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Conservation of ecdysis-triggering hormone signalling in insects

D. itan^1,*, I. itanová^1,2, I. Spalovská^1,3, P. Taká¹, Y. Park⁴ and M. E. Adams⁴

¹ Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84206 Bratislava, Slovakia
² Institute of Medical Chemistry and Biochemistry, School of Medicine, Comenius University, Sasinkova 2, 81108 Bratislava, Slovakia
³ Department of Zoology, Comenius University, Mlynská dolina B2, 84215 Bratislava, Slovakia
⁴ Departments of Entomology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, CA 92521, USA

View larger version (50K): [in a new window]   Fig. 1. Schematic drawings of the organization and shape of Inka cells detected with the PETH antiserum in representatives of different insect orders.

View larger version (117K): [in a new window]   Fig. 2. Inka cells of primitive aquatic insects stained with the PETH antiserum. A very large number of small Inka cells (stained orange) of the dragonfly Sympetrum was located on the surface of narrow tracheae branching from the major lateral tracheal trunk (A). A lower density of cells was found on the broad longitudinal tracheal trunk (B). (C) Numerous small Inka cells were scattered throughout the tracheal surface of the mayfly Epheorus. (D) Larger single or coupled Inka cells with cytoplasmic processes were present on the narrow tracheae of the stonefly Perla. Scale bar, 200 µm in A and 50 µm in B—D.

View larger version (105K): [in a new window]   Fig. 3. PETH-immunoreactive Inka cells (stained orange/red) in pharate larval stages of hemimetabolous insects. In the cockroach Nauphoeta, tracheae on the surface of gonads contained Inka cells with narrow processes (A), while a different type of numerous single or coupled Inka cells lacking cytoplasmic processes were scattered throughout major broad tracheae (B). In the cricket Acheta, small Inka cells with few cytoplasmic processes were located on narrow tracheae (C), or more abundant larger cells with very prominent branching processes were found on the surface of broad tracheae (D). Numerous Inka cells with thick processes were distributed throughout the surface of narrow tracheae of the bugs Triatoma (E) and Pyrrhocoris (F,G). (H) PETH staining disappeared from all Inka cells of Pyrrhocoris after larval ecdysis. Scale bar, 50 µm in A—F; 100 µm in G,H.

View larger version (88K): [in a new window]   Fig. 4. PETH-immunoreactive Inka cells (stained orange/red) in different developmental stages of Holometabola. Numerous small, simple Inka cells attached to tracheae of pharate larva of the alderfly Sialis (A) and pharate pupa of the antlion Myrmeleon (B). (C,D) Pharate larvae of the water beetles Dytiscus and Laccophilus contained a large number of mostly coupled Inka cells with cytoplasmic processes. (E,F) Small and large types of Inka cells in the mealworm beetle Tenebrio. Small cells were scattered throughout tracheae of pharate adults (E); broad trachea near the abdominal spiracle contained two large cells with processes and two small cells in one-week-old adults (F). (G,H) Epitracheal glands, each containing one immunoreactive Inka cell and two smaller cells in dipteran pharate larvae of the mosquito Aedes (G) and cranefly Tipula (H). Note that Sialis, Myrmeleon and Tenebrio contain many variable Inka cells scattered throughout the tracheae, while only 18 pairs of oval epitracheal glands, each containing one Inka cell, are segmentally distributed along the major trunks of lateral tracheae in Diptera. Scale bar, 50 µm in A—D and G—H; 100 µm in E.

View larger version (187K): [in a new window]   Fig. 5. PETH-immunoreactive Inka cells of holometabolous pharate adults. Only one large Inka cell attached to tracheae near each spiracle was found in (A) the Colorado potato beetle Leptinotarsa (Coleoptera), (B) the sawfly Trichiocampus (Hymenoptera) and (C) the ant Myrmica (Hymenoptera). (D) Tracheae of another hymenopteran, the honey bee Apis, contained a large number of small cells. (E,F) Single Inka cells were attached to longitudinal tracheae near each spiracle in dipterans: the black fly Simulia (E) and the fruit fly Drosophila (F). Scale bar, 50 µm.

View larger version (21K): [in a new window]   Fig. 6. RP-HPLC chromatogram of a Nauphoeta tracheal extract showing four PETH-immunoreactive (PETH-IR) fractions (grey columns).

View larger version (18K): [in a new window]   Fig. 7. RP-HPLC chromatogram of Pyrrhocoris tracheal extract showing two PETH-immunoreactive (PETH-IR) fractions (grey columns).

View larger version (71K): [in a new window]   Fig. 8. Organization and structure of the putative eth gene from Anopheles gambiae. (A) The promoter region contains the putative ecdysone receptor response element (EcRE) followed by the eth open reading frame, interrupted by an intron. The signal peptide (open box) is followed by ETH1 and ETH2 (hatched boxes), separated by GRR-processing sites for dibasic cleavage and amidation. (B) The non-coding nucleotide sequence of Anopheles eth is shown in lower-case letters. The upstream putative pulindromic EcRE (aggtcaattcacct) is bold and underlined, the intron donor and acceptor motifs are underlined and in bold and italic, and the poly-A signal is underlined. Predicted nucleotide sequence of an open reading frame and deduced amino acid sequence are indicated by upper-case letters. Putative active peptides ETH1 and ETH2 are boxed, and amidation and processing signals (GRR) are underlined.

View larger version (32K): [in a new window]   Fig. 9. Alignment of the predicted protein precursors deduced from the eth genes of Anopheles and Drosophila. Mature ETH1 and ETH2 are underlined with solid lines; cleavage and amidation sites are underlined with broken lines.

View larger version (164K): [in a new window]   Fig. 10. Neuropeptide immunoreactivity in Inka cells of Bombyx before and after larval, pupal and adult ecdysis. Strong double staining with antibodies to small cardioactive peptide B (SCPB) and PBAN (pheromone biosynthesis activating neuropeptide; dark brown colour) in pharate 5th instar larvae (A) was depleted 5 min after ecdysis (B; arrow). PG, prothoracic gland. Strong reaction with the antiserum to FMRFamide in pharate pupae (C; blue colour) diminished at the onset of ecdysis (D). Intense staining with antibodies to SCPB and vasopressin (dark brown colour) in pharate adults (E) decreased considerably 5 min after eclosion (F). Scale bar, 200 µm.

View larger version (24K): [in a new window]   Fig. 11. Chromatogram of Manduca Inka cell extracts showing that myomodulin (MM) and FMRFamide (FMRF) antisera cross-react with fractions containing PETH (pre-ecdysis-triggering hormone) and ETH (ecdysis-triggering hormone). Note the very weak immunoreactivities of myomodulin and FMRFamide antisera.

View larger version (132K): [in a new window]   Fig. 12. Comparison of myomodulin-IR in Inka cells of the Drosophila control line (CantonS) and the eth deletion mutant (eth25b), which lacks ETH. (A) The CantonS line shows strong myomodulin-IR in Inka cells (arrows), but (B) no immunoreactivity was detected in Inka cells of the eth deletion mutant (arrows).