Fig 1.
Isolation of metacyclic promastigotes from the sand fly gut.
(A) Promastigote stages during development within the sand fly gut. Adapted from [14]. (B) Location of metacyclic promastigotes in the anterior pole of the PSG-promastigote plug in contact with the SV. Reproduced from [7]. (C) In vitro infectivity of sand fly–derived L. infantum metacyclic promastigotes (LiPro-Pper) compared with metacyclic promastigotes from culture (LiPro-PNA−) in the human cell line U937. Reproduced from [25]. LiPro-PNA−, L. infantum metacyclic promastigotes from culture obtained by the peanut agglutinin negative selection method; LiPro-Pper, L. infantum metacyclic promastigotes from the P. perniciosus stomodeal valve; PNA, peanut agglutinin; PSG, promastigote secretory gel; SV, stomodeal valve.
Fig 2.
Strategies for DGE analysis of sand fly–derived promastigotes.
Only transcriptomics strategies are feasible to date for DGE analysis for very-low-input samples such as sand fly–derived promastigotes. In slRNA-seq strategies, the SL sequence is used in second-strand cDNA synthesis (#), thus increasing specificity when analyzing samples containing genetic material from the host. A cross-hybridization control should be included in microarray experiments to avoid biased results due to noise of the host genetic material. The RNA-seq strategies allow for multiplexed analysis by including indexing sequences during PCR amplification (†). Mapping to genome and alignment to transcript annotations is required during microarray hybridization experiments only when the DNA probes spotted on the slides have not been identified before the experiment (*). An example is shotgun genome DNA microarrays, in which only the clones of interest containing DEGs are sequenced and aligned to identify those genes [24]. aRNA, amplified RNA; Cy3, cyanine 3; Cy5, cyanine 5; DEG, differentially expressed gene; DGE, differential gene expression; IVT, in vitro transcription; NGS, Next Generation Sequencing; RNA-seq, RNA sequencing; SL, spliced leader sequence; slRNA-seq, spliced-leader RNA sequencing.
Table 1.
Transcriptome studies and sample abbreviations.
Table 2.
Functional genomics in sand fly–derived promastigotes: Main findings.