Deformed Wing Virus- Pathogenesis

The Deformed Wing Virus (DWV) is the most serious pathogenic virus affecting the health and sustainability of managed honey bees. The virus, which likely has been on Earth for tens of millions of years [1], appears in many arthropods without significant impact on their populations. However, the varroa destructor mite has become the ideal vector for the virus. Thus, following the global spread of varroa, the DWV became virulent and associated with the collapse of honeybee populations, especially in places where there is no resistance to the mite. The mite plays a crucial role in the spread of the virus, as well as in its evolutionary path [2]. Information on the relationship between varroa and viral diseases was provided in a previous article. This article will discuss the pathogenesis of the Deformed Wing Virus with the help of observations made under the dissecting microscope [3]. The term pathogenesis refers to the mechanism of disease development and describes its appearance, persistence, exacerbation, etc.

Note: The article contains images of insect dissections, which may be unpleasant. Reader discretion is advised.

Observations
The obvious symptom of the disease caused by the virus is, as its name suggests, the deformed wings. Examination under the dissecting microscope reveals a series of additional symptoms not visible to the naked eye. The wing deformity has gradations related to the viral load, as well as to how this load is synchronized with the developmental stages of the pupa. A five-stage scale of deformity has been proposed to describe the severity of the disease [4],[5]. The virus attacks the bee during its development, while it is inside its cell. The mature insect is also affected by the virus but without physical deformities [15].

Picture 1: High grade deformities (left) and lower grade deformities (right). In the lower grade one pair of wings is intact, while in the high grade, all the wings appear thread-like. The virus tends to affect the forewings more than the hindwings. While initially the deformed wings are soft, the deformation later becomes permanent due to the hardening of the wing with chitin. Due to the preservation method the sample on the right has darkened and discoloured because of oxidation.

The possible process deformed wings formation is as follows. Prior to the final molting stage, each pair of wings is folded inside the limb. Normally, the wings are being formed while folded, and later during the final molt, the wings are filling with hemolymph. During the wing formation, the epidermal tissues change and compose the basic structure of the wing, forming the characteristic wing venation. When the venation finally fills with hemolymph, the wing unfolds [6]. The “mechanism” that causes deformation may be atrophy or/and incomplete venation, resulting in the wing not properly unfolding. The wing hardening that occurs slightly later fixes the deformation [16].

Another characteristic of symptomatic bees is their lighter color compared to normal and asymptomatic insects. This is due to the slowing down of the melanization process, that is, the deposition of melanin on the exoskeleton. Melanins are dark pigmented substances deposited in areas around wounds or where pathogenic organisms are found. They are also the natural pigments of the exoskeleton. Eumelanin is responsible for the orange hues, while pheomelanin is responsible for the darker colors [7]. Melanins are produced through the processes of melanogenesis. the amino acid tyrosine is converted into the polymers of melanin through enzymes and biochemical reactions.

Picture 2: Certain translucent areas on the exoskeleton can be distinguished (e.g. hind legs and sternites), indicating a low rate of melanization (i.e. accumulation and oxidation of melanins), or a low rate of melanogenesis (production of melanins) and sclerotization (production of chitin). Although the bee had emerged from the cell several hours ago, its exoskeleton could not darken enough. The sample appears darker because the examination is carried out by immersion in water (hence the bubbles), while it is a dark-colored honeybee ecotype.

The slower melanization of the exoskeleton during the virus disease is visually apparent compared to the controls and has also been reported in other organs [8], [9]. The virus seems to slow down these processes in the pupae’s body and cause immunosuppression, but not to an absolute degree. Inside honeybees with DWV virus, white nodules were observed, characteristic of stressed bees and pupae when they have an immune response (picture 3). As mentioned earlier, tyrosine is the precursor compound of melanins, which ultimately do not form sufficiently in symptomatic bees. It is noted that these nodules remain white and do not darken.

Something else observed in bees infected by the virus is the immature muscles. The muscle fibers of the thorax have a bright white color and resemble the muscles of a pupae. This explains why bees with DWV are lethargic and of smaller size (abdomen is less extended). The occasional incomplete myogenesis in symptomatic bees and pupae is described in the literature [9].

Picture 3: Dorsal part of the abdomen. The aorta is visible (appearing as a channel in the center), as well as the tracheols (white), some muscle fibers, and the “immature” fatty bodies. The scattered white granules (nodules) are formed by the bee’s immune response to the virus. They likely consist of the amino acid Tyrosine [10]. Due to low level of melanization, these tissues have cold and gray color tones, despite the warm lighting (halogen lamp).

Picture 5: Additional developmental deformation was found aside from the wings: a malformed right antenna. Due to the delay and hindrance of the developmental processes, it may have been affected either at one of the stages of the final molting [11], or at the post-ecdysis stage of sclerotization and melanization of the exoskeleton.

Picture 5: Protruding proboscis. Bees with DWV have been observed to have their proboscis projected (also reported here [5]). This is a secondary phenomenon with a simple explanation. The bee’s proboscis is held in by groups of muscles. Otherwise, the tongue and proboscis would unfold. The muscles of the bee that is sick from the virus are so weak and immature that the bee cannot completely hold its tongue folded. The sticking out tongue in symptomatic bees is less than shown here.

Seasonality and Virus Types
There are two prevalent types of the virus in the world: the older type A and the newer type B. A study from 2022 suggests that the type B virus is dominant in Europe [12]. However, the virus type does not have significant beekeeping importance, as the differences are small and practical management of DWV is the same.

In the case of the DWV strain A, the viral load in the colony is likely increased in each cycle of varroa feeding [13] and with abrupt feedback. The virus appears dynamically as long as the varroa is fed by bees with high virus loads. In type B DWV, the varroa mite itself has been infected by the virus. The appearance of deformed bees may be more frequent, but not as massive as in type A. A gene expression study [16] found that certain immune responses of bees to both types A and B DWV are the same, which reinforces that not much changes in everyday practice.

In Mediterranean and dry ecosystems, the symptomatic bees make their appearance at the end of spring, disappear in summer, and reappear in autumn. This is primarily due to the significant reduction in colony brood during the dry months. The higher average temperature of the brood nest may also be inhibitory to the virus. In colder climates, on the other hand, the symptomatic manifestation of the virus is more continuous.

Timing
It has been found that subclinical levels of Deformed Wing Virus are detectable throughout the year, even when the Varroa load of the colony is very small [14]. The continuous presence of the virus, however, is not a major burden on the colony, but only when varroa mite populations are present and the two pathogens display synergy. The conditions for the virus to cause extensive damage are the presence of the varroa mite and the synchronization of its multiplication with specific developmental stages of the bee. This is evident when noting how rare the symptomatic drones and queens are in relation to the workers.

Picture 6: Drone with symptoms. The occurrence of the virus in a drone or a queen bee is rare, and when it happens, the deformities are milder. The reason is that there is no synchronization (timing) of the peak virus population with the wing formation time. The chronic effect of the varroa has favored virus strains that are synchronized with the ontogenesis of the worker and not the drone.

Treating DWV
Finally, I will briefly refer to the therapeutic management of DWV virus. In general, the occurrence of the virus has significant impacts on hney production. There is no direct treatment for the virus, and the proposed actions are usually indirect, through varroa management. However, this approach takes a long time to reduce varroa populations, allowing the virus to persist for many weeks. An ideal treatment for the virus would involve interrupting the brood cycle (special queen cage or splits) for approximately 15 days, along with varroa treatment. Interrupting the brood cycle shows that it breaks the virus’s feedback loop (likely regardless of whether the virus is type A or B). However, because interrupting the brood cycle is a time-consuming process (requires spotting the queen), I prefer a different method with three main points:

1. Reducing high varroa loads with treatment.
2. Help of thermal homeostasis and spatial confinement of the colony: removal of extra frames, addition of plywood board, or vertical foam insulation inside the hive.
3. Prolonged feeding with small quantities of diluted syrup. Addition of an essential oil formulation.

The recipe and manipulations will be described in a future article. Also, the temporary withdrawal of the DWV infested hives from production is a key action for a rapid recovery. In the presence of deformed wing virus, treatments for varroa that disrupt the the colony homeostasis should be avoided (e.g. high dose of formic acid, oxalic acid-glycerin strips, oxalic acid dribble, etc.). These treatments may cause temporary disorganization and ultimately lead to colony collapse.

George Mitsikas (Amateur Beekeeper, Chemist)

August 25, 2024

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REFERENCES

1. Vršanský, P., Vršanská, L., Beňo, M., Bao, T., Lei, X. J., Ren, X. J., … & Jarzembowski, E. (2019). Pathogenic DWV infection symptoms in a Cretaceous cockroach. Palaeontogr Abt A, 314, 1-10.
2. Optimizes the virulence and synchronization of the virus. High viral loads enhance the phenomenon of “evolutionary bottleneck” for infections. See: Woodford L, Evans DJ. Deformed wing virus: using reverse genetics to tackle unanswered questions about the most important viral pathogen of honey bees. FEMS Microbiol Rev. 2021 Aug 17;45(4):fuaa070. doi: 10.1093/femsre/fuaa070. PMID: 33320949.
3. The samples were honeybees sick with the DWV virus, which were collected in front of beehives. The bees were euthanized in a special way so that death occurred immediately and their suffering was minimized.
4. The scale was designed to describe DWV expression in queens, not in workers which express more deformities: Williams, G. R., Rogers, R. E., Kalkstein, A. L., Taylor, B. A., Shutler, D., & Ostiguy, N. (2009). Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen. Journal of invertebrate pathology, 101(1), 77-79.
5. Koziy, R. V., Wood, S. C., Kozii, I. V., van Rensburg, C. J., Moshynskyy, I., Dvylyuk, I., & Simko, E. (2019). Deformed wing virus infection in honey bees (Apis mellifera L.). Veterinary Pathology, 56(4), 636-641.
6. Comstock, J. H., & Needham, J. G. (1898). The wings of insects. The American Naturalist, 32, 33
7. The study was on bumblebees, however, they belong to the same family with the honeybees (corbiculates). Polidori, C., Jorge, A., & Ornosa, C. (2017). Eumelanin and pheomelanin are predominant pigments in bumblebee (Apidae: Bombus) pubescence. PeerJ, 5, e3300.
8. Di Prisco, G., Annoscia, D., Margiotta, M., Ferrara, R., Varricchio, P., Zanni, V., … & Pennacchio, F. (2016). A mutualistic symbiosis between a parasitic mite and a pathogenic virus undermines honey bee immunity and health. Proceedings of the National Academy of Sciences, 113(12), 3203-3208.
9. Power, K., Martano, M., Altamura, G., Piscopo, N., & Maiolino, P. (2021). Histopathological features of symptomatic and asymptomatic honeybees naturally infected by deformed wing virus. Pathogens, 10(7), 874.
10. Erickson, E., Cohen, A., Brummett, D., Lusby, W., & Cameron, B. (1997). Tyrosine nodules in the gasters of adult honeybees. Journal of Invertebrate Pathology, 70(1), 27-32.
11. Molting includes 8 stages.
12. Paxton, R. J., Schäfer, M. O., Nazzi, F., Zanni, V., Annoscia, D., Marroni, F., … & Shafiey, H. (2022). Epidemiology of a major honey bee pathogen, deformed wing virus: potential worldwide replacement of genotype A by genotype B. International Journal for Parasitology: Parasites and Wildlife, 18, 157-171.
13. Posada-Florez, F., Childers, A. K., Heerman, M. C., Egekwu, N. I., Cook, S. C., Chen, Y., … & Ryabov, E. V. (2019). Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner. Scientific reports, 9(1), 12445.
14. Locke, B., Semberg, E., Forsgren, E., & De Miranda, J. R. (2017). Persistence of subclinical deformed wing virus infections in honeybees following Varroa mite removal and a bee population turnover. PloS one, 12(7), e0180910.
15. Although the insect does not have deformities etc, its immune system works harder, so the insect expends additional energy. Also, some impact on behavior (ex. higher colony drift) is expected.
16. Norton, A. M., Buchmann, G., Ashe, A., Watson, O. T., Beekman, M., & Remnant, E. J. (2023). Deformed wing virus genotypes A and B do not elicit immunologically different responses in naïve honey bee hosts. Insect Molecular Biology.
17. There are rare occurrences of badly unfolded wings, which does not mean a DWV symptomatic bee. DWV symptoms produce a more ‘cloudy’ wing.