Fig 1.
Synthesis of cis-9-tetradecen-1-ol.
a. Ag2O, BnBr, DCM, RT, 18 h; b. TsCl, anhydrous pyridine, RT, 1 h; c. NaI, DMF, 50°C, 4 h; d. hexyne, n-BuLi, HMPA, THF, -78°C to RT, 96 h; e. Lindlar cat., quinoline, H2, 8 h. For more detail see S1 Text.
Fig 2.
Behavioral bioassay apparatuses used in this study.
Panel a: binary choice chamber; Panel b: orientation double chamber, with oviposition substrate uncovered; Panel c: Petri dish assay.
Fig 3.
The behavioral responses of gravid S. vittatum to fresh conspecific eggs in the binary choice chamber assay.
Panel A: Oviposition preference of gravid S. vittatum on a substrates containing or lacking eggs. Values significantly different from the control are denoted by an asterisk [*] (Wilcoxon matched-pairs signed rank test, P = 0.002). Panel B: Orientation response represented the number of flies located after landing on a substrate with eggs or lacking eggs. Values significantly different from the control are denoted by an asterisk [*] (t-test, P < 0.05).
Fig 4.
The behavioral responses of gravid S. vittatum to fresh conspecific eggs in the orientation double chamber assay.
Assays were conducted in the presence or absence of fresh eggs, in the lower chamber, as described in Materials and Methods. Values that were significantly different from the control are indicated by an asterisk [*] (unpaired t-test, P < 0.05) and those which were not significantly different values are indicated by NS (P > 0.05).
Fig 5.
The behavioral responses of gravid S. vittatum to uncovered or covered fresh conspecific eggs.
Values represent the number of flies present on the substrate or mesh screen after 20 minutes. Significantly different values are indicated by an asterisk [*] (unpaired t-test, P < 0.05) and values that were not significantly different are indicated by NS (P > 0.05).
Fig 6.
Oviposition responses of gravid S. vittatum egg extracts in the Petri dish assay.
The response values of the crude extracts were compared to their respective solvent controls. A significant difference between the solvent control and the extract is indicated by an asterisk [*] (Fisher’s exact test, P < 0.05). Each column represents an assay run on 60 individual flies.
Fig 7.
Isolation and characterization of compounds present in hexane crude extracts of S. vittatum eggs.
Panel A: Gas chromatographic analysis of hexane crude extracts. Numbers highlight the major peaks. The minor peaks were identified in blank runs as well as in the egg extract and were therefore attributed to contamination. Panel B: Oviposition response in the Petri dish assay of gravid S. vittatum to the individual compounds identified in the hexane crude extracts (1-hexadecene, 1-pentadecene, 1-tridecene, and cis-9-tetradecen-1-ol). Numbers under the x- axis correspond to the peaks shown in Panel A. Compounds were tested at 3 different dilutions (1:1000, 1:100, 1:10 w/v). Response values of the individual compounds that were significantly different than the solvent control are indicated by a single asterisk [*](Fisher’s exact test, P < 0.05) or a double asterisk [**](Fisher’s exact test, P < 0.005). Because different numbers of flies were used in the tests of the individual compounds and the blends, the data are presented as the percentage of flies ovipositing on the substrate. Blend = a mixture of pentadecene, hexadecene, cis-9-tetradecen-1-ol and tridecene at a ratio of 7.8: 2.3: 2.3: 1.0 (the ratio found in the hexane extract; see S1 Text).
Fig 8.
EAG responses of gravid S. vittatum to individual compounds identified from the crude extract of conspecific eggs.
Assays were carried out on a live restrained fly as described in Materials and Methods. Responses that are significantly different from the solvent control are denoted by the asterisks * and ** (Fisher’s LSD, P < 0.005 and P < 0.0001, respectively).
Fig 9.
Compounds identified as oviposition pheromones in Dipteran species.
Oviposition pheromones were identified in the following sources: Simulium vittatum, this work; Culex spp. [14]; Aedes aegypti [19]; Lutzomyia longipalpis [5]; Glossina morsitans morsitans and G. m. centralis [20].