Morphological characterization and staging of bumble bee pupae

Abstract

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.

Keywords: Bumble bee; Development; Pupa; Pupal duration; Staging.

Figure 1. Cocoons of B. impatiens at different stages.

Stages include a prepupa (A), pupa (B) and (C) and a newly eclosed adult bee (D).

Figure 2. Characteristics unique to pupal stages.

Metasomal tergal spicules are present in bumble bee pupae (A), but absent in honey bee pupae (B). (C) The trochanteral protuberance (Trp) braces the basitarus (Btar) of the foreleg (Flg) and midleg (Mlg), suggesting a function as a leg hook that maintains proper folding of pupal legs. The tibial spine (Tbsp) of the hind leg is also shown. (D) A schematic illustration of the bumble bee pupal leg showing coxal (Cxp) and trochanteral (Trp) protuberances and tibial (Tbsp) spines. Black lines, pupal cuticle; gray lines, developing adult cuticle; gray shades, developing setae. Illustration is modified from Michener (1954). (E) Trochanteral protuberance with developing adult hair bundle (broken line) encased in pupal cuticle. (F–H), Coxal and trochanteral protuberances of mid and hind legs of early, mid, and late-stage pupae, respectively. The tibia and basitarsus are removed to reveal the protuberances. Note the degeneration of these protuberances in the late pupa (H). Mst, Metasomal tergite; Mtsp, Metasomal tergal spicules; Cx, coxa; Tr, trochanter; Fm, Femur; Tb, tibia; Btar, basitarsus. Scale bar unit: mm.

Figure 3. Morphological criteria marking transitions to P0–P8.

(A & B) Illustration and photo for transition from P0 to P1. Note the retraction of A1 and A8–A10. (C & D) Illustration and photo for changes in shapes of basitarsi of the hind leg (broken lines) of B. impatiens workers in the transition from P1 to P2. (E–G) Illustration (E) and photo (F) for color shift of the CE from P3 to P8. (G) shows zoomed view of the CE revealing appearance of ommatidial units at different stages, including dotted (P3 and P4), then hexagonal (P5–P7) and finally, a filled pattern (P8-eclosion). (H) and (I) Black triangles indicate a slightly tanned suture at forewing-tegula junction (H) at P3, and between the mesepisternum and mesocoxa (I) at P7. Teg, tegula; Fw, forewing; Msetm, mesepisternum; Msc, meso-coxa. Scale bar unit: mm.

Figure 4. Morphological criteria marking the transition to P9.

(A & B) The black triangle indicates the tanned suture in the lateral posterior thorax. (C & D) The black triangle indicates a slightly tanned tarsal claw. Sct, scutum; Stm, scutellum; Tc, Tarsal claw. Scale bar unit: mm.

Figure 5. Morphological criteria marking the transition to P10–P16.

(A & B) Photo and schematic illustration of melanization patterns of abdominal cuticle. Melanization first appears as peppered stripes on the apical margin of each metasomal tergite/sternite disc (P10–P12L), then expands anteriorly to cover the whole disc (P13–P16). The orange tinge at P12L/P13 demonstrates the beginning of tanning of the cuticle. Orange at P14–P16 indicates partial pigmentation of black setae, while the white in T2 (P14–P16) indicates the white color of the future yellow hairs, as the underlying cuticle at these stages is mostly or all black. (C & D) Photo and schematic illustration of pigmentation on the head region including the face, mouthparts and antennae. Mb, mandible; Th, teeth; Fl, frontal line; Sp, Scape; Lb, labrum; Pb, proboscis; Pa, paraocular area; Clp, clypeus; Pd, pedicle; Fg, flagellomeres; Pbl, lateral-basal proboscis. Black triangle: broken melanization stripes at stage P11. Scale bar unit: mm.

Figure 6. Temporal changes in setal color in pupal (A) and callow (B) stages.

For (A) metasomal setal color development at late pupal stages of B. impatiens visible upon removal of the pupal cuticle. For (B) prospective yellow, black, and red hairs are indicated by yellow, black, and red arrows, respectively. Note the shift in setal color as the callow gets older, as well as the difference between yellow, black, and red setae in the timing of pigmentation. Yellow setae appear to be white until 24–48 h after eclosion, when black and red hairs are nearly fully pigmented.

Figure 7. Photographic guide to developmental stages of B. impatiens male pupa.

Figure 8. Variation in timing of melanization among body parts in B. impatiens males.

Relative darkness = gray value of measured area/255 × 100%. The dotted lines indicate background darkness value of the white body, above which the tanning is visible.

Figure 9. Summary of stage duration and pigmentation changes in B. impatiens worker pupae and callows.

Colored bars illustrate timing of pigmentation on different body parts. Bumble bee pupal staging applying the honey bee criteria (Rembold, Kremer & Ulrich, 1980) is compared to the newly developed staging system. Details on the timing of bumble bee stages relative to the honey bee are provided in Table S2.

Figure 10. Bumble bee pupal duration.

(A) B. impatiens pupal duration of various castes and body sizes reared at two different temperatures. (B) Pupal duration of B. impatiens and B. vosnesenskii male pupae reared at different temperatures. S-W, M-W, L-W, small, medium, and large workers, respectively; S-M, M-M, small, and medium males, respectively. Q, queen. Error bars are standard error of the mean (SE). Means with the same letter are not significantly different, One-way ANOVA followed by Tukey’s HSD post hoc test, p < 0.05; *p < 0.05, two-sample t-test.