Male Accessory Glands of Blister Beetles and Cantharidin Release: A Comparative Ultrastructural Analysis

Abstract

Members of the family Meloidae are known to produce cantharidin, a highly toxic monoterpene found in their hemolymph and exuded as droplets capable of deterring many predators. As a nuptial gift, males transfer large amounts of cantharidin to females via a spermatophore, which is formed by specific accessory glands containing high concentrations of this terpene. Using light, electron and ion beam microscopy, the ultrastructural features of the three pairs of male accessory glands as well as the glandular part of the vasa deferentia were comparatively investigated in seven species of blister beetles belonging to five different tribes and two subfamilies. All gland pairs examined share common features such as mesodermal derivation, the presence of muscle sheath, a developed rough endoplasmic reticulum, abundant mitochondria, secretory vesicles, and microvillated apical membranes. Within the same species, glands exhibit distinctive features, suggesting that each pair is responsible for the formation of a specific substance. The vasa deferentia, while showing many similarities within the family, often exhibit features unique to each of the individual species investigated, whereas the accessory glands of the first and second pairs display the highest degree of ultrastructural variability. A comparison across the species shows an interesting constancy limited to ultrastructural features in the third pair of accessory glands. The similarities and differences among the species are discussed in the light of the available literature and in relation to the potential role that blister beetles' male accessory glands could play in the storage and management of cantharidin.

Keywords: FIB/SEM; Meloidae; accessory glands; electron microscopy; male reproductive system; vas deferens.

Figure 1

Photos of the different species of blister beetles analyzed comparatively in this work. From top to bottom and from left to right: Mylabris variabilis; M. flexuosa; M. pusilla; Lydus trimaculatus; Epicauta rufidorsum; Cerocoma schreberi; Zonitis immaculata and Meloe proscarabaeus.

Figure 2

General structure of the male reproductive systems of the investigated Meloidae species. The different components are colored using specific colors: First pair of accessory glands in red, second pair of accessory glands in light blue, third pair of accessory glands in yellow and internal reproductive system in green. (A) Light micrograph of the dissected system of M. variabilis in ventral view; inset displaying the first pair of glands distended and separated from the reproductive system. Asterisk indicates the bifurcation of the third pair of glands, located near the point of insertion. (B) Light micrograph of the dissected system of L. trimaculatus in ventral view, the gland of the third pair on the left and the vas deferens on the right have been purposely removed; inset presenting a close-up of the expanded horn-like region of the vas deferens. (C) Schematic drawings of the systems of the different species illustrated in ventral view (diagrams are not in scale). The same colors were used as previously presented; ejaculatory duct was not colored. Scale bars: (A) = 1 mm; (B) = 2 mm.

Figure 3

Ultrastructure of the first pair of male accessory glands in M. variabilis. (A) Cross section of the gland showing the epithelium pseudostratification, central lumen containing the secretions and a developed muscular sheath encasing the cells. (B) Close-up of the former micrograph showing muscles and basal regions of the cells with indented nuclei, mitochondria and developed rough endoplasmic reticulum stacks. (C) Longitudinal section of the glands showing polygonal appearance of the cells and apical microvillated plasma membrane. (D) Micrograph towards the cell apex depicting cytoplasm with numerous mitochondria, flattened rough endoplasmic reticulum stacks and different vesicles. (E) Plasma membrane developing an abundant network of crowded and overlapping microvilli. Note the presence of numerous mitochondria. (F) Micrograph of the interface between lumen and secretory cells showing numerous lucid vesicles and a few small secretory granules near the microvilli. (G) Close up of the cytoplasm illustrating slender mitochondrial morphology, small Golgi complexes, flattened rough endoplasmic reticulum and a pale inclusion. (H) Detail of the apical region showing connections between two adjacent cells, small electrolucent vesicles, endoplasmic reticulum, and free ribosomes. BL, basal lamina; GO, Golgi complex; LU, lumen; LV, large vesicles; MU, muscles; MS, muscular sheath; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SC, secretory cells; SG, secretory granule; VE, vesicles. Scale bars: (A) = 30 μm; (B,C) = 10 μm; (D) = 5 μm; (E,F) = 3 μm; (G,H) = 2 μm.

Figure 4

Ultrastructure of the second pair of male accessory glands in M. variabilis. (A) Cross section of the gland showing tissue pleats towards the lumen. (B) Micrograph showing the basal region of the cells with moderate folds of the basal membrane (indicated by the asterisks) and muscle fibers surrounding the secretory cells. (C) Close up at the level of the involution, displaying abundance of mitochondria and vesicles near the microvillated apical membrane. Some electrondense granules can also be observed in the cytoplasm. (D) Nucleus with obvious nucleolus surrounded by abundant flattened stacks of rough endoplasmic reticulum. (E) Apical region of the cell bearing microvilli. Note the sinuous contours of the lateral membranes of two neighboring cells, slender mitochondria and vesicles. (F) Close up showing flattened Golgi complex, thin mitochondria, dark granules and rough endoplasmic reticulum with both flattened and expanded cisterns. BL, basal lamina; GO, Golgi complex; LU, lumen; MS, muscular sheath; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SC, secretory cells; SEC, secretions; VE, vesicles. Scale bars: (A) = 50 μm; (B) = 20 μm; (C) = 10 μm; (D) = 5 μm; (E,F) = 2 μm.

Figure 5

Ultrastructure of the third pair of male accessory glands in M. variabilis. (A) Cross section of the gland; note the particulate secretion inside the lumen. Inset depicting one of the autophagic vacuoles commonly encountered in these cells. (B) Micrograph of the secretory cell showing the lacunar system, Golgi complexes with very small cisternae, and flattened rough endoplasmic reticulum. Note the apical microvilli and the ampullaceous expansions. (C) Developed nucleus surrounded by small Golgi complex and mitochondria. In the lumen, the particulate secretion is clearly visible. (D) Micrograph displaying developed rough endoplasmic reticulum, Golgi with small dictyosomes, several vesicles (some of which are approaching the microvilli) and expanded region of the thin microvilli (E) Cell displaying multilamellar bodies, small Golgi complex, and electrondense inclusions. (F,G) Details of the vesicles, Golgi complex and mitochondria. AE, ampullaceous expansions; BL, basal lamina; BLS, basal lacunar system; EI, electrondense inclusion; GO, Golgi complex; LU, lumen; MLB, multilamellar body; MU, muscles; MT, mitochondria; MV, microvilli; NU, nucleus; RER, rough endoplasmic reticulum; SC, secretory cells; SEC, secretory products; TR, trachea; VE, vesicles. Scale bars: (A) = 20 μm; (B,D) = 5 μm; (C) = 5 μm; (E) = 4 μm; (F,G) = 2 μm.

Figure 6

Ultrastructure of the vasa deferentia in M. variabilis. (A) Epithelium of the vasa deferentia encircled by developed muscles. Note the arrangement of the nuclei and the lumen containing bundles of spermatozoa. (B) Epithelium with abundant mitochondria and slightly indented nucleus, basally located. Note bigger mitochondria with angular shape (indicated by asterisks). (C) Basal region showing moderate intercellular spaces, polymorphic mitochondria and secretory granules. (D) Apical region of the cell with cytoplasm rich in mitochondria of various sizes and shapes, especially located near the microvilli. Note the fibrillar content of the lumen. (E) Detail of rough endoplasmic reticulum with slightly vacuolized cisternae. (F) Close up presenting Golgi complex, mitochondria, very small electronlucent vesicles and an electrondense inclusion. BL, basal lamina; EI, electrondense inclusions; GO, Golgi complex; IS, interecellular space; MU, muscles; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SB, sperm bundles; SC, secretory cells; SEC, secretions; Sg, secretory granule; VE, vesicles. Scale bars: (A) = 20 μm; (B) = 10 μm; (C,D) = 5 μm; (E,F) = 2 μm.

Figure 7

Ultrastructural features of M. pusilla and M. flexuosa male accessory glands and glandular region of the vasa deferentia. (A,B) First pair of male accessory glands of M. pusilla exhibiting a reduced level of pseudostratification. (B) Cytoplasm with small Golgi complexes, mitochondria and vacuolar bodies. (C,D) Second pair of male accessory glands of M. flexuosa with developed rough endoplasmic reticulum. Note the peculiar appearance of the content of the secretory vesicles located near the microvilli. (E,F) Third pair of male accessory glands of M. flexuosa exhibiting basal lacunar system, autophagic vacuoles, inclusions, numerous mitochondria, small Golgi complexes, minute electronlucid vesicles and microvilli with ampullaceous expansions. (G,H) Vasa deferentia of M. pusilla. Details of the rough endoplasmic reticulum (G) and apical microvillar region facing the lumen, displaying small vesicles. AB, autophagic body; AE, ampullaceous expansions; AV, autophagic vacuole; BL, basal lamina; BLS, basal lacunar system; EI, electrondense inclusion; GO, Golgi complex; LU, lumen; ML, multilamellar body; MU, muscles; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; VE, vesicles. Scale bars: (A) = 10 μm; (B) = 2 μm; (C) = 10 μm; (D) = 2 μm; (E) = 5 μm; (F) = 3 μm; (G) = 4 μm; (H) = 1 μm.

Figure 8

Ultrastructural features of the first pair of male accessory glands in L. trimaculatus. (A) Oblique section of the gland showing the high level of pseudostratification. (B) Longitudinal section illustrating the basal region of the cells, lying on the basal lamina. The cytoplasm contains regular oval nuclei, abundant rough endoplasmic reticulum and lucent vesicles. Note the presence of tracheae between the muscles. (C) Cross section of the gland in the medial region. Notable is the polygonal shape of the cells and the presence of secretory vesicles and granules. Some exhausted cells, indicated by asterisk, show a more electrondense cytoplasm. (D) Micrograph of the apical region of the cells where, in addition to the presence of vesicles and granules, the cell projections and their budding towards the lumen can be appreciated. (E) Detail of the apical region with microvilli and budding. (F) Close up showing membrane folding, mitochondria, vesicles and granules. BL, basal lamina; LU, lumen; MS, muscular sheath; MU, muscles; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SC, secretory cells; SG, secretory granules; VE, vesicles. Scale bars: (A) = 50 μm; (B–D) = 10 μm; (E,F) = 2 μm.

Figure 9

Ultrastructural features of L. trimaculatus’ second and third pair of male accessory glands and glandular region of the vasa deferentia. (A) Transverse section of second pair gland showing monolayer of microvillated columnar cells and lumen containing dark spheroidal secretions immersed in a lighter matrix. (B) Apical region of the former bearing microvilli and exhibiting irregular vesicles and numerous mitochondria. (C–F) Sections of the gland of the third pair. (C) Note the increased height of the cells and the presence of autophagic vacuoles. (D) Microphotograph illustrating the basal lacunar system, numerous mitochondria, thin and elongated microvilli with expanded ampulla-like projections and particulate secretion within the lumen. (E,F) Details of mitochondria, vacuoles, multilamellar bodies, inclusions, small Golgi complex, microvilli and electronlucent vesicles. (G,H) Pictures of vasa deferentia cytoplasm showing typical, distinctive-looking secretory vesicles as well as rough endoplasmic reticulum in both flattened and expanded forms. AE, ampullaceous expansions; AV, autophagic vacuole; BL, basal lamina; BLS, basal lacunar system; EI, electrondense inclusion; GO, Golgi apparatus; LU, lumen; MLB, multilamellar body; MS, muscular sheath; MU, muscles; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SC, secretory cells; SEC, secretory products; SV, secretory vesicles; TR, trachea; VE, vesicles. Scale bars: (A) = 40 μm; (B) = 5 μm; (C) = 10 μm; (D,E) = 5 μm; (F) = 3 μm; (G,H) = 2 μm.

Figure 10

Ultrastructural features of E. rufidorsum male accessory glands and glandular region of the vasa deferentia. (A,B) First pair of glands. (A) Micrograph showing the pseudostratification of the monolayer, firmly adherent to the basal lamina, and the presence of well-developed muscles surrounding the epithelium. (B) Apical region of cells showing rounded inclusions and rough endoplasmic reticulum with enlarged forms. Note the appearance of the aggregates contained in the lumen. (C,D) Second pair of glands. (C) Cells have abundant mitochondria and show several cytoplasmic inclusions. Note the presence within the lumen of conspicuous laminar structures and a dark region near the microvilli. (D) Cytoplasm view showing rough endoplasmic reticulum, irregularly shaped inclusions with areas of increased electrondensity, mitochondria and microvilli. Note, also, the projections of the cell towards the lumen. (E,F) Gland of the third pair. (E) Cells exhibiting a developed lacunar basal system and bearing ampullaceous expansions at the level of the microvilli facing the lumen filled with particulate matter. (F) Apical region with microvilli bearing expansions. Note small Golgi complex, and inclusions of various sizes. (G,H) Vasa deferentia with high development of the wrinkled endoplasmic reticulum. Note the presence of vacuolar structures of increased size. AE, ampullaceous expansions; BLS, basal lacunar system; EI, electrondense inclusion; GO, Golgi complex; LU, lumen; MU, muscles; MT, mitochondria; MV, microvilli; NU, nucleus; RER, rough endoplasmic reticulum; SEC, secretory products; VA, vacuoles. Scale bars: (A) = 10 μm; (B) = 4 μm; (C) = 5 μm; (D) = 3 μm; (E,F) = 5 μm; (G) = 10 μm; (H) = 2 μm.

Figure 11

Ultrastructural features of C. schreberi male accessory glands and glandular region of the vasa deferentia. (A,B) Gland of the first pair. (A) Oblique section of the accessory gland showing the cells contours appearing moderately sinuous and only slightly polygonal. Note the lumen filled with electrondense irregular secretions immersed in a uniform matrix of lesser electrondensity. (B) Detail of cells of the first pair of glands showing elongated mitochondria, flattened rough endoplasmic reticulum, abundant Golgi complex, electron lucent vesicles and autophagic vacuoles. (C,D) Micrographs of the second pair of accessory glands displaying the ovoid nucleus surrounded by mitochondria and a rough reticulum. (C) The apical region with numerous mitochondria near the thin microvilli that face a glandular lumen with a pale and uniform content (D). (E) Gland of the third pair exhibiting particulate secretion stored in the lumen, slender apical microvilli with scarce ampullaceous expansion and developed basal lacunar system. (F) Close up of the basal lacunar system of the third pair of glands, note the presence of mitochondria in proximity to the lacunar spaces and invaginations of the basal membrane. (G,H) Glandular part of vasa deferentia. (G) Transverse section showing muscles, basal lamina and the basal region of the cells rich in rough endoplasmic reticulum with swollen cisternae and some electrondense granule. (H) Apical region with the peculiar secretory vesicles approaching to the short microvilli. AE, ampullaceous expansions; AV, autophagic vacuole; BL, basal lamina; BLS, basal lacunar system; GO, Golgi apparatus; LU, lumen; MU, muscles; MT, mitochondria; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; SEC, secretory products; VE, vesicles. Scale bars: (A) = 10 μm; (B–F) = 2 μm; (G) = 5 μm; (H) = 1 μm.

Figure 12

Ultrastructural features of Z. immaculata male accessory glands and glandular region of the vasa deferentia. (A,B) Gland of the first pair. (A) Polygonal appearance of cells in oblique section. The cytoplasm shows numerous vesicles. (B) Secretory vesicles in the apical region, near the microvilli. (C,D) Second pair of glands. (C) Abundant rough endoplasmic reticulum in proximity to a nucleus. (D) Apparent structures as multivesicular bodies. (E,F) Glands of the third pair. (E) Cells showing a basal lacunar system and numerous mitochondria clustered towards the apical region, near the microvilli. (F) Close up of the previous image, note a large inclusion. (G,H) Vasa deferentia with mostly flattened rough endoplasmic reticulum. BLS, basal lacunar system; EI, electrondense inclusion; GO, Golgi complex; MT, mitochondria; MU, muscles; MVB, multivesicular body; MV, microvilli; NC, nucleolus; NU, nucleus; RER, rough endoplasmic reticulum; VE, vesicles. Scale bars: (A) = 5 μm; (B) = 2 μm; (C) = 4 μm; (D) = 1 μm; (E) = 10 μm; (F) = 4 μm; (G) = 5 μm; (H) = 2 μm.