Tyrosine Hydroxylase is crucial for maintaining pupal tanning and immunity in Anopheles sinensis

Abstract

Tyrosine hydroxylase (TH), the initial enzyme in the melanin pathway, catalyzes tyrosine conversion into Dopa. Although expression and regulation of TH have been shown to affect cuticle pigmentation in insects, no direct functional studies to date have focused on the specific physiological processes involving the enzyme during mosquito development. In the current study, silencing of AsTH during the time period of continuous high expression in Anopheles sinensis pupae led to significant impairment of cuticle tanning and thickness, imposing a severe obstacle to eclosion in adults. Meanwhile, deficiency of melanin in interference individuals led to suppression of melanization, compared to control individuals. Consequently, the ability to defend exogenous microorganisms declined sharply. Accompanying down-regulation of the basal expression of five antimicrobial peptide genes resulted in further significant weakening of immunity. TH homologs as well as the composition of upstream transcription factor binding sites at the pupal stage are highly conserved in the Anopheles genus, implying that the TH-mediated functions are crucial in Anopheles. The collective evidence strongly suggests that TH is essential for Anopheles pupae tanning and immunity and provides a reference for further studies to validate the utility of the key genes involved in the melanization pathway in controlling mosquito development.

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

“…According to our observations of pupae phenotype in different developmental stages, the cuticle tanning pattern is the same in the male and female pupae [12]. The cuticle colour of the female pupae is a little transparent with a slight yellowish tint within 24 h after pupation (Fig.…”

“…2b). Interestingly, a retarded expression pattern of AsLac2 was observed when compared with the upstream, rate-limited gene AsTH [12], indicating that the catalytic function of AsLac2 occurred after the accumulation of catecholamine. Furthermore, we found that the expression intensity of AsLac2 in the pupae (from the early-middle to the last stage) was significantly stronger than that in the adults (Fig.…”

“…The cuticle, fat body, and hemolymph of the pupae were dissected at 32 h after pupation for tissue expression pattern analysis. Gene expression was determined by qRT-PCR as previously described [12]. Each sample was used to perform three biological repeats.…”

“…The tanning degree of the pupae was checked at 38 h after ds Lac2 or ds Red injection, while the checking time for adults was 3 h after the eclosion. Three pupae or adults in the ds Lac2- and ds Red- injected groups at each sampling point were also collected for qRT-PCR analyses [12]. Each sample was used to perform three biological repeats.…”

“…All of the tissue treatments and slice preparations were carried out as previously described [12]. The thickness of each cuticle layer was measured with Image J software (https://imagej.net/Welcome) as previously described [12]. Four equal spaced measurements for each dorsal plate section, and the average value was calculated as the mean thickness in one biological repeat.…”