Table 1.
Primer sequences for the qRT-PCR assays.
Figure 1.
Analysis of sNPF receptor sequence conservation.
Multiple sequence alignment of amino acid sequences of insect sNPF receptors, performed with Clustal Omega software, available from the European Bioinformatics Institute. Default parameters were used. Anoga: Anopheles gambiae (ABD96049.1), Drome: Drosophila melanogaster (AAF49074.2), Solin: Solenopsis invicta (AAY88918.1), Schgr: Schistocerca gregaria (JX855828). ‘*’ identities, ‘:’ conservative substitutions, ‘.’ semi-conservative substitutions.
Figure 2.
Dose-response curves for bioluminescence induced by Schgr-sNPF and Schgr-sNPF4–11 in Schgr-sNPFR-expressing cells.
A. Aequorin bioluminescence induced in CHO-WTA11-Schgr-sNPFR cells, stably expressing the promiscuous Gα16 subunit. Data represent the average ± SEM of three independent measurements performed in duplicate and given in percentage of the maximum response. The zero response level corresponds to treatment with BSA solution. B. Aequorin bioluminescence induced in CHO-PAM28-Schgr-sNPFR cells. Data represent the average ± SEM of three independent measurements performed in duplicate and given in percentage of the maximum response. The zero response level corresponds to treatment with BSA solution. C. Luciferase bioluminescence induced in HEK-293T-Schgr-sNPFR cells. Data represent the average ± SEM of three independent measurements performed in duplicate and given in percentage of the maximum response. The 100% response level corresponds to treatment with DMEM/F12 containing 200 µM IBMX and 20 µl NKH477 (a water-soluble analogue of forskolin).
Figure 3.
Relative levels of the sNPF receptor transcript four and ten days after adult eclosion.
Expression of the sNPF receptor transcript seems limited to neural tissue. Br: brain; OL; optic lobes; CC: corpora cardiac; CA: corpora allata; SOG: suboesophageal ganglion; SaGl: salivary gland; TG1: prothoracic ganglion; TG2: metathoracic ganglion: TG3: metathoracic ganglion; AG4–5: abdominal ganglia 4 and 5; AG6–7: abdominal ganglia 6 and 7; AG8: abdominal ganglion 8; Test: testes; Ova: ovaries; Fb: fatbody; FG: foregut; MG; midgut; HG: hindgut; AcGm: male accessory gland; Mscl: flight muscle. Data represent the average ± SEM (n = 6).
Figure 4.
Relative levels of the sNPF receptor transcript upon starvation and after feeding.
A. Expression of the Schgr-sNPFR transcript in the brain of adult locusts is significantly lower after five days of starvation. Data represent the average ± SEM (n = 10). Results were analysed using Student’s t-test. * p<0.05. B. Expression of the Schgr-sNPFR transcript in the brain of male adult locusts peaks 30 minutes after feeding, after which it returns to a baseline level. Data represent average ± SEM (n = 6). Results were analysed using ANOVA in combination with Tukey’s Multiple Comparison Test. * p<0.05.
Figure 5.
Injection of 200 pg Schgr-sNPF or Schgr-sNPF4–11 in adult locusts and its effect on food uptake, normalized to the body mass of the locusts.
A significant decrease in food uptake is observed for both sNPF and sNPF4−11. Data represent mean values ± SEM (n = 20). Results were analysed using ANOVA in combination with Tukey’s Multiple Comparison Test. ** p<0.01; *** p<0.001.
Figure 6.
RNAi mediated knockdown of the mRNA encoding the Schgr-sNPF receptor and its effect on food uptake.
Starved control animals show a higher food uptake than fed controls. Knockdown of the sNPFR transcript mimics the starved state in fed animals. Data represent mean values ± SEM (n = 20). Results were analysed using ANOVA in combination with Tukey’s Multiple Comparison Test. * p<0.05, *** p<0.001.