1. Andres Sandoval-Mojica and Michael Scharf
Peritrophins of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae): An approach
Andres Sandoval-Mojica and Michael Scharf
Entomology and Nematology Department, University of Florida. PO Box , Gainesville, Florida
Background
Approaches
Objective 1.
The peritrophic matrix (PM) is an acellular semipermeable membrane that lines the midgut of most orders within the class Insecta. The PM is conformed of proteins, glycoproteins and chitin microfibrils and can occur in two forms (1):
Type I PM: Concentric lamellae are delaminated from the entire midgut epithelium. It could be produced only in response to feeding and the type of meal ingested.
Type II PM: Produced by the cardia, an specialized region of the anterior midgut. One to three laminated layers are secreted, forming a continuous sleeve that is constantly present.
The functions of the PM confer to this matrix a vital role in the intestinal biology of insects (1):
Protection of the midgut epithelium from mechanical and/or biochemical damage, caused by abrasive food particles and/or digestive enzymes.
Insulation of the midgut epithelium from pathogens and toxins.
Compartmentalization of the digestive process, improving nutrient acquisition and reprocess of hydrolytic enzymes.
Because of its close involvement in digestive processes and its role as a barrier against pathogens an toxins, the PM is an attractive target for pest management strategies.
Proteins are main components of PMs and chitin-binding proteins represent the majority of PM polypeptides. PM’s chitin-binding proteins are collectively known as peritrophins and are characterized by the presence of one to 19 Peritrophin-A domains. This motif belongs to the CBM14 family of carbohydrate binding domains, also known as the type 2 chitin-binding domain (ChtBD2). However, this domain is present in proteins that are found in locations other than the PM (2, 3).
CX13-30CX5-6CX9-19CX10-14CX4-14C
The goal of this study was to identify genes that encode peritrophins in the lower termite Reticulitermes flavipes, and reveal their gut expression profile as well as their responsiveness to starvation. This information will be essential in order to select the appropriate methodology for disrupting the formation of the PM in this species of termite.
Identify and characterize genes encoding proteins with the type 2 chitin–binding domain (ChtBD2) in R. flavipes.
Compare the relative expression of peritrophins genes among the gut sections of R. flavipes.
Evaluate the effect of starvation and feeding in the relative expression of the peritrophins genes.
Figure 2. Amino acid sequence alignment of a ChtBD2 of the peritrophic matrix proteins in R. flavipes. (PI: Rf Peri-I, PIII: Rf Peri-III, PIV: Rf Peri-IV).
The relative expression of the three genes was compared between the three sections of the R. flavipes gut (Figure 3). The expression in the Mid gut for Rf Peri-I and Rf Peri-IV was highly superior than in the other two gut sections. Rf Peri-III, although considerable expressed in the Mid gut, was expressed in higher proportion in the Fore gut. The results support the hypothesis of these genes being part of the lower termite peritrophic matrix.
Objective 2.
Fore gut
+ s.g
R. flavipes:
25 workers/colony
4 colonies (K5, K9, B2, W1)
Total RNA
extraction A B
Gut dissections
Mid gut
Hind gut
qRT-PCR:
Ct values
Relative mRNA expression
2-ΔΔCt method (5)
Reference gene: NADH-dh
cDNA synthesis
Figure 3. A: Diagram of the termite gut (E: esophagus; SG: salivary glands; FG: fore gut; MG: mid gut; MT :Malpighian tubules; HG: hind gut; R: rectum).
B: Mean fold Change in the expression of Rf Peri-I, Rf Peri-III and Rf Peri-IV across the gut of R. flavipes. The (*) represents the highest fold change in boxes of same color.
Objective 3.
Duration
cDNA synthesis
3 replicates
The three peritrophin genes were not responsive to starvation (Fig 4) during a 15 day bioassay. There were no difference in relative expression level of Rf Peri-I (p= 0.67), Rf Peri-III (p=0.30) or Rf Peri-IV (p= 0.75) between starved or fed termites. Results suggest a Type II PM for R. flavipes.
Figure 1. ChtBD2 present in peritrophins.
Results
Three genes were sequenced from a normalized R. flavipes gut cDNA library (4). The genes are referred to as Rf (R. flavipes) Peri (peritrophin) genes I, III and IV (Table 1). Each of the genes presented one or more sequences containing six cysteines, the characteristic motif of a protein that attaches to chitin (Figure 2).
Objectives
Table 1. R. flavipes peritrophins genes.
Figure 4. Expression of peritrophin genes across 15 days in response to starvation/feeding. Verticals bars denote 0.95 confidence intervals.
References
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Livak, K., Schmittgen, T. (2001). Methods. 25: