Mosquito transcriptome changes and filarial worm resistance in Armigeres subalbatus

Abstract

Background: Armigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it rapidly and proficiently kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, the Armigeres-Brugia system serves as a valuable model for studying the resistance mechanisms in mosquito vectors. We have initiated transcriptome profiling studies in Ar. subalbatus to identify molecular components involved in B. malayi refractoriness.

Results: These initial studies assessed the transcriptional response of Ar. subalbatus to B. malayi at 1, 3, 6, 12, 24, 48, and 72 hrs after an infective blood feed. In this investigation, we initiated the first holistic study conducted on the anti-filarial worm immune response in order to effectively explore the functional roles of immune-response genes following a natural exposure to the parasite. Studies assessing the transcriptional response revealed the involvement of unknown and conserved unknowns, cytoskeletal and structural components, and stress and immune responsive factors. The data show that the anti-filarial worm immune response by Ar. subalbatus to be a highly complex, tissue-specific process involving varied effector responses working in concert with blood cell-mediated melanization.

Conclusion: This initial study provides a foundation and direction for future studies, which will more fully dissect the nature of the anti-filarial worm immune response in this mosquito-parasite system. The study also argues for continued studies with RNA generated from both hemocytes and whole bodies to fully expound the nature of the anti-filarial worm immune response.

Figure 1

Functional composition of transcripts significantly affected by parasite challenge based on abundant and immunity-related EST clusters observed from Ar. subalbatus cDNA libraries. Transcripts with a detectable increase in abundance (top) and with a detectable decrease in abundance (bottom) 1, 3, 6, 12, 24, 48, and 72 hours after exposure to a B. malayi infective blood meal.

Figure 2

Further functional dissection of immunity-related transcripts significantly affected by parasite challenge at 1, 3, 6, and 12 hours post infection. Graphical representation of the percentage of immunity-related transcripts with a detectable increase or decrease in abundance (y-axis) at a given time (x-axis).

Figure 3

12 K-means Cluster Analysis of features changing in response to B. malayi infection (y-axis, log-scale) over time (x-axis) colored by significance (red = more significant; green = less significant). K-means clustering was used to group genes with similar transcriptional patterns. Cluster six (numbered) contains 517 features, including 26 features with known immune activity, e.g., AMPs, pattern recognition molecules, serine proteases, etc. Clusters were constructed so that the average behavior in each group was distinct from any other group. This serves as a means to implicate unknowns in processes based on temporal transcriptional behavior, and based on semblance to other transcripts/pathways that maintain a similar profile.

Figure 4

The number of transcripts with unknown function found within cluster six. Cluster six contained 517 features, 303 of which were unknowns or conserved unknowns. Of these 303 transcripts, 41 of them showed significantly different transcriptional behavior over the time course (at a 95% confidence interval over two-fold values). By narrowing down the number of candidates selected for further study, it becomes a very tractable problem to elucidate the probable function of a number of these unknown features. And, this can be accomplished by the mining of these unknown features based on similarities in transcriptional patterns with those of known genes.

Figure 5

Shared transcripts between hemocyte vs. whole body RNA sources. Four transcripts with a significant detectable increase or decrease in abundance were shared between hemocyte RNA and whole Body RNA. 107 unique transcripts were attributed to hybridizations done with a hemocyte RNA source. 115 unique transcripts were attributed to hybridizations done with a whole body RNA source. Of the 4 shared transcripts, 1 is unknown [GenBank:EU206282], 2 are conserved unknowns [GenBank:EU205740 and GenBank:EU207864], and 1 is a protein kinase [GenBank:EU209290].

Figure 6

Functional composition of genes significantly affected by parasite challenge based on abundant and immunity-related EST clusters observed from Ar. subalbatus cDNA libraries. Transcripts showing significantly different behavior when array hybridizations were done with RNA generated from whole bodies (top) and hemocytes (bottom) 24 hours following parasite challenge.