| Octodonta nipae(Maulik)is an important invasive pest of palm plants in southern China,and the endoparasitoid Tetrastichus brontispae Ferrière shows great potential in biological control of O.nipae pupae.Fat body,serving as major sites for innate immunity and nutrient storage,are main targeted tissues regulated by endoparasitoids.Basement membranes make diverse regulatory contributions to fat body,and also serve as tissue boundary,which covers the entire surface of fat body.Thus,decomposition of basement membrane is a precondition for lysis of host fat body by endoparasitoids.Laminins are major components of basement membranes,comprising of the combination of ?,β and γ chains.Mi RNA has emerged as a critical regulator in insect physiology,and its possible function in the alternation of host physiology during parastism attracts researchers’ attention.Previously,we found that the expression of O.nipae laminin β and γ(Onlamininβ and Onlamininγ)was significantly inhibited after the parasitization by T.brontispae using RNA sequencing.Similarily,the expression of two mi RNAs,Oni-mi R-263-5p and Oni-mi R-305-5p,was markedly affected using mi RNA sequencing.Oni-mi R-263-5p and Oni-mi R-305-5p are targeted to Onlamininβand Onlamininγ,respectively.Therefore,in the present study,the regulation of Onlamininβ and Onlamininγ by T.brontispae was investigated,further,the involvement of Oni-mi R-263-5p and Oni-mi R-305-5p in this process was analyzed.The main results are as follows.(1)The full-length sequences of Onlamininβ and Onlamininγ encoded 1791 and1629 amino acids,respectively.Blast analyses indicated that both Onlamininβ and Onlamininγ possessed the typical characteristics of laminin protein family,containing a short arm on the N-terminal end(Lam NT),rodlike EGF-like tandems(EGFs),and alpha helical coils(LCC).Onlamininβ has 12 EGFs,one less than that in most coleopterous insects,and does not contain laminin β-knob domain(Lβ).Onlamininγhas a special Lam B domain compared with Onlamininβ.The special characteristics of Onlamininβ and Onlamininγ may indicicate their special function.(2)q RT-PCR results showed that Onlamininβ and Onlamininγ were expressed highest in haemocytes.Using indirect immunofluorescence analysis with lamininβ and lamininγ specific antibodies,it was found that Onlamininβ and Onlamininγ were mainly produced in granulocytes.The expression of Onlamininβ and Onlamininγ was significantly inhibited after the parasitization by T.brontispae,especially at 48 h,with a decrease by two folds.(3)RNA interference results showed that O.nipae adults presented a deformed wing after loss-of-function of Onlamininβ.After knockdown of Onlamininγ,melanotic nodules appeared on the surface of pupae,and pupae died with blackened body in severe cases.Silencing of Onlamininβ or Onlamininγ could lead to the lysis of fat body.The above results suggest that Onlamininβ and Onlamininγ play key roles in maintaining the structure and function of fat body.(4)Silencing of dicer-1 resulted in the decreased expression of Onlamininβ and increased expression of Onlamininγ,with the change fold of about 1.5.Dual luciferase experiments showed that Oni-mi R-263-5p and Oni-mi R-305-5p could bind to the 3’-UTR regions of Onlamininβ and Onlamininγ,respectively.Further,it was found that the expression of Oni-mi R-263-5p was inhibited after parasitization,especially at 96 h,with the change fold of about 1.7;Oni-mi R-305-5p expression was increased,especially at 72 h,with the changed fold of 2.8.Theses results indicated that Onlamininβ was positively regulated by Oni-mi R-263-5p,and Onlamininγ was negatively regulated by Oni-mi R-305-5p.In summary,Onlamininβ and Onlamininγ play important roles in maintaining of the structure and functions of fat body.Suppression of Onlamininβ and Onlamininγmay be an important strategy used by T.brontispae for the destruct of basement membrane and the lysis of fat body,and further for the regulation of host physiology.Oni-mi R-263-5p and Oni-mi R-305-5p are involved in this process. |
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