into empty cells.     

Nocturnal orientation by the Asian honey bee, Apis dorsata

Honey bees have been observed to forage and dance on moonlit nights, but it has never been established whether the moon serves as a reference in orienting nocturnally active bees. The present study, of the Asian honey bee Apis dorsata, suggests that although the moon's illumination is essential for nocturnal flight, the moon itself is ignored for orienting the dances. Rather, bees probably use the sun's position as a reference point for their dances, even though the sun is below the horizon. This ability may involve an extension of the mechanism that honey bees employ to find the sun on overcast days.     

Parental investment in nest defence by stonechats (Saxicola torquata)

Breeding stonechats (Saxicola torquata) made mixed sequences of two calls when a human intruder entered territories. ‘Whits’ are modulated notes with a small frequency range, and in laboratory tests caused nestlings to stop begging. ‘Chacks’ cover a wide range of frequencies, and in the field were combined with flights made so as to distract an intruder from the nest. On average male and female call-rates were similar, but varied greatly according to the intruder's distance from the nest, and at different stages of the nesting cycle. Rates increased rapidly after hatching, and this correlated most closely with the cumulative total of parents' visits to feed nestlings. This suggests that the level of defence may be adjusted to the value of the offspring to their parents. Call-rates declined about one week after fledging. A smaller peak by some pairs at the start of incubation was apparently related to probable poor condition after a previous breeding attempt, and after laying large clutches. Rates of Whits were higher at nests with larger broods, up to an asymptote, but rates of Chacks were independent of brood size. Birds suffering nest-predation showed lower call-rates before the event than equivalent successful birds, suggesting that the calls do reduce the risk of predation.     

Honeydew feeding in the solitary bee Osmia bicornis as affected by aphid species and nectar availability

Like honey bees (Apis mellifera), non-Apis bees could exploit honeydew as a carbohydrate source. In addition to providing carbohydrates, this may expose them to potentially harmful plant products secreted in honeydew. However, knowledge on honeydew feeding by solitary bees is very scarce. Here we determine whether the polylectic solitary bee Osmia bicornis (=O. rufa) collects honeydew under semi-field conditions, and whether this is affected by aphid species and presence of floral nectar. Bees were provided with oilseed rape plants containing flowers and/or colonies of either Myzus persicae or Brevicoryne brassicae. We used the total sugar level of the bee crop as a measure of the individual's nutritional state and the oligosaccharide erlose as indicator for honeydew consumption. Erlose was present in honeydews from both aphid species, while absent in oilseed rape nectar, nor being synthesized by O. bicornis. When bees were confined to a single honeydew type as the only carbohydrate source, consumption of M. persicae honeydew was confirmed for 47% of the bees and consumption of B. brassicae honeydew for only 3%. Increased mortality in the latter treatment provided further evidence that B. brassicae honeydew is an unsuitable food source for O. bicornis. All bees that were given the choice between honeydew and floral nectar showed significantly increased total sugar levels. However, the fact that no erlose was detected in these bees indicates that honeydew was not consumed when suitable floral nectar was available. This study demonstrates that honeydew exploitation by O. bicornis is dependent on honeydew type and the presence of floral nectar.     

Experimental evidence for state-dependent nest weight in the blue tit, Cyanistes caeruleus

Parental investment in reproduction is generally limited by food availability, and so avian life-history research has traditionally focused on the brood rearing phase, when food requirements are greatest. Only relatively recently has the focus extended to the incubation phase, and even more recently to the nest-building phase, where observational and comparative evidence suggest that avian nest building is an energetically expensive and time-consuming activity. We aimed to experimentally test the limitations on this cost in a hole-breeding passerine, the blue tit (Cyanistes caeruleus), by providing supplementary food to experimental pairs during the nest-building period. In comparison with control females, that did not receive supplementary food, experimental females constructed heavier nests, with greater amounts of moss base but similar amounts of cup lining, despite there being no differences in the time taken to build the nest. This study provides empirical support for the hypothesis that avian nest building is a costly behaviour, limited by food availability.     

Caenorhabditis elegans TLK-1 controls cytokinesis by localizing AIR-2/Aurora B to midzone microtubules

Defects in chromosome condensation, segregation or cytokinesis during mitosis disrupt genome integrity and cause organismal death or tumorigenesis. The conserved kinase AIR-2/Aurora B is required for normal execution of all these important mitotic events in Caenorhabditis elegans. TLK-1 has been recently shown to be a substrate and activator of AIR-2 in the presence of another AIR-2 activator ICP-1/INCENP, and to cooperate with AIR-2 to ensure proper mitotic chromosome segregation. However, whether TLK-1 may contribute to chromosome condensation or cytokinesis is unclear. A time-lapse microscopy analysis showed that tlk-1 mutants are defective in chromosome condensation and cytokinesis, in addition to chromosome segregation, during mitosis. Our data indicate that TLK-1 contributes to chromosome condensation and segregation, at least in part, in a manner that is distinct from the ICP-1-mediated mechanism and does not involve loading AIR-2 or condensin proteins to mitotic chromosomes. Moreover, TLK-1 functions in cytokinesis by localizing AIR-2 to the midzone microtubules. The localization pattern of TLK-1 is different from those of ICP-1 and AIR-2, revealing differences in dynamic regulation and association of TLK-1 and ICP-1 towards AIR-2 in vivo. Interestingly, human TLK2 could functionally substitute for tlk-1, suggesting that the mitotic roles of TLK members might be evolutionarily conserved.     

Flower color change accelerated by bee pollination in Tibouchina (Melastomataceae)

Floral color changes are common among Melastomataceae and have been interpreted as a warning mechanism for bees to avoid old flowers, albeit increasing long-distance flower display. Here the reproductive systems of Tibouchina pulchra and T. sellowiana were investigated by controlled pollinations. Their pollinators were identified, and experiments on floral color and fragrance changes were conduced to verify if those changes affect the floral visitation. Both Tibouchina species are self compatible. The flowers lasted three days or more, and the floral color changed from white in the 1st day to pink in the following days. Pollen deposition on stigma induced floral color change. The effectiveness of the pollination is dependent on bees’ size; only large bees were regarded as effective pollinators. In experimental tests, the bees in T. pulchra preferred the natural white flowers while the visitors of T. sellowiana were attracted by both natural and mimetic 1st-day flowers (2nd-day flowers with experimentally attached 1st-day flower petals). During the experiments on floral fragrance, the bees visited both natural and mimetic 1st-day flowers (2nd-day flowers with 1st-day flower scents). In both experiments, the bees avoided natural 2nd-day flowers, but seldom visited modified 2nd-day flowers. The attractiveness of T. pulchra and T. sellowiana flowers cannot be attributed exclusively to the color or the fragrance separately, both factors seemingly act together.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Timing of eclosion affects diapause development, fat body consumption and longevity in Osmia lignaria, a univoltine, adult-wintering solitary bee

Most insects from temperate areas enter diapause ahead of winter. Species diapausing in a feeding stage and accumulating metabolic reserves during permissive pre-wintering conditions are expected to enter diapause shortly before the onset of winter. In contrast, species diapausing in a non-feeding stage are expected to lower their metabolism as soon as possible to avoid excessive consumption of metabolic reserves. The solitary bee Osmia lignaria winters as a non-feeding adult within its cocoon, but previous studies show important weight losses and increased winter mortality in populations pre-wintered for extended periods. We measured respiration rates to assess diapause initiation and maintenance during pre-wintering, and tested whether timing of adult eclosion affected fitness by measuring fat body depletion, winter mortality and post-winter longevity. We worked with different cohorts of a population reared under natural conditions, and manipulated pre-wintering duration in a population reared under artificial conditions. In agreement with our expectation, O. lignaria lower their metabolic rates within a few days of adult eclosion, but nonetheless suffer strong weight loss during pre-wintering. Early developing individuals suffer greater weight loss and fat body depletion, and have short post-winter longevity. Although, we found no differences in winter mortality among treatments, our results indicate that increased mortality may occur in years with late winter arrivals. We discuss fundamental ecophysiological differences between adult and prepupal diapause within the Megachilidae, and hypothesize that species wintering as adults will be more negatively affected by a situation of extended summers under a scenario of global warming.