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Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

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The characterization of the Phlebotomus papatasi transcriptome.

J Abrudan¹, M Ramalho-Ortigão, S O'Neil

¹Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, USA.

Insect Molecular Biology

|February 13, 2013

Summary

Phlebotomine sand flies transmit diseases like leishmaniasis. This study presents the first global transcriptome of Phlebotomus papatasi, revealing novel genes involved in digestion and immunity, crucial for understanding disease transmission.

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Area of Science:

* Entomology
* Molecular Biology
* Parasitology

Background:

* Phlebotomine sand flies are critical vectors for numerous human diseases globally.
* Leishmaniases, transmitted by sand flies, cause substantial mortality and morbidity.
* Understanding sand fly biology is key to controlling disease transmission.

Purpose of the Study:

* To perform the first global transcriptome analysis of *Phlebotomus papatasi*, an Old World vector of cutaneous leishmaniasis.
* To compare the *P. papatasi* transcriptome with that of *Lutzomyia longipalpis*, a New World vector of visceral leishmaniasis.
* To identify genes involved in key physiological processes relevant to sand fly vector competence.

Main Methods:

* Construction of a normalized cDNA library from *Phlebotomus papatasi* across various life stages and feeding conditions (sugar, blood, infected blood).
* High-throughput sequencing, data cleaning, and assembly of unique transcripts.
* Gene Ontology (GO) classification and in-depth analysis of identified sequences.

Main Results:

* Generation and assembly of 17,120 unique transcripts from *P. papatasi*.
* 50% of sequences were classified using GO terms, indicating a comprehensive dataset.
* Identification of 245 sequences with homology to proteins involved in blood/sugar digestion, immune response, and peritrophic matrix formation.
* Discovery of 12 novel genes (trypsins, peptidoglycan recognition proteins (PGRP), chymotrypsins) with higher expression in larval stages.
* Identification of two novel chymotrypsins and one novel PGRP with high expression after blood feeding.

Conclusions:

* This study provides a comprehensive transcriptome resource for *Phlebotomus papatasi*, significantly enhancing genomic information.
* Identified genes offer insights into the physiological adaptations of sand flies for vector competence.
* Findings are crucial for future genome annotation and for developing strategies to control leishmaniasis transmission.