AUTHOR=Firmino Alexandre Augusto Pereira , Pinheiro Daniele Heloísa , Moreira-Pinto Clidia Eduarda , Antonino José Dijair , Macedo Leonardo Lima Pepino , Martins-de-Sa Diogo , Arraes Fabrício Barbosa Monteiro , Coelho Roberta Ramos , Fonseca Fernando Campos de Assis , Silva Maria Cristina Mattar , Engler Janice de Almeida , Silva Marília Santos , Lourenço-Tessutti Isabela Tristan , Terra Walter Ribeiro , Grossi-de-Sa Maria Fátima 

TITLE=RNAi-Mediated Suppression of Laccase2 Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis)

JOURNAL=Frontiers in Physiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.591569

DOI=10.3389/fphys.2020.591569

ISSN=1664-042X

ABSTRACT=<p>The cotton boll weevil, <italic>Anthonomus grandis</italic>, is the most economically important pest of cotton in Brazil. Pest management programs focused on <italic>A. grandis</italic> are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, <italic>A. grandis</italic> has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the <italic>Laccase2</italic> (<italic>AgraLac2</italic>) gene in <italic>A. grandis</italic> and its potential as an RNAi target for the control of this insect pest. We found that <italic>AgraLac2</italic> is expressed throughout the development of <italic>A. grandis</italic> with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of <italic>AgraLac2</italic> in <italic>A. grandis</italic> resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with ds<italic>AgraLac2</italic> exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of ds<italic>AgraLac2</italic>-treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that <italic>AgraLac2</italic> is involved in the cuticle tanning process in <italic>A. grandis</italic> and may be a promising target for the development of RNAi-based technologies.</p>