Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa

Abstract

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

Keywords: Ecdysteroid hormone; Neuropeptide; Neurosecretory cells; Ovary.

Figure 1

Sequence of HPLC steps for the purification of native AsILP3 from the two aqueous cuts of the extract of An. stephensi adult heads. HPLC fractions with ILP3 immunoreactivity were pooled for each next step and loaded onto the column and subjected to the elution conditions given. Heptafluorobutyric acid (HFBA) or trifluoroacetic acid (TFA) were used as ion pairing agents in the mobile phases, and elution was monitored at 275 or 206 nm, respectively.

Figure 2

Immunocytochemistry with AaILP3 antibody revealed cells in the nervous system of An. stephensi adult females. A. Brain with stained medial neurosecretory cells (arrowhead). B. Ventral nerve cord ganglion (dotted gray outline) with stained cell bodies (arrowhead) and axon processes extending along body wall (arrows). C Anterior midgut with axon processes extending from the MNCs of the brain (arrows). Autofluorescence of sphere-like midgut contents is evident. Scale bars = 100 µm.

Figure 3

Elution of the native AsILP3 as a single peak in HPLC step 4 (top) and corresponding fractions (bottom) with the amount of immunoreactive ILP3-like peptide (gray bars) in an aliquot of 2,000 An. stephensi head equivalents, as estimated by radioimmunoassay.

Figure 4

MALDI-TOF mass spectroscopy readout of native AsILP3 showing the mass of the non-reduced form (top) and resultant B and A chains of the reduced forms (bottom).

Figure 5

Structure of the AsILP3 prepropeptide, the predicted mature processed form of AsILP3, and the reduced B and A chain with masses. Black bars between cysteines represent disulfide bonds.

Figure 6

Native (F 51),synthetic AsILP3 (ILP3), and AsILP4 (ILP4) stimulated ecdysteroid production by ovaries of non-blood fed females representing the three medically important taxa: Anopheles, Culex, and Aedes. Ovaries (two pairs in 60 µl of media) were incubated with doses of native AsILP3 (represented as head equivalents/60 µl) or synthetic AsILP3 and AsILP4 (pmol/60 µl) for 6 h, 27 °C. Secreted ecdysteroids in the collected media were quantified with the ecdysteroid RIA. Bars represent mean ecdysteroid pg ± standard error (SE) for triplicate samples for each of three experiments with different female cohorts for each species (N = 9). Control ovaries were incubated in media only. Results represent significant differences between control and treatments (Dunnett’s multiple comparisons test, *P < 0.05; **P < 0.001; ***P < 0.0001).

Figure 7

Immunoblot detection of native and synthetic AsILP3 and AaILP3. A. Representative blot of synthetic AaILP3 and AsILP3 (0.25–2 µg) separated in a Tris-tricine gel, stransferred to nitrocellulose, treated to reducing conditions, and subjected to chemiluminescent detection. B. Blot of native AsILP3 (F51) detected after treatment of an aliquot (~16,000 head equivalents) as above but transferred to PVDF and not subjected to reduction treatment. C. Representative blot of synthetic AaILP3 (2.5 µg), AaILP4 (5 µg), AaILP8 (5 µg), and AsILP4 (4 µg), along with human insulin (hIns, 0.6–10 µg) processed and detected as described in A. Molecular weight markers in kilodaltons (kDa) are indicated on left side of blots shown in A and C. D. Amino acid alignment of An. stephensi and Ae. aegypti ILPs and human insulin used in blots A and C to highlight amino acid regions identical to the AaILP3 antigen. AsILP3 is most similar and the others are less so, which may explain the lack of antiserum recognition on immunoblots.