Localization and functional characterization of a novel adipokinetic hormone in the mollusk, Aplysia californica

Abstract

Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.

Figure 1. Nucleotide and deduced amino acid sequences of prepro ap-AKH.

Full-length nucleotide sequence (lower case letters) and deduced amino acid sequence (upper case letters) of prepro ap-AKH (GenBank Accession #JQ929303.2). The signal peptide is underlined with a solid line; the mature ap-AKH decapeptide is boxed, and the associated peptide is underlined with a dashed line. The dibasic cleavage site and α-amidation signal are highlighted in grey. The asterisk denotes the stop codon and the polyadenylation site is underlined with double dotted lines.

Figure 2. RT-PCR of ap-AKH in central and peripheral tissues of A. californica.

ap-AKH is expressed in the CNS but not peripheral tissues of A. californica (top panel). Negative control (middle panel) was RNA with no RT, and positive control (bottom panel) used ap-Actin primers. Abbreviations: BCN, bag cell neurons; Abd, abdominal ganglion; Cer, cerebral ganglia; Ped/Pleu, pedal/pleural ganglia; Buc, buccal ganglia; Tail, tail muscle; Osph, osphradium; SHD, small hermaphroditic duct; Hrt, heart; OVT, ovotestis; Water, no template.

Figure 3. ISH and ICC of A. californica CNS.

Representative photomicrographs of ISH (purple; B, D, F, J) or ICC (brown; A, C, E, G, H, I, K, L, M) staining in the CNS of A. californica, including abdominal (A, B, L, M), buccal (K), cerebral (C, D, I, J), pleural (E, F), and pedal (H) ganglia, and bag cell neurons (G). Preadsorption with ap-AKH (M) completely abolished ap-AKH-ir signal compared to an adjacent section (L). Green = methyl green nuclear counterstain. Solid arrows denote ap-AKH-ir or ap-AKH transcript-positive neuronal cell bodies; arrow pairs of the same shape in panels A/B, C/D, E/F, and L/M point to identical neurons in adjacent sections. Dashed arrows in panels A, C, E, G, H, I, and K point to ap-AKH-ir fibers in the neuropil regions of ganglia. Dashed arrow in panel J points to ap-AKH transcript-positive fibers. Dashed outline in panel L surrounds ap-AKH-ir fibers in the neuropil region of the abdominal ganglia, and in M surrounds the same region of neuropil devoid of signals. Scale bars = 50 µm.

Figure 4. Diagrammatic representation of ap-AKH transcript and peptide in A. californica CNS.

The relative abundance and location of neurons and fibers positive for ap-AKH transcript (purple dots and lines; left) or ap-AKH peptide (tan dots and lines; right) are shown.

Figure 5. Hemolymph glucose was not acutely altered by a single ap-AKH injection.

Hemolymph glucose was normalized against baseline levels at Time 0. ASW or ap-AKH was injected immediately after Time 0. No significant differences were detected between ASW-injected and ap-AKH-injected animals. Data are mean ± SEM; n = 5/group.

Figure 6. Long-term effects of ap-AKH injections on body mass.

A) Time course of body mass change in response to ap-AKH injections over time. Daily body mass was presented as a percentage of baseline body mass. Hatched zones with arrows indicate injection days, and white zones indicate non-injection days. B) Initial body mass was not different between ASW- and AKH-injected groups. C) Terminal body mass was significantly reduced in ap-AKH-injected group. * = p<0.05, ** = p<0.01, *** = p<0.001, and **** = p<0.0001 compared to ASW-injected group. Data are mean ± SEM; n = 4–5/group.

Figure 7. Long-term effects of ap-AKH injections on feeding, metabolic parameters, and OVT.

A) Time course of food consumption in response to ap-AKH injections. Hatched zones with arrows indicate injection days, and white zones indicate non-injection days. The dotted line at 20 g indicates maximum food consumption possible per day. B) Total food consumption over the 20-day experimental period was significantly reduced in ap-AKH injected animals. C) HP glycogen stores at the end of the experiment were not altered by ap-AKH. D) Hemolymph glucose levels were higher at sacrifice in the ap-AKH-injected group. E) Mean oocyte diameter and F) OVT mass at sacrifice were reduced in the ap-AKH-injected group. * = p<0.05, ** = p<0.01, *** = p<0.001, and ****p<0.0001 compared to ASW-injected group. Data are mean ± SEM; n = 4–5/group.

Figure 8. Acute effect of ap-AKH on excretion of feces.

ap-AKH injection significantly increases fecal pellet mass within one hour of injection. * = p<0.05 compared to ASW-injected group. Data are mean ± SEM; n = 7/group.

Figure 9. A single injection of ap-AKH induced rapid and sustained loss of body mass.

Significant reductions in body mass were found at 2, 3, 4, 6, and 24 hours in ap-AKH-injected group. * = p<0.05, ** = p<0.01, and *** = p<0.001 compared to ASW-injected group. Data are mean ± SEM; n = 5/group.

Figure 10. Comparisons of in vivo effects of ap-AKH and ap-GnRH revealed no overlap.

A) A single injection of ap-GnRH, but not ap-AKH, stimulated parapodial opening at 5 and 10 minutes post-injection. B) A single injection of ap-AKH, but not ap-GnRH, reduced body mass between 2 and 24 hours post-injection. C) A single injection of ap-AKH, but not ap-GnRH, suppressed food intake at 1 and 3 hours post-injection. * = p<0.05, ** = p<0.01, *** = p<0.001, ****p<0.0001. In panel B, # denotes differences between ap-AKH and ap-GnRH groups while *denotes differences between ap-AKH and ASW groups. Data are mean ± SEM; n = 5/group.