Fig. 1. Amino acid alignment of the eight AaegILPs separated into putative signal, B, C, and A peptides. Identical amino acids are highlighted in black and similar amino acids in grey. Introns are indicated with a triangle (). Both AaegILP6 splice variants are represented by the split amino acid sequence at the C-terminus. The dibasic processing sites flanking the C peptide are underlined. GenBank accession numbers—AaegILP1: DQ845750, AaegILP2: DQ845752, AaegILP3: DQ845751, AaegILP4: DQ845753, AaegILP5: DQ845758, AaegILP6a: DQ845755, AaegILP6b: DQ845756, AaegILP7: DQ845757, and AaegILP8: DQ845754.

Fig. 2. Phylogenetic tree of the known dipteran ILPs generated with ClustalW alignment and neighbor-joining tree with bootstrap analysis. Bootstrap values are shown at the base of the branches and represent the percentage of times that grouping was supported. Representatives from the silkworm ILP families were used as an outgroup. The putative mosquito IGFs are highlighted with a light grey circle. ILPs in the putative ILP operons in Ae. aegypti and An. gambiae are highlighted by dark and light grey boxes, respectively. Aaeg = Aedes aegypti, Ag = Anopheles gambiae, Asub = Aedes subalbatus, DILP = Drosophila melanogaster, Bmor = Bombyx mori.

Fig. 3. Transcript expression of AaegILP genes in life stages of Ae. aegypti. (A) Total RNA extracted from eggs (E) and the heads (H), thoraces (T) and abdomina (A) of early fourth instar larvae (4L), early pupae (P), males (M), and females (F; 3–5 days old, non-blood fed) was used to make cDNA that then served as template for RT-PCR. (B) Total RNA extracted from heads (H), anterior midguts (AM), posterior midguts (PM), thorax walls without midgut (TW), abdomen walls without midgut (AW), and ovaries (O) of non-blood fed females (3–5 days old) was used to make cDNA that then served as template for RT-PCR. For (A) and (B), three different cohorts of females were separately processed and used for RT-PCR. Below each gel photo of the AaegILP PCR products (expected size without intron—right) is the summary of RT-PCR for the three cohorts: +, transcripts present in majority of body region/life stage samples or −, transcripts absent in majority of samples. NT—control PCR with primers and no template. No genomic DNA contamination was evident when the above total RNA was used as template for PCR with ILP primers. Also, the above cDNAs were checked for integrity by PCR amplification of actin transcripts.

Fig. 4. Whole tissue immunocytochemistry with bombyxin II and locust ILP antibodies in immature and male Ae. aegypti. (A) Bilateral clusters of neurosecretory cells (arrows) immunostained with BII MAb in the brain of a fourth instar larvae (dorsal, posterior brain region, top). (B) Axons (arrows) immunostained with BII MAb pass through the gastric caecae (anterior, right) and along the midgut of a fourth instar larvae. (C) Endocrine cells (arrows) immunostained with LIRP Ab in the midgut of a mid-stage pupa. (D) Lateral neurosecretory cells (arrows) immunostained with LIRP Ab in a male brain (dorsal, top).

Fig. 5. Whole tissue immunocytochemistry with bombyxin II and locust ILP antibodies in adult female Ae. aegypti. (A) Medial neurosecretory cells (MNCs) and their axons in a female brain (dorsal, top) were immunostained with BII MAb. The axons (arrows) extend from the cell bodies, cross-over, exit the brain in the nervi corporis, and pass into the corpus cardiacum. (B) Axons (axons) from the MNCs immunostained with BII MAb branching along the anterior midgut of a female (anterior, top). Cardiac valve—CV. (C and D) Endocrine cells (arrows) immunostained with LIRP Ab in the cardiac valve (CV) and anterior midgut of a female. (E) Lateral neurosecretory cells (arrows) immunostained with LIRP Ab in a female brain; other less stained neurosecretory cells in the medial region are present (anterior, top). (F) Pair of cells (arrows) in the terminal abdominal ganglion (anterior, right) of a female immunostained with BII MAb. (G) Double immunostaining of the MNC with BII MAb (star; yellow-green) and the LNC with LIRP Ab (arrows; red) in a female brain. (H) Double immunostaining of axons with BII MAb (star; yellow-green) and endocrine cells with LIRP Ab (arrows; red) in the anterior midgut of a female. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 6. Composite distribution of immunoreactive cells and axons in the central nervous system and midgut (drawn to scale) in female Ae. aegypti to bombyxin II (A) and locust ILP (B) antibodies.

Primers used for AaegILP RT-PCR expression studies

Primers were designed to amplify a product which spanned an intron to differentiate transcript expression from genomic DNA contamination.

Changes in the pattern of insulin-like immunostaining in the nervous system and midgut of mosquitoes at different times/stages during metamorphosis

+ = majority of tissues with stained cells or axons;  = half or fewer tissues with stained cells or axons; 0 = no stained cells or axons. F, female; M, male; SNS, stomatogastric nervous system; ^*number of tissues with stained cells or axons/total number of tissues processed and observed.