Adipokinetic hormone signaling mediates the fecundity of Diaphorina citri infected by ‘Candidatus Liberibacter asiaticus’

Reviewer #1 (Public Review):

Summary:
The manuscript by Jiayun Li and colleagues aims to provide insight into adipokinetic hormone signaling that mediates the fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Using FISH, qRT-PCR, WB, RNAi, and miRNA-related methods, authors found that knockdown of DcAKH and DcAKHR not only resulted in triacylglycerol accumulation and a decline of glycogen but also significantly decreased fecundity and CLas titer in ovaries. miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Most of the methods and results are solid and valuable, but I have a number of concerns with this paper, relating to the writing and lack of sufficient information about data analysis.

Reviewer #2 (Public Review):

Diaphorina citri is the primary vector of Candidatus Liberibacter asiaticus (CLas), but the mechanism of how D. citri maintains a balance between lipid metabolism and increased fecundity after infection with CLas remains unknown. In their study, Li et al. presented convincing methodology and data to demonstrate that CLas exploits AKH/AKHR-miR-34-JH signaling to enhance D. citri lipid metabolism and fecundity, while simultaneously promoting CLas replication. These findings are both novel and valuable, not only have theoretical implications for expanding our understanding of the interaction between insect vectors and pathogenic microorganisms but also provide new targets for controlling D. citri and HLB in practical implications. The conclusions of this paper are mostly well supported by data, but some aspects of phrasing and data analysis need to be further clarified and extended.

Key Considerations:

There are specific instances where additional information would enhance comprehension of the results and their interpretation.

There seem to be two inconsistencies related to some results depicted in Figures 1, 2, 3 and 5.

Firstly, Figure 1 shows the effect on CLas infection (CLas+) compared to the control (CLas-), where results show an increase of TAG, Glycogen, lipid droplet size, oviposition period, and fecundity. In Figures 2, 3, and 5, the authors establish the involvement of the genes DcAKH, DcAKHR, and miR34 in this process, by showing that by preventing the function of these three factors the effects of CLas+ are lost. However, while Figure 1 shows the increase of TAG and lipid droplet size in CLas+, Figures 2, 3, and 5 do not show a significant elevation in TAG when comparing CLas- and CLas+.

Secondly, in addition to the absence of statistical difference in TAG and lipid droplet size observed in Figure 1, Figures 2, 3, and 5 show an increase in TAG and lipid droplet size after dsDcAKH (Figure 2), dsDcAKHR (Figure 3) and agomiR34 (Figure 5) treatments. Considering that AKH, AKHR, and miR34 are important factors to CLas-induce increase in TAG and lipid droplet size, one might expect a reduction in TAG and lipid droplet size when CLas+ insects are silenced for these factors, contrary to the observed results.