Enzymatic assay performed in solution with trypsin synthetic substrate BAρNA.

<p>Horizontal axis indicates insect midgut samples obtained at different hours ABF. Vertical axis indicate enzyme units detected per midgut. <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF. White bars indicate samples obtained from non infected insect midguts. Gray bars indicate samples obtained from infected insect midguts. Samples corresponding to half of one midgut were used in each assay. Statistically significant difference (<i>P</i><0.05) is marked with an asterisk.</p

Alignment of deduced amino acid sequences of <i>L. longipalpis</i> trypsins (Lltryp1 and Lltryp2).

<p>Conserved residues are shown as white letter on black background. Non-conserved residues are shown as black letters on a white background. Black letters on gray background indicate conserved substitutions. The predicted secretory signal peptides for Lltryp1 (residues 2–17) and Lltryp2 (residues 1–19) are indicated by a traced line. Lltryp1 and Lltryp2-specific peptides used for immunization procedures are boxed (residues 89 to 111).</p

Zymography performed with gelatin co-polymerized gel, incubated in different pH defined buffers.

<p><b>4</b> to <b>12</b>: pH of buffers. Samples corresponding to 1/50 of one insect midgut obtained at 12 h ABF were loaded in lanes. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Western blot performed with anti-Lltryp1-peptide antibody.

<p><b>0</b>: non-fed female; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF; <b>B</b>: 2 µL of hamster blood. Samples corresponding to 2 insect midguts were loaded in lanes <b>0</b> to <b>72</b>. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Zymography performed with gelatin co-polymerized gel incubated at pH 8 with samples obtained at different times ABF.

<p>M: males, <b>F</b>: females; <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF; <b>B</b>: 2 µL of hamster blood. Samples corresponding to 1/50 of 1 insect midgut were loaded in lanes <b>M</b> and <b>0</b> to <b>72</b>. A control sample contained 1/50 of 2 µL of hamster blood. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Zymography performed with gelatin co-polymerized gel, incubated at pH 8 with different protease inhibitors, as indicated.

<p>Samples corresponding to 1/50 of 1 insect midgut obtained at 12 h ABF were loaded in the gel. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Zymography performed with gels co-polymerized incubated at pH 8 with different protein substrates.

<p><b>G</b>: gelatin; <b>C</b>: casein; <b>H</b>: hemoglobin; <b>B</b>: BSA. Samples corresponding to 1/50 of 1 insect midgut obtained at 12 h ABF were loaded in lanes <b>G</b> and <b>C</b>, and 1/10 of 1 insect midgut obtained at 12 h ABF in lanes <b>H</b> and <b>B</b>. Molecular mass standard values (kDa) are indicated by numbers and arrows on the left side of the figure.</p

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

<div><p> BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.</p></div