The Transcriptome of Lutzomyia longipalpis (Diptera: Psychodidae) Male Reproductive Organs

BACKGROUND: It has been suggested that genes involved in the reproductive biology of insect disease vectors are potential targets for future alternative methods of control. Little is known about the molecular biology of reproduction in phlebotomine sand flies and there is no information available concerning genes that are expressed in male reproductive organs of Lutzomyia longipalpis, the main vector of American visceral leishmaniasis and a species complex. METHODS/PRINCIPAL FINDINGS: We generated 2678 high quality ESTs ("Expressed Sequence Tags") of L. longipalpis male reproductive organs that were grouped in 1391 non-redundant sequences (1136 singlets and 255 clusters). BLAST analysis revealed that only 57% of these sequences share similarity with a L. longipalpis female EST database. Although no more than 36% of the non-redundant sequences showed similarity to protein sequences deposited in databases, more than half of them presented the best-match hits with mosquito genes. Gene ontology analysis identified subsets of genes involved in biological processes such as protein biosynthesis and DNA replication, which are probably associated with spermatogenesis. A number of non-redundant sequences were also identified as putative male reproductive gland proteins (mRGPs), also known as male accessory gland protein genes (Acps). CONCLUSIONS: The transcriptome analysis of L. longipalpis male reproductive organs is one step further in the study of the molecular basis of the reproductive biology of this important species complex. It has allowed the identification of genes potentially involved in spermatogenesis as well as putative mRGPs sequences, which have been studied in many insect species because of their effects on female post-mating behavior and physiology and their potential role in sexual selection and speciation. These data open a number of new avenues for further research in the molecular and evolutionary reproductive biology of sand flies

Classification of ESTs in Gene Ontology category.

<p>The ESTs of <i>L. longipalpis</i> were submitted to a search against the three categories of Gene Ontology (NCBI). The e-value cutoff was 1.0e-5.</p

Thioredoxin sequence analysis.

<p>(A) Neighbor-joining tree of putative thioredoxin <i>L. longipalpis</i> (RAAPBAR020D12 male reproductive organs cDNA libraries), <i>A. aegypti</i> (AAEL010777), <i>A. gambiae</i> (AGAP009584-PA) and <i>Tribolium castaneum</i> (XM_962894.2). Bootstrap percentage values indicated in nodes are based on 1000 replicates. (B) Multiple alignment of putative thioredoxin of male reproductive tracts from <i>L. longipalpis</i> and its orthologues in Diptera. Conserved amino acids are indicated by (*).</p

Putative <i>L. longipalpis</i> mRGPs.

<p><b>N-</b> Number of reads. <b>DB</b>- Database. PTN- Protein. COEBE4D- carboxylesterase, beta esterase. Crisp- Cysteine-rich secreted proteins.</p>*<p>ESTs that have yielded best matches to mRGPs/Acps from protein databases (three against <i>A. aegypti</i> and two against <i>A. gambiae</i>). AGAP00 Sequences come from AgamP3.6_vectorbase and AAEL0 Sequences come from AaegL1.2_vetorbase.</p

Cyclophilin sequence analysis.

<p>(A) Neighbor-joining tree of putative cyclophilin <i>L. longipalpis</i> (RAAPBAR022E08/AM092289, male reproductive organs and whole female cDNA libraries), <i>A. gambiae</i> (AGAP007088-PA), <i>A. aegypti</i> (AAEL013279), <i>D. melanogaster</i> (FBpp0071844/CG2852) and <i>A. mellifera</i> (NP_001229473). Bootstrap percentage values indicated in nodes are based on 1000 replicates. (B) Multiple alignment of putative cyclophilin of male reproductive tracts from <i>L. longipalpis</i> and its orthologues in Diptera. Conserved amino acids are indicated by (*).</p

β-defensin sequence analysis.

<p>(A) Neighbor-joining tree of putative β-defensins: <i>L. longipalpis</i> 1 (BAR005E03/AM091821, male reproductive organs and whole female cDNA libraries), <i>L. longipalpis</i> 2 (EU124626, midgut female library) and <i>L. longipalpis</i> 3 (EX211140, midgut female library), <i>A. aegypti</i> (AEL009861), <i>A. gambiae</i> (AGAP007049), <i>D. melanogaster</i> (CG10433), and <i>B. mori</i> (NP_001106745). Bootstrap percentage values indicated in nodes are based on 1000 replicates. (B) Multiple alignment of putative β-defensin of male reproductive tracts from <i>L. longipalpis</i> and its orthologues in Diptera. Conserved amino acids are indicated by (*).</p

ESTs with other specific function.

<p><b>N-</b> Number of reads. OBP-Odorant Binding Protein.</p

Summary of L. longipalpis cDNA library sequencing results.

<p>bp- base pair.</p

Protease inhibitor sequence analysis.

<p>(A) Neighbor-joining tree of putative protease inhibitor <i>L. longipalpis</i> (RAAPBAR023H02/EW989852 B male reproductive organs and midgut female cDNA library), <i>A. aegypti</i> (AAEL000551), <i>A. gambiae</i> (AGAP011319), and Apis mellifera (XP_003250953). Bootstrap percentage values indicated in nodes are based on 1000 replicates. (B) Multiple alignment of putative protease inhibitor of male reproductive tracts from <i>L. longipalpis</i> and its orthologues in Diptera. Conserved amino acids are indicated by (*).</p

Astacin metalloprotease sequence analysis.

<p>(A) Neighbor-joining tree of putative astacin from <i>L. longipalpis</i> (RAAPBAR022F08 male reproductive organs cDNA libraries), <i>L. longipalpis</i> 2 (AM088883 whole female cDNA libraries) and <i>L. longipalpis</i> 3 (Lulo-Astacin A8CW49_LUTLO, midgut female library) <i>A. aegypti</i> (AAEL013449), <i>A. gambiae</i> (AGAP010764), <i>D. melanogaster</i> (FBpp0080341/CG15254) and <i>Nasonia vitripenis</i> (NV12552). Bootstrap percentage values indicated in nodes are based on 1000 replicates. (B) Multiple alignment of putative astacin of male reproductive tracts from <i>L. longipalpis</i> and its orthologues in Diptera. Conserved amino acids are indicated by (*).</p