Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania-major-infected sandflies.
BACKGROUND
In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR.


RESULTS
Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major.


CONCLUSIONS
This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by real-time PCR, suggesting that this customized bioinformatic analysis is a powerful and accurate tool useful in analysing expression profiles from different cDNA libraries. Additionally, the findings presented in this work suggest that the Leishmania parasite is modulating key enzymes or proteins in the gut of the sandfly that may be beneficial for its establishment and survival.
Ramalho-Ortigão
Marcelo
M
Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. m_ortigao@nd.edu
Jochim
Ryan C
RC
Anderson
Jennifer M
JM
Lawyer
Phillip G
PG
Pham
Van-My
VM
Kamhawi
Shaden
S
Valenzuela
Jesus G
JG
D000818:Animals; D001769:Blood; D003199:Computer Systems; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D015723:Gene Library; D019476:Insect Proteins / Q000096:biosynthesis* / Q000235:genetics; D007303:Insect Vectors / Q000235:genetics* / Q000469:parasitology / Q000502:physiology; D007413:Intestinal Mucosa / Q000378:metabolism*; D018320:Leishmania major / Q000502:physiology; D009928:Organ Specificity; D010691:Phlebotomus / Q000235:genetics* / Q000469:parasitology / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D019518:Postprandial Period / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013395:Sucrose; D014158:Transcription, Genetic
D018076:DNA, Complementary; D019476:Insect Proteins; D012333:RNA, Messenger; D013395:Sucrose