Suppression of Laccase 2 severely impairs cuticle tanning and pathogen resistance during the pupal metamorphosis of Anopheles sinensis (Diptera: Culicidae)

Abstract

BackgroundPhenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear.MethodsThe association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls.ResultsDuring the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage.ConclusionsTo our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2118-4) contains supplementary material, which is available to authorized users.

Functional characterization of the horizontally transferred 4,5‐DOPA extradiol dioxygenase gene in the domestic silkworm, Bombyx mori

“…BmDODA1 is most likely involved in other biological pathways. In insects, dopa, which is an important pigment precursor, is also involved in melanization immune responses (Yu et al, 2011;Du et al, 2017). This finding indicates that functional horizontally transferred BmDODA1 may play a role in pigment biosynthesis or immunization.…”

“…To date, betalain pigments have been thought to exist only in plants and fungi (Celli and Brooks, 2017). In insects, dopa, which is an important pigment precursor, is also involved in melanization immune responses (Yu et al, 2011;Du et al, 2017). BmDODA1 is most likely involved in other biological pathways.…”

Multiple roles for laccase2 in butterfly wing pigmentation, scale development, and cuticle tanning

“…The clearest mutant phenotypes we observed manifested as color and structural defects to cuticular features, including wings, wing scales, the larval head capsule, and larval spines. Previous research shows that melanin pigmentation and cuticularization are often linked through a process called tanning, and can share a common set of genes and gene products (Hopkins & Kramer, 1992;Arakane et al, 2005;Du et al, 2017;Moussian, 2010). The cuticle is made up of several layers which follow an ordered synthesis.…”

“…Stinkbugs , honey bees (Elias-Neto et al, 2010), mosquitos (Du et al, 2017), pine sawyer beetles (Niu et al, 2008), and red flour beetles (Arakane et al, 2005) that experienced laccase2 knockdown were unable to properly develop pigmented cuticle, resulting in soft, demelanized bodies. Drosophila melanogaster affected by laccase2 knockdown fail to melanize and also improperly develop wing cuticle and wing sensory bristles (Riedel et al, 2011).…”

Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendagess

“…We expect that our method also works with other, non-chemosensory genes regardless of whether they encode soluble proteins or membrane receptors, or whether they are lowly or highly expressed. Moreover, since RNAi-based suppression of Lac2 or TH decreases cuticular pigmentation in different coleopteran 18,[26][27][28] and several other non-coleopteran insect species [29][30][31][32][33][34] , our method can be adapted to many other hexapod species enabling investigations beyond transparent embryos 35 or ovaries 36 . Imaging beneath the RNAi-cleared cuticle may further benefit from light sheet microscopy that minimizes fluorophore bleaching and phototoxic effects.…”