Outbreak of densovirus with high mortality in a commercial mealworm (Tenebrio molitor) farm: A molecular, bright-field, and electron microscopic characterization

Abstract

Mealworms are one of the most economically important insects in large-scale production for human and animal nutrition. Densoviruses are highly pathogenic for invertebrates and exhibit an extraordinary level of diversity which rivals that of their hosts. Molecular, clinical, histological, and electron microscopic characterization of novel densovirus infections is of utmost economic and ecological importance. Here, we describe an outbreak of densovirus with high mortality in a commercial mealworm (Tenebrio molitor) farm. Clinical signs included inability to prehend food, asymmetric locomotion evolving to nonambulation, dehydration, dark discoloration, and death. Upon gross examination, infected mealworms displayed underdevelopment, dark discoloration, larvae body curvature, and organ/tissue softness. Histologically, there was massive epithelial cell death, and cytomegaly and karyomegaly with intranuclear inclusion (InI) bodies in the epidermis, pharynx, esophagus, rectum, tracheae, and tracheoles. Ultrastructurally, these InIs represented a densovirus replication and assembly complex composed of virus particles ranging from 23.79 to 26.99 nm in diameter, as detected on transmission electron microscopy. Whole-genome sequencing identified a 5579-nucleotide-long densovirus containing 5 open reading frames. A phylogenetic analysis of the mealworm densovirus showed it to be closely related to several bird- and bat-associated densoviruses, sharing 97% to 98% identity. Meanwhile, the nucleotide similarity to a mosquito, cockroach, and cricket densovirus was 55%, 52%, and 41%, respectively. As this is the first described whole-genome characterization of a mealworm densovirus, we propose the name Tenebrio molitor densovirus (TmDNV). In contrast to polytropic densoviruses, this TmDNV is epitheliotropic, primarily affecting cuticle-producing cells.

Keywords: Parvoviridae; Tenebrio molitor; densovirus; electron microscopy; epitheliotropic virus; histopathology; mealworm; mortality; virus assembly; virus replication.

Figure 1.

Tenebrio molitor larvae infected with densovirus. (a) On the right, infected larvae at different development instars (A) presenting variable degrees of dehydration and generalized dark discoloration compared with one unaffected individual on the left. (b, c) Cross sections of larvae infected with densovirus. Subgross images at the levels of the proximal (b) and distal (c) midgut depict the retention of an unshed cuticle (arrowhead), which is above a thin and undulate new cuticle. Open arrows indicate tracheal tubes; Sm, skeletal muscle; fb, fat body; pco, pseudocoelom; g, midgut. Boxes indicate areas highlighted in (e), (f), and (g). Hematoxylin and eosin (HE). (d) Unaffected integument with an eosinophilic cuticle (C) and columnar epidermal epithelium (E). These cells have a centrally located nucleus with coarse marginated chromatin (black arrow), eosinophilic secretory granules (white arrow), and basilar-located cytoplasmic vacuoles. HE. (e)–(h) Progression of epidermal cell lesions in larvae infected with densovirus; magnification of areas of the integument highlighted in (b) and (c). HE. A variably thin and undulate cuticle (C) is overlying infected epidermal cells (E). Infected epidermal cells are detached and enlarged, mildly in (e) to severely in (f) and (g). As is obvious in (h), individualized, infected cells between the cuticle (C) and basal lamina (black arrow) are suspended in proteinaceous fluid. Note the thick, unshed old cuticle (black arrowhead) compared with a thin, undulate new cuticle (open arrowhead). The nuclei of infected epidermal cells are enlarged due to eosinophilic and basophilic intranuclear inclusions. While eosinophilic inclusions (black arrowheads) predominate in (e) and (f), basophilic inclusions (black arrows) are present in (g) and (h). (e)–(g) Eosinophilic inclusions are rounded to polyhedrals surrounded by a clear hollow with chromatin displaced to the margin (arrowheads). (f) Inset: magnification of eosinophilic inclusion. (g) Basophilic inclusions occupy the full area of the nucleus with indistinct margins of chromatin (black arrows). Inset: magnification of basophilic inclusion. (i) Unaffected integument, centrally located nuclei of epidermal cells display a bright pink reaction. Inset: magnification of 2 nuclei. Feulgen reaction correlates with a coarse distribution of the chromatin. Feulgen stain. (j) Infected integument, consecutive section of (g) stained with Feulgen. A significant number of inclusions have a homogeneous, positive bright pink reaction, which correlates with basophilic inclusions (black arrows in (g) and (j)). In contrast, a faint Feulgen reaction correlates with eosinophilic inclusions (black arrowheads in (f) and (j)). C, cuticule; fb, fat body lobe. Inset: magnification of a positive Feulgen reaction of a basophilic inclusion (right) in contrast to a faint reaction of an eosinophilic inclusion (left).

Figure 2.

Tenebrio molitor larvae infected with densovirus. Leg preparations embedded in plastic and stained with toluidine blue. (a) Diffusely, epidermal cells are infected with densovirus. Note the interstitial space filled with proteinaceous exudate and hemocytes. (b) Magnification of the box in (a). The infected epidermal cells (E) are detached from the cuticle. A detachment of an intrinsic leg muscle due to death of epithelial cells is highlighted with black arrowheads. The black arrow shows inserted muscle at the button. Hypercontracted and hyperstretched muscle fiber segments are indicated by a white arrowhead and open arrowhead, respectively. The basement lamina is intact (white arrow).

Figure 3.

Tenebrio molitor larvae. (a) Unaffected larvae, sections included in the hematoxylin and eosin (HE) histological panel: 1, head; 2, thorax; 3, abdomen. (b) Infected larvae, section 1 in (a), subgross image of the head; the box indicates an area highlighted in (c). HE. (c) Diffusely, infected epidermal epithelial cells contain basophilic intranuclear inclusions (black arrow). The detached cuticle is not present. Skeletal muscles are segmentally necrotic (Sm). HE. (d) Section 2 in (a), esophagus, epithelial cells have eosinophilic intranuclear inclusions (black arrow). The cuticle is detached (black arrowhead). M, tunica muscularis; white arrowheads, tracheole; white arrows, basal lamina; pco, pseudocoelom; fb, fat body. HE. (e) Tracheal tubes, infected epithelial cells are cuboidal. Enlarged nuclei present basophilic inclusions (black arrows). The cuticle is detached (arrowheads). pco, pseudocoelom; L, tracheal tube lumen. Inset: Unaffected tracheal tube. Black arrow, epithelial cells; black arrowhead, cuticle; pco, pseudocoelom; L, tracheal tube lumen. HE. (f) Midgut, infected epithelial cells of tracheoles are hypertrophied, nuclei have basophilic inclusions (black arrows). L, tracheoles lumen; pco, pseudocoelom; M, tunica muscularis; ME, mucosal epithelium; gL, midgut lumen. HE.

Figure 4.

Densovirus virions recovered from Tenebrio molitor larvae. Transmission electron microscopy. Negative-contrasted, nonenveloped, round virus particles ranging from 23.79 to 26.99 nm in diameter. Comparable proportions of spheres (sealed, black arrow) and hollow (contrast-permeable, white arrow) virus particles are presented. Phosphotungstic acid. Bar = 100 nm.

Figure 5.

Tenebrio molitor larvae infected with densovirus. Transmission electron microscopy. (a) The epidermal epithelial cell contains a large nuclear densovirus replication and assembly complex (DRAC). Other cytopathic effects include endocuticular detachment due to the degradation of the anchoring junctions (black arrowhead) and cytoplasmic formation of circular and lamellar membranous bodies (black arrows). White arrowhead, plasma membrane; white arrow, nuclear membrane and chromatin; EC, endocuticle. Bar = 5 μm. (b) Epidermal epithelial cells are detached from the endocuticle and adjacent cells. The plasma membrane is indicated with a white arrowhead. Within its cytoplasm, there are electron-dense phagolysosomes (black arrow) and degenerated mitochondria (white arrow). Bar = 5 μm. (c) Infected epithelial cells at the “cuticle-epithelial cell-skeletal muscle insertion” are detached due to the disintegration of the muscle attachment fiber-hemidesmosome junction (black and white arrows, respectively). The nuclear chromatin is replaced by a DRAC. Degenerated tonofibrils and mitochondria are depicted with white and black arrowheads, respectively. Bar = 5 μm. (d) Tracheole, epithelial cells contain a nuclear DRAC that triplicates the size of a normal nucleus. Dead epithelial cells (DC) with nuclear fragments (arrowheads) are detaching from the cuticle (white arrow). L, lumen; black arrow, basement lamina; pco, pseudocoelom; Sm, skeletal muscle. Bar = 5 μm.

Figure 6.

Tenebrio molitor larvae infected with densovirus. Transmission electron microscopy. (a) Nucleus of an epidermal epithelial cell contains a densovirus replication and assembly complex (DRAC), which has displaced the chromatin to the margin (black arrows). These DRACs are composed of anastomosing streams of viral matrices of variable electron densities. On the left is a compacted virus matrix radiating cordons of assembled densovirus particles. N, nucleolus. Bar = 2 μm. (b) Magnification of black box in (a). Densovirus particles (white arrowheads) are assembled at the margin of electron-dense fibrillar matrix (em) streams. Bar = 200 nm. (c) Magnification of white box in (a). Densovirus particles (white arrowhead) forming long cords are assembled at the margin of the nucleolus-like compacted aggregate of electron-dense fibrillar matrix (em). Bar = 200 nm. (d) Nucleus of an epidermal epithelial cell displaying DRAC with paracrystal formation (Cry) that comprises approximately 40% of the nuclear volume. em, electron-dense matrix. Bar = 200 nm. (e) Magnification of the box in (d). Densovirus particles (white arrowhead) are assembled at the margin of an electron-dense fibrillar matrix (em) stream. Cry, paracrystal formation. Bar = 500 nm. (f) Magnification of the box in (e). Paracrystalline formation. Bar = 200 nm.

Figure 7.

Tenebrio molitor larvae infected with densovirus. Transmission electron microscopy. (a–f) Different conformations of the densovirus replication and assembly complex (DRAC). Note the variation of electron density, texture of the virus matrix, and chromatin at the periphery (white arrow). A nucleolus (N) is present only in (c) and (e). A nucleolus-like aggregation of dense matrix (nl) is present in (c)–(e). Densovirus virions are marked with black arrows in (c), (d), and (f). Paracrystal (Cry) formation is obvious in (f). Bars = 2 μm.

Figure 8.

Tenebrio molitor larvae infected with densovirus. Transmission electron microscopy. (a) In the cytoplasm of an apparently uninfected epidermal cell are small and large phagolysosome (PhL)-containing densovirus particles. Its oval nucleus has clumped and marginated heterochromatin. Black arrow, mitochondrion. Bar = 1 µm. Inset: Magnification of a phagolysosome containing densovirus particles lined by membrane (black arrow). Bar = 125 nm. (b) The epidermis is expanded by up to 5 layers of nuclei, which contain intranuclear, heterogeneous densovirus replication and assembly complexes (DRACs). While numerous cells have small nuclear/DRAC fragments (white arrowheads) within their cytoplasm, larger fragments of nuclei/DRAC with vestigial amounts of cytoplasm are free (white arrow). Some cells have swollen cytoplasm and nuclear lysis (black arrows). Bar = 10 µm. (c) Nuclear/DRAC fragments of various sizes are present within the cell cytoplasm (white arrows). Black arrow, basal lamina; pco, pseudocoelom. Bar = 5 µm. (d) Tracheolar epithelial cells undergoing death are detaching from the cuticle (black arrows). The cytoplasm contains nuclear fragments in which the chromatin was replaced by virus particles (white arrows). L, lumen; pco, coelom; black arrowhead, basement lamina. Inset: Magnification of the white box in (d), nuclear fragment containing myriad virus particles. Bar = 500 nm.

Figure 9.

Genomic organization of Tenebrio molitor densovirus (TmDNV) indicating the location and direction of transcription for each of the 5 open reading frames (ORFs).

Figure 10.

Phylogenetic analysis of the mealworm densovirus (TmDNV) whole-genome sequence in relation to other insect densoviruses. The numbers at the nodes represent bootstrap confidence values for 1000 replicates based on maximum likelihood analysis.