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Pattern of cuticle pigmentation and sclerotization in adults. Whole mount preparations (at the left) and histological sections (at the right) of thoracic (above) and abdominal (below) integuments of an adult worker bee (shown in the center of the figure). cut – cuticle; ep – epidermis; mus – muscle.
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The changing pattern of developing cuticle and associated epidermis is described during the imaginal molt in the honey bee. Observations began immediately after the pupal molt, and included histological analyses of the integument during apolysis and the subsequent deposition and differentiation of the adult cuticle. Apolysis coincides with a marked...
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Context 1
... Thoracic Fig. 2D) muscles apparently that marks initially the were transition only from loosely cuticle associated deposition with to its the di ff erentiation. integument As (Fig. 2D), thoracic now cuticle become pigmentation tightly intensifies connected (as (Figs. represented 2E, F). At in the the end schemes of the pharate at the adult left side development of Figs. (Figs. 2E, F), 2E, the F), epidermis the thoracic becomes cuticle very is characterized thin, mainly by in two the di thorax, ff erentially where stained it can hardly be visualized. Epidermis is also con- verted into a thin layer in the abdominal region (Figs. 2E, F). Thoracic muscles that initially were only loosely associated with the integument (Fig. 2D), now become tightly connected (Figs. 2E, F). At the end of the pharate adult development (Figs. 2E, F), the thoracic cuticle is characterized by two di ff erentially stained layers Figure 3 (pink shows and blue) the onset suggesting and progress distinct of chemical cuticle pigmentation compositions. in whole Both layers mount are prepa- part of rations. the procuticle, Between the which pink-eyed is the (Figs. major 3A, cuticle B) component and the early formed brown-eyed mainly pharate by adult interlaced phases proteins (Fig. 3C), and pigments chitin. could not yet be discerned macroscopically in the thoracic or abdominal cuticle, but they became detectable in the thoracic cuticle of the subsequent pharate adult phase (Fig. 3D), with a rapid increase in the amount of pigmented areas as development Figure 3 shows the onset and progress of cuticle pigmentation in whole mount preparations. Between the pink-eyed (Figs. 3A, B) and the early brown-eyed pharate adult phases (Fig. 3C), pigments could not yet be discerned macroscopically in the thoracic or abdominal cuticle, but they became detectable in the thoracic cuticle of the subsequent pharate adult phase (Fig. 3D), with a rapid increase in the amount of pigmented areas as development advances (Figs. 3E, F). Pigmentation initiates later in abdominal cuticle, only when the thorax is already darkened (compare Figs. 3D, E). Cuticle tanning also continues in the honey bee after adult ecdysis. Whole mount preparations of adult cuticle (Fig. 4, left side) clearly show that it is much darker in comparison to the cuticle of later pharate adults (Fig. 3F). In histological sections of the adult cuticle we could observe the presence of a very dark layer (Fig. 4, right side), not seen in the cuticle of later pharate adults (Fig. 2F). The temporal sequence of integument sections revealed considerable changes in epidermis and cuticle structure during the imaginal molt. Pupal epidermis contains large clear areas in the basal cytoplasm, but following pupal / adult apolysis, clear inclusions also appeared pulverized throughout the apical cytoplasm. Studies carried out in the epidermis of a lepidopteran, Calpodes ethlius , revealed apical and basal peptide tra ffi cking routes for secretion into the cuticle and hemolymph, respectively. However, it appears that basally localized peptides may be firstly secreted api- cally, and then endocytosed into vesicles to be transported via a basal route to the hemolymph (Locke, 2003). Our preparations do not make possible to identify the nature and destination of the material forming the clear areas in the epidermal cell cytoplasm. Considering that a new cuticle is being deposited following apolysis, it is possible that such material is being transported from the basal to the apical cytoplasm to be used in cuticle formation. Alter- natively, as this material is abundantly present in epidermal cells before and during formation of the ecdysial space, it is possible that it is discharged to fill this space as molting fluid. At a molting stage that precedes the detach- ment of epidermis in the crab Carcinus mae- nas , similar clear areas appear at the apices of the epidermal cells. They were identified as membrane-bounded large vacuoles opened at the cell surface, and their profiles strongly suggested that they contribute to molting fluid formation (Compère et al., 1998). Our results show that apolysis is marked by an increase in the thickness of the epidermal layer, which also change cell shape. In fact, in insects in general, apolysis has been associated with mitosis and expansion of the volume of epidermal cells (Hepburn, 1985). Following the period of cuticle deposition during the first half of pharate adult development in worker bees, the epidermis becomes very thin indicating that its biosynthetic activity for cuticle formation is subsiding. This change in morphological pattern reflects the transition from the period of biosynthesis of the adult cuticle to its di ff erentiation, which is visually marked by a progressive pigmentation. As cuticle is deposited in the dark-pink- eyed pharate adults, a mass of muscle cell precursors (myocytes) appears in the thoracic sections. In brown-eyed pharate adults, muscle formation is in progress and fibers derived from myocytes come in contact with the epidermis for attachment on the cuticle. The thoracic integument sections of developing pharate adults (see Fig. 2) show the re- building of the thoracic musculature in sub- stitution of the larval, which is completely histolyzed during honey bee metamorphosis (Oertel, 1930). Muscle fibers are not seen in abdominal (see Fig. 2) sections because histo- logical preparations did not include the inter- segmental grooves where the abdominal longi- tudinal muscle fibers are attached (Snodgrass, 1956), but only the medial region of the seg- ment (3rd tergite), deprived of attached muscles. Cuticle is a multilayered structure formed by an outermost envelope (which serves to protect the epidermis from molting fluid en- zymes during cuticle renewal), an intermediary chitin-free epicuticle and the most in- ternal procuticle, the thickest layer, made up mainly of proteins and chitin (Locke, 2001; Willis et al., 2005). This layer is in part secreted during the pharate stage, and in part after ecdysis. Pre-ecdysially secreted procuticle is termed exocuticle, and the post-ecdysial secretion forms the endocuticle (Andersen et al., 1995). Our integument preparations do not permit us to distinguish the thinner envelope and the epicuticle, but the pink and blue layers seen in histological sections (Figs. 1, 2) are both components of the procuticle. The great increase in procuticle thickness during honey bee pharate adult development indicates that a very major part is deposited before adult ecdysis, mainly during the first half of pharate development. We observed that the process of procuticle melanization, which accompanies the harden- ing (sclerotization) of the newly forming exoskeleton, continues and intensifies even after the eclosion of the adult. This is in accordance with studies on chemical and mechanical properties of the honey bee cuticle (Thompson and Hepburn, 1978; Andersen et al., 1981), which demonstrated that both deposition of materials, including melanin, and reactions for cuticle stabilization continue well into the adult stage. However, when the degree of sclerotization was estimated by measuring the amounts of ...
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... Thoracic Fig. 2D) muscles apparently that marks initially the were transition only from loosely cuticle associated deposition with to its the di ff erentiation. integument As (Fig. 2D), thoracic now cuticle become pigmentation tightly intensifies connected (as (Figs. represented 2E, F). At in the the end schemes of the pharate at the adult left side development of Figs. (Figs. 2E, F), 2E, the F), epidermis the thoracic becomes cuticle very is characterized thin, mainly by in two the di thorax, ff erentially where stained it can hardly be visualized. Epidermis is also con- verted into a thin layer in the abdominal region (Figs. 2E, F). Thoracic muscles that initially were only loosely associated with the integument (Fig. 2D), now become tightly connected (Figs. 2E, F). At the end of the pharate adult development (Figs. 2E, F), the thoracic cuticle is characterized by two di ff erentially stained layers Figure 3 (pink shows and blue) the onset suggesting and progress distinct of chemical cuticle pigmentation compositions. in whole Both layers mount are prepa- part of rations. the procuticle, Between the which pink-eyed is the (Figs. major 3A, cuticle B) component and the early formed brown-eyed mainly pharate by adult interlaced phases proteins (Fig. 3C), and pigments chitin. could not yet be discerned macroscopically in the thoracic or abdominal cuticle, but they became detectable in the thoracic cuticle of the subsequent pharate adult phase (Fig. 3D), with a rapid increase in the amount of pigmented areas as development Figure 3 shows the onset and progress of cuticle pigmentation in whole mount preparations. Between the pink-eyed (Figs. 3A, B) and the early brown-eyed pharate adult phases (Fig. 3C), pigments could not yet be discerned macroscopically in the thoracic or abdominal cuticle, but they became detectable in the thoracic cuticle of the subsequent pharate adult phase (Fig. 3D), with a rapid increase in the amount of pigmented areas as development advances (Figs. 3E, F). Pigmentation initiates later in abdominal cuticle, only when the thorax is already darkened (compare Figs. 3D, E). Cuticle tanning also continues in the honey bee after adult ecdysis. Whole mount preparations of adult cuticle (Fig. 4, left side) clearly show that it is much darker in comparison to the cuticle of later pharate adults (Fig. 3F). In histological sections of the adult cuticle we could observe the presence of a very dark layer (Fig. 4, right side), not seen in the cuticle of later pharate adults (Fig. 2F). The temporal sequence of integument sections revealed considerable changes in epidermis and cuticle structure during the imaginal molt. Pupal epidermis contains large clear areas in the basal cytoplasm, but following pupal / adult apolysis, clear inclusions also appeared pulverized throughout the apical cytoplasm. Studies carried out in the epidermis of a lepidopteran, Calpodes ethlius , revealed apical and basal peptide tra ffi cking routes for secretion into the cuticle and hemolymph, respectively. However, it appears that basally localized peptides may be firstly secreted api- cally, and then endocytosed into vesicles to be transported via a basal route to the hemolymph (Locke, 2003). Our preparations do not make possible to identify the nature and destination of the material forming the clear areas in the epidermal cell cytoplasm. Considering that a new cuticle is being deposited following apolysis, it is possible that such material is being transported from the basal to the apical cytoplasm to be used in cuticle formation. Alter- natively, as this material is abundantly present in epidermal cells before and during formation of the ecdysial space, it is possible that it is discharged to fill this space as molting fluid. At a molting stage that precedes the detach- ment of epidermis in the crab Carcinus mae- nas , similar clear areas appear at the apices of the epidermal cells. They were identified as membrane-bounded large vacuoles opened at the cell surface, and their profiles strongly suggested that they contribute to molting fluid formation (Compère et al., 1998). Our results show that apolysis is marked by an increase in the thickness of the epidermal layer, which also change cell shape. In fact, in insects in general, apolysis has been associated with mitosis and expansion of the volume of epidermal cells (Hepburn, 1985). Following the period of cuticle deposition during the first half of pharate adult development in worker bees, the epidermis becomes very thin indicating that its biosynthetic activity for cuticle formation is subsiding. This change in morphological pattern reflects the transition from the period of biosynthesis of the adult cuticle to its di ff erentiation, which is visually marked by a progressive pigmentation. As cuticle is deposited in the dark-pink- eyed pharate adults, a mass of muscle cell precursors (myocytes) appears in the thoracic sections. In brown-eyed pharate adults, muscle formation is in progress and fibers derived from myocytes come in contact with the epidermis for attachment on the cuticle. The thoracic integument sections of developing pharate adults (see Fig. 2) show the re- building of the thoracic musculature in sub- stitution of the larval, which is completely histolyzed during honey bee metamorphosis (Oertel, 1930). Muscle fibers are not seen in abdominal (see Fig. 2) sections because histo- logical preparations did not include the inter- segmental grooves where the abdominal longi- tudinal muscle fibers are attached (Snodgrass, 1956), but only the medial region of the seg- ment (3rd tergite), deprived of attached muscles. Cuticle is a multilayered structure formed by an outermost envelope (which serves to protect the epidermis from molting fluid en- zymes during cuticle renewal), an intermediary chitin-free epicuticle and the most in- ternal procuticle, the thickest layer, made up mainly of proteins and chitin (Locke, 2001; Willis et al., 2005). This layer is in part secreted during the pharate stage, and in part after ecdysis. Pre-ecdysially secreted procuticle is termed exocuticle, and the post-ecdysial secretion forms the endocuticle (Andersen et al., 1995). Our integument preparations do not permit us to distinguish the thinner envelope and the epicuticle, but the pink and blue layers seen in histological sections (Figs. 1, 2) are both components of the procuticle. The great increase in procuticle thickness during honey bee pharate adult development indicates that a very major part is deposited before adult ecdysis, mainly during the first half of pharate development. We observed that the process of procuticle melanization, which accompanies the harden- ing (sclerotization) of the newly forming exoskeleton, continues and intensifies even after the eclosion of the adult. This is in accordance with studies on chemical and mechanical properties of the honey bee cuticle (Thompson and Hepburn, 1978; Andersen et al., 1981), which demonstrated that both deposition of materials, including melanin, and reactions for cuticle stabilization continue well into the adult stage. However, when the degree of sclerotization was estimated by measuring the amounts of ketocatechols released from abdominal cuticle by acid hydrolysis, the highest amount was found in the late pharate adults, and these levels did not increase after eclosion (Andersen et al., 1981). Although ketocatechol ...
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... tanning also continues in the honey bee after adult ecdysis. Whole mount prepara- tions of adult cuticle (Fig. 4, left side) clearly show that it is much darker in comparison to the cuticle of later pharate adults (Fig. 3F). In histological sections of the adult cuticle we could observe the presence of a very dark layer (Fig. 4, right side), not seen in the cuticle of later pharate adults (Fig. ...
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... tanning also continues in the honey bee after adult ecdysis. Whole mount prepara- tions of adult cuticle (Fig. 4, left side) clearly show that it is much darker in comparison to the cuticle of later pharate adults (Fig. 3F). In histological sections of the adult cuticle we could observe the presence of a very dark layer (Fig. 4, right side), not seen in the cuticle of later pharate adults (Fig. ...
Citations
... Physiologically, the cuticle pigmentation of honeybees begins at the pupal stage [22] and extends to the adult stage [23] and is regulated by hormones [24] and correlated with genes involved in cuticle development, such as AmproPO [25], Amlac2 [26], AmelTwdl1, AmelTwdl2 and Ampxd [27], and AmBurs α and AmBurs β [28]. ...
Simple Summary
This study examined the genetic basis of a mutation in cuticle color in the honeybee Apis cerana cerana using genome resequencing of wild−type and mutant drones produced by a single virgin queen. A candidate locus was identified by calculating the Euclidean distance between mutants and wild types at each SNP, performing Lowess regression to fit a curve to these data, and setting a threshold of the top 0.5% Euclidean distance for candidate region selection. From this, genes with synonymous substitutions became candidate genes. One of these genes, the yellow gene, had a 2 bp deletion causing a frameshift mutation. RT−qPCR of this gene was performed on RNA extracted from mutant and wild−type drones; gene expression was only significantly different between wild types and mutants at the yellow gene. Finally, RNA interference silencing of the yellow gene was used to reduce yellow gene expression in workers and putatively result in a lighter coloration. These results indicate that the yellow gene participated in the body pigmentation, and its defect was responsible for the brown mutation. It promotes the understanding of the molecular basis of body coloration in honeybees, enriching the molecular mechanisms underlying insect pigmentation.
Abstract
The honeybee, Apis cerana cerana (Ac), is an important pollinator and has adapted to the local ecological environment with relevant coloration. The cuticle coloration of the brown (br) mutant is brown instead of black in wild−type individuals. Therefore, this study aimed to identify and characterize the gene responsible for the br mutation. Genome resequencing with allele segregation measurement using Euclidean distance followed by Lowess regression analysis revealed that the color locus linked to the mutation was located on chromosome 11. A 2−base deletion on exon 4 was identified in the g7628 (yellow) gene after genome assembly and sequence cloning. In addition, the cuticle color of the abdomen of worker bees changed from black to brown when a defect was induced in the yellow gene using short interfering RNA (siRNA); however, the survival rate did not decrease significantly. These results indicate that the yellow gene participated in the body pigmentation, and its defect was responsible for the br mutation. This study promotes the understanding of the molecular basis of body coloration in honeybees, enriching the molecular mechanisms underlying insect pigmentation.
... Young bees were removed, and we recorded their age, survival, parasitism status and DWV symptoms (normal or deformed wings). Bees were aged based on morphological characteristics [47]. All brood-parasitising foundress mites were collected from uncapped cells. ...
The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives showed evidence of an elevated RNAi response, although no significant difference in DWV levels was found. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency.
... As a consequence of the accelerated development, the transition to the pupal stage starts earlier in queens [18]. The last apolysis event [21], also triggered by an ecdysteroid pulse [22,23], induces the detachment of the pupal cuticle and onset of adult cuticle synthesis. Thenceforward, the adult development occurs inside the pupal cuticle, thus characterizing the sequential pharateadult phases. ...
... After pupal cuticle apolysis, the epidermis produces the adult cuticle, thus characterizing the PB phase. The cuticle then turns intensely pigmented and sclerotized at the PBD phase [21]. Our distance analysis also clearly separated the adult wings and thoracic dorsum from the pupal ones in terms of gene expression. ...
... The pharate-pupal development ends when the larval cuticle is discarded (ecdysis), thus revealing the pupa (PW phase) with its newly formed wings and thoracic dorsum. A new apolysis after the end of PW phase marks the beginning of adult cuticle synthesis, and subsequently, its pigmentation and sclerotization [21]. At adult ecdysis time, the wing disc-derived structures phenotypically differ between workers and queens (Additional file 14: Supplementary Figure 7) [35,43]. ...
Background
Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation.
Results
We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development.
Conclusion
Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis.
... ELPs, which are described by Fine et al. (2018), consist of artificial plastic comb designed for the collection of fertilized eggs from a mated honey bee queen. In a colony, pharate adults do not exit their cells until their cuticle has sufficiently hardened (Elias-Neto et al., 2009). Bees reared using standard in vitro methods typically eclose in 48 or 24 well plates (Crailsheim et al., 2013;Schmehl et al., 2016), and in this work, they were transferred to cup cages immediately after they were observed to have eclosed as pharate adults. ...
As social insects, honey bees (Apis mellifera) rely on the coordinated performance of various behaviors to ensure that the needs of the colony are met. One of the most critical of these behaviors is the feeding and care of egg laying honey bee queens by non-fecund female worker attendants. These behaviors are crucial to honey bee reproduction and are known to be elicited by the queen’s pheromone blend. The degree to which workers respond to this blend can vary depending on their physiological status, but little is known regarding the impacts of developmental exposure to agrochemicals on this behavior. This work investigated how exposing workers during larval development to chronic sublethal doses of insect growth disruptors affected their development time, weight, longevity, and queen pheromone responsiveness as adult worker honey bees. Exposure to the juvenile hormone analog pyriproxyfen consistently shortened the duration of pupation, and pyriproxyfen and diflubenzuron inconsistently reduced the survivorship of adult bees. Finally, pyriproxyfen and methoxyfenozide treated bees were found to be less responsive to queen pheromone relative to other treatment groups. Here, we describe these results and discuss their possible physiological underpinnings as well as their potential impacts on honey bee reproduction and colony performance.
... Of the obtained queen cells, 135 contained chitin remnants sufficient to proceed with the extraction. Using sterile spatula and forceps, we have removed the paper-like, white to yellow structures (remnants of cuticle after the last molt of the queen; [19,27], Figure 1) deposited on the inner cell walls and transferred them into fresh 2 mL tube. In some cells, there was a yellowish mass present at the bottom, which was collected separately to extract DNA. ...
In traditional bee breeding, the honeybee queen is chosen for breeding based on the performance of the colony produced by its mother. However, we cannot be entirely certain that a specific queen will produce offspring with desirable traits until we observe the young queen’s new colony. Collecting the queen’s genetic material enables quick and reliable determination of the relevant information. We sampled exuviae, feces, and wingtips for DNA extraction to avoid fatally injuring the queen when using tissue samples. Quantity and purity of extracted DNA were measured. Two mitochondrial markers were used to determine the lineage affiliation and exclude possible contamination of DNA extracts with non-honeybee DNA. dCAPS (derived Cleaved Amplified Polymorphic Sequences) markers allowed detection of single nucleotide polymorphisms (SNPs) in nuclear DNA regions presumably associated with Varroa sensitive hygiene and set the example of successful development of genotyping protocol from non-destructive DNA sources. One of the logical future steps in honeybee breeding is introducing genomic selection and non-destructive sampling methods of genetic material may be the prerequisite for successful genotyping. Our results demonstrate that the extraction of DNA from feces and exuviae can be introduced into practice. The advantage of these two sources over wingtips is reducing the time window for processing the samples, thus enabling genotyping directly after the queen’s emergence.
... The onset of pupal apolysis in the P3 (Pd) phase (Elias-Neto et al. 2009) defines all subsequent phases up to the adult ecdysis as the pharate-adult stage of development. The testiolar tubule structure and progressing spermiogenesis in early pharate adults (Pdl) are depicted in longitudinal sections through the apical (Figure 9a) and basal region of a testiolar tubule (Figure 9b), respectively. ...
Like Apis mellifera queens and different from all other bees, drones also have an exaggerated gonad phenotype, with over 150 serial units in each gonad. Yet, compared with the ovaries of the female castes, little is known about the development of the honey bee testis. Here we present a histological atlas on postembryonic testis development and spermatogenesis. Already in the first instar larvae, the testioles composing each testis can be distinguished. The testioles then grow along their apical-basal axis by mitotic divisions of the spermatogonia, which eventually form germ cell clusters. Meiosis starts when brood cells are capped, and it ends with the appearance of spermatids in red-eyed pupae. Subsequently, spermiogenesis takes place, and all spermatozoa are formed before adult emergence. We also present the first data on juvenile hormone levels in drone larvae. With this, we provide a database for future research on gonad development in honey bee drones.
... Among the genes expressed in the integument, we focused on those involved in the melanization/sclerotization pathway, related to chitin, genes encoding structural cuticular proteins, regulators of cuticle renewal and tanning, desaturase and elongase genes potentially involved in CHC biosynthesis, circadian clock genes that could determine the rhythm of cuticle layers deposition [39,40], and genes encoding pigments other than melanin. Our study included bees at three different developmental phases: pharate-adults at the Pbm phase (classified according to Michelette and Soares [41]), where the adult cuticle in process of pigmentation is apparent underneath the disintegrating pupal cuticle [42], newlyemerged (newly-ecdysed) bees, and foragers. During this time interval, the remarkable developmental events are the intensification of pigmentation/sclerotization of the adult cuticle (cuticle maturation) that can extend through early adulthood, the imaginal molting culminating in the ecdysis, and the emergence of the adults from the brood cells. ...
Differences in the timing of exoskeleton melanization and sclerotization are evident when comparing eusocial and solitary bees. This cuticular maturation heterochrony may be associated with life style, considering that eusocial bees remain protected inside the nest for many days after emergence, while the solitary bees immediately start outside activities. To address this issue, we characterized gene expression using large-scale RNA sequencing (RNA-seq), and quantified cuticular hydrocarbon (CHC) through gas chromatography-mass spectrometry in comparative studies of the integument (cuticle plus its underlying epidermis) of two eusocial and a solitary bee species. In addition, we used transmission electron microscopy (TEM) for studying the developing cuticle of these and other three bee species also differing in life style. We found 13,200, 55,209 and 30,161 transcript types in the integument of the eusocial Apis mellifera and Frieseomelitta varia, and the solitary Centris analis, respectively. In general, structural cuticle proteins and chitin-related genes were upregulated in pharate-adults and newly-emerged bees whereas transcripts for odorant binding proteins, cytochrome P450 and antioxidant proteins were overrepresented in foragers. Consistent with our hypothesis, a distance correlation analysis based on the differentially expressed genes suggested delayed cuticle maturation in A. mellifera in comparison to the solitary bee. However, this was not confirmed in the comparison with F. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated between A. mellifera and F. varia, and negatively or non-correlated with C. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species and C. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except for F. varia, the TEM analysis is consistent with cuticle development timing adapted to the social or solitary life style. In support to our hypothesis, the absolute quantities of n-alkanes and unsaturated CHCs were significantly higher in foragers than in the earlier developmental phases of the eusocial bees, but did not discriminate newly-emerged from foragers in C. analis. By highlighting differences in integument gene expression, cuticle ultrastructure, and CHC profiles between eusocial and solitary bees, our data provided insights into the process of heterochronic cuticle maturation associated to the way of life.
... Elias-Neto, Soares & Bitondi, 2009). Thus far, attempts to separate bumble bee pupa by morphology has involved either using a modification of the same six stages for honey bees or a more simplified model (Dong et al., 2017;Li et al., 2010;Mänd et al., 2005). ...
... This morphological key, annotated with high resolution images, is intended for use in any study on developing bumble bees requiring precise pupal staging. The characterization of bumble bee pupal stages presented here differs substantially from those described in honey bees and other bee species, with the exception of eye pigmentation (Elias-Neto, Soares & Bitondi, 2009). It includes more stages than defined in previous studies on bees, both in eye color and body pigmentation stages ( Fig. 9 and Table S2). ...
Bumble bees (Hymenoptera: Apidae, Bombus ) are important pollinators and models for studying mechanisms underlying developmental plasticity, such as factors influencing size, immunity, and social behaviors. Research on such processes, as well as expanding use of gene-manipulation and gene expression technologies, requires a detailed understanding of how these bees develop. Developmental research often uses time-staging of pupae, however dramatic size differences in these bees can generate variation in developmental timing. To study developmental mechanisms in bumble bees, appropriate staging of developing bees using morphology is necessary. In this study, we describe morphological changes across development in several bumble bee species and use this to establish morphology-based staging criteria, establishing 20 distinct illustrated stages. These criteria, defined largely by eye and cuticle pigmentation patterns, are generalizable across members of the subgenus Pyrobombus , and can be used as a framework for study of other bumble bee subgenera. We examine the effects of temperature, caste, size, and species on pupal development, revealing that pupal duration shifts with each of these factors, confirming the importance of staging pupae based on morphology rather than age and the need for standardizing sampling.
... Novak [27] reported that activated epidermal cells secrete molting fluid before apolysis; Klowden [10] and Elias-Neto et.al. [35] stated that shape change and volume increase in epidermal cells resulted in apolysis, separation of old and new cuticle from each other, and filling of the space created by apolysis with molting fluid; and Chapman et all. [12] expressed that molting fluid enzymes became activated after new epicuticle formation. ...
The cycle of sixth instar larval integument of a Lepidoptera example, Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) was investigated by light microscopy. Two phases of larval integument change of G. mellonella larvae were observed via histological and histometrical measurements performed over 12-108 hours. The first of these phase changes was observed between 12 and 84 hours with changes to epidermal cells and procuticle formation resulting in interzone cuticle. The second phase change occurred in cuticle and epidermal cells with digestion of the old procuticle and formation of a new procuticle, which was observed between 96 and 108 hours commencing with apolysis at 96 hours. During the first period complete epicuticle formation was detected at 12 hours, the development of procuticle continued with lamellae addition between 12 and 84 hours. The procuticle development was completed with formation of the interzone cuticle at 84 hours. Epidermal cells underwent mitosis at 72 hours and at 84 hours formed the interzone cuticle by secreting a small quantity of seventh instar cuticle. In the second stage, apolysis occurred at 96 hours. The observed effects of molting fluid were, firstly, the separation of endocuticle lamellae, followed by their dissolution and, finally, absorption by epidermal cells. The procuticle thickness decreased from 16.9 urn to 7.5 urn at this stage. Epidermal cells took on a prismatic shape at this stage and started to form seventh instar epicuticle and procuticle.
... The changes physiological functions have been extensively studied in worker honeybees through the ages. In recents years, significant interests have been shown in studying honeybee humoral, cellular immune response (Bedick et al., 2001;Amdam et al., 2004Amdam et al., , 2005Klaudiny et al., 2004;Lourenço et al., 2005;Yang and Cox-Foster, 2005;Evans, 2006;Wilson-Rich et al., 2008;Alaux et al., 2010) and characteristcs of integument (Thompson and Hepburn, 1978;Nemtsev et al., 2001;Elias-Neto et al.,2009, 2014Seehuus et al. 2013;Kaya et al., 2015). We have a few information about the component of cuticle (Thompson and Hepburn, 1978) and cellular immune component of honeybee age-related division labor (Amdam et al., 2005;Schmid et al., 2008). ...
The aim of this work is to study the difference of physiology between the worker bee nurse and forager (Apis mellifera intermissa). The chosen physiological characteristics were the component of the cuticle (protein-chitin content) and the measure of the efficiency of immune system (the total number of haemocytes (THC), the normal haemocytes and the relative mass of fat body). The THC is widely used as an indicator of cellular immunocompetence of insects. The normal haemocytes, also referred to immunocytes, indicate the integrity of cellular immune system. The fat body is an indirect measurement of induced humoral immunocompetence. The THC and the normal haemocytes were determined by the method described by Amdam et al., (2004). For the estimation of the cuticular abdominal protein-chitin content, the method described by Berghiche et al., (2007) was employed. The relative mass of fat body was determined using an ether extraction method according to Doums et al., (2002) and Wilson-Rich et al., (2008). The results show that a considerable percentage of a cuticular protein and a decrease of chitin was observed in nurse compared to forager. The older bees exhibited a strong reduction in the immun parameters.
