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First published online 30 June 2004
doi: 10.1242/dev.01237

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Apical sensory neurones mediate developmental retardation induced by conspecific environmental stimuli in freshwater pulmonate snails

Elena E. Voronezhskaya¹, Marina Yu. Khabarova² and Leonid P. Nezlin^1,*,

¹ Institute of Developmental Biology, Russian Academy of Science, Moscow 117808, Russia
² Tula State Pedagogical University, Tula 300026, Russia

View larger version (28K): [in a new window]   Fig. 1. Normal larval development of Helisoma trivolvis (n=215) and Lymnaea stagnalis (n=340) from the trochophore stage till the adult-like form showing interrelations between timing of embryogenesis in percents (lower x-axis), developmental stages after Mescheriakov (Mescheriakov, 1990) (upper x-axis), and the length of embryos (y-axis).

View larger version (79K): [in a new window]   Fig. 2. Effects of conditioned water (CW) on larval development of Helisoma (A,C,D) and Lymnaea (B,E,F). (A,B) Incubation in CW induced developmental retardation in both Helisoma and Lymnaea. The effect disappeared after washout (CW-wash; the beginning of washout is indicated by arrows). Water conditioned by juveniles raised with plentiful food (CW-f) was ineffective. At the time when control animals developed into postmetamorphic adult like forms (C,E), the animals in CW were still at the veliger stage (D,F). Scale bars: 100 µm.

View larger version (22K): [in a new window]   Fig. 7. Effect of the tested drugs on the duration of larval development in Helisoma (A) and Lymnaea (B) measured from stage 19 till stage 27, and normalized by controls (*significantly different from control; P<0.05). CW, conditioned water; CW-f, water conditioned by fed juveniles; 5-HTP, 5-hydroxy-L-tryptophan; LDOPA, L-3,4-dihydroxyphenylalanine; 5-HT, 5-hydroxytryptamine creatine sulphate; DA, dopamine; -m-T, -methyl-tryptophan; -m-DOPA, -methyl-DOPA; NSD, 3-hydroxybenzilhydrazine; PCPA, L-p-chlorophenylalanine; EM, ergometrine maleate.

View larger version (34K): [in a new window]   Fig. 3. Effects of monoamine precursors on larval development. (A) In Helisoma, 5-HTP (1 mM) retarded the development similar to CW, while L-DOPA (10 mM) was ineffective. (B) In Lymnaea, L-DOPA (1 mM) retarded the development, while 5-HTP (10 mM) insignificantly slowed the development at the late stages only. (C,D) In both species, the effect of the precursors disappeared after washout (the beginning of washout is indicated by arrows).

View larger version (32K): [in a new window]   Fig. 4. Decarboxylase inhibitor NSD-1015 (10 µM) significantly attenuated the retarding effect of CW in both Helisoma (A) and Lymnaea (B). When added together with 1 mM 5-HTP in Helisoma (C) or 1 mM L-DOPA in Lymnaea (D), NSD-1015 also attenuated their effects.

View larger version (27K): [in a new window]   Fig. 5. In Helisoma, 5-HT synthesis inhibitor PCPA (5 µM) accelerated larval development (*P<0.05) (A) and reduced the retarding effect of CW (B). The antagonist of monoamine receptors, ergometrine (EM, 10 µM) attenuated the effect of CW (C) and 1 mM 5-HTP (D).

View larger version (31K): [in a new window]   Fig. 6. In Lymnaea, ergometrine (10 µM) accelerated normal development (*P<0.05) (A), and attenuated the retarding effect of CW (B) and L-DOPA (C). The rescue effect of ergometrine was concentration dependent (D). Arrow shows the start of ergometrine administration.

View larger version (93K): [in a new window]   Fig. 8. The first monoaminergic neurones in trochophores of Helisoma and Lymnaea at stage 19 (20%). (A,C-F) Anti-tubulin Immunostaining (red). (A) Helisoma; anti-5-HT immunolabelling (green). Anterior sensory neurones (arrows) are located on both sides of the mouth (m), and project to the pedal ciliary band (pb); pn, cilia in protonephridia (LSM projection, 48x0.6 µm). (B) Lymnaea; glyoxylic acid-induced fluorescence of catecholamines; conventional epifluorescence. The pair of the first anterior cells (arrows) is located above the mouth (m). (C) Lymnaea; anti-tyrosine hydroxylase immunolabelling (green). Anterior neurones (arrows) make a varicose network underneath the apical ciliary plate (LSM projection, 26x1 µm). (D) Lymnaea; anti-5-HT immunolabelling (green). High power image of the anterior neurone with a short thick ciliated apical fibre, thin basal fibre and two short lateral fibres (arrows) emanating from the soma (LSM projection, 54x0.35 µm). (E) Helisoma; anti-5-HT immunolabelling (green). The anterior neurone also has a thick ciliated apical fibre, a thin basal fibre and two short lateral fibres (arrows) (LSM projection, 43x0.3 µm). (F) Helisoma; anti-5-HT immunolabelling (green). Varicose network made by the fibres of the anterior neurones underneath the pedal ciliary band (pb) (LSM projection, 26x0.4 µm). Scale bars: 20 µm in A,B; 10 µm in C-F.

View larger version (70K): [in a new window]   Fig. 9. (A) Changes of average anti-5-HT immunofluorescence in the anterior neurones of Helisoma, after 6 hours of incubation with 5-HTP and -m-T; normalized to controls. (B) Changes of average glyoxylic acid-induced fluorescence of catecholamines in the anterior neurones of Lymnaea after 6 hours of incubation with CW, L-DOPA and -m-DOPA normalized to controls. *Significantly different from control, P<0.05. (C) Representative micrographs of glyoxylic acid-induced fluorescence in the anterior neurones of Lymnaea, showing increase after incubation with L-DOPA, and reduction after incubation with -m-DOPA. Scale bar: 20 µm.

View larger version (92K): [in a new window]   Fig. 10. Representative fluorescence micrographs of PCPA induced reduction of anti-5-HT immunofluorescence (green) in the anterior neurones of Helisoma; cilia are labelled in red (LSM projections; 20x0.5 µm). (A) Control. (B) After 3 days of incubation with 5 µM PCPA. Scale bar: 10 µm.