Serotonin- and two putative serotonin receptors-like immunohistochemical reactivities in the ground crickets Dianemobius nigrofasciatus and Allonemobius allardi.

Serotonin (5-hydroxytryptamine; 5-HT)- and two putative serotonin receptors, 5-HT1A- and 5-HT1B-like, immunohistochemical reactivities were investigated in the cephalic ganglia of two ground crickets, Dianemobius nigrofasciatus and Allonemobius allardi. 5-HT-ir was strongly expressed in the central body, accessory medulla region of the optic lobe, frontal ganglion, posterior cortex of the protocerebrum, dorsolateral region of the protocerebrum, and the suboesphageal ganglion (SOG) in both crickets. However, 5-HT1A-ir and 5-HT1B-ir showed quite mutually distinct patterns that were also distinct from 5-HT-ir. 5-HT1A-ir was located in the pars intercerebralis, dorsolateral region of the protocerebrum, optic tract, optic lobe, and the midline of the SOG in both crickets. 5-HT1B-ir was located in the pars intercerebralis and dorsolateral region of the protocerebrum, and detected weakly in the optic lobe, tritocerebrum, and the midline of the SOG in both crickets. Interspecific differences were observed with 5-HT1A-ir. 5-HT1A-ir was expressed weakly in two neurons in the mandibular neuromere of the SOG in D. nigrofasciatus, while it was expressed strongly in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG in A. allardi and co-localized with CLOCK-ir (CLK-ir). 5HT-1B-ir was co-localized with CLK-ir in the tritocerebrum, mandibular neuromere, and maxillary neuromere of the SOG when double-labeling was conducted in both crickets. These results indicated that 5-HT and both types of 5-HT receptors may regulate circadian photo-entrainment or photoperiodism in A. allardi, while only 5-HT1B may be involved in circadian photo-entrainment or photoperiodism in D. nigrofasciatus.

Precursor structure, distribution and possible functions of pigment-dispersing hormone (PDH) in the terrestrial isopod Armadillidium vulgare (Latreille).

Pigment-dispersing hormone (PDH) is an 18 amino acid neuropeptide that induces pigment migration in Decapoda and serves as a circadian neurotransmitter in the locomotor activity rhythm in Drosophila. In this study, a cDNA encoding PDH was cloned from adult brains of the pill bug, Armadillidium vulgare (Av). The cDNA comprising 529 bp encodes a peptide (AvPDH) that consists of a putative 26 amino acid signal peptide, and a 34 amino acid PDH-precursor-related peptide containing an 18 amino acid mature peptide. The peptide shows a high sequence identity (55-77%) to crustacean ß-PDHs and insect PDFs. The tissue-specific expression pattern was examined by reverse transcription PCR. The transcript is expressed in the brain strongly and ventral nerve cord weakly, but the signal was not detected in the intestinal tract. A similar expression profile appeared in Western blot analyses. Western blot analyses with timed samples showed more intense expression of PDH-like antigen at night. PDH-like immunohistochemical reactivity (PDH-ir) was detected in the optic lobe, anteromedian protocerebrum, accessory lobe, tritocerebrum, and suboesophageal ganglion but the reactivity was faint or nil in the pseudofrontal organ (sinus gland). These results were substantiated by in situ hybridization. Co-localization using anti-Gryllus bimaculatus (Gb)-PDF, anti-Bombyx mori (Bm)-CLK, and anti-Bm-CYC showed a co-localization of these antigens in the optic lobe and SOG. The results provide the first structural and immunocytochemical identification of PDH neurons in terrestrial isopods, and the co-localization of PDH with CLK and CYC supports its possible involvement in circadian clock. A day/night rhythm of PDH content is also a new feature.

Molecular structure, expression patterns, and localization of the circadian transcription modulator CYCLE in the cricket, Dianemobius nigrofasciatus.

CYCLE (CYC), also known as BMAL1 in vertebrate nomenclature, is a transcription modulator of the circadian genes period and timeless of Drosophila melanogaster. We cloned a cDNA encoding a CYC homologue from the head of the ground cricket, Dianemobius nigrofasciatus (Dncyc), the first CYC from Hemimetabola. The deduced sequence corresponded to a 601 amino-acid polypeptide, with well-defined bHLH, PAS-A, PAS-B, PAC, and BTCR domains. The amino-acid sequence showed 70.7% identity with the CYC protein of Athalia rosae, 63.8% with D. melanogaster, and 52% identity with the human homologue. A cyc transcript of around 3.6kb occurs in the brain, midgut, testis, fatbody, and muscle. An additional band of around 1.1kb gave a hybridization signal in the head. No temporal oscillation in cyc mRNA abundance was observed in the head of the adult cricket when investigated by Northern blot analysis. CYC-like immunohistochemical reactivity (ir) and its dimerization partner CLOCK (CLK)-ir appeared in the pars intercerebralis (PI), tritocerebrum, dorsolateral protocerebrum, and subesophageal ganglion (SOG), but no CYC-ir was observed in the optic lobe (OL) that showed CLK-ir. The deutocerebrum showed a unique CLK-ir but no CYC-ir pattern. Double-labelling experiments showed that both antigens were co-localized in the mandibular and maxillary neuromeres of the SOG. CYC-ir showed no daily oscillation in intensity and the staining pattern was always cytoplasmic. CLK-ir occurred in the nucleus at ZT 16, but was cytoplasmic at other ZT times. A neuronal network equivalent to adult system occurred in the second nymphal stadium.

Co-localization and unique distributions of two clock proteins CYCLE and CLOCK in the cephalic ganglia of the ground cricket, Allonemobius allardi.

CYCLE (CYC) and CLOCK (CLK) are transcriptional activators of the circadian clock genes, period (per) and timeless (tim), binding at E-boxes of their upstream regulatory region in Drosophila. CYC-like and CLK-like immunohistochemical reactivities (CYC-ir and CLK-ir) were investigated in the ground cricket, Allonemobius allardi, in which immunohistochemical reactivities for three circadian clock proteins (PERIOD, Doubletime, and Cryptochrome), two neuropeptides (crustacean cardioactive peptide and diapause hormone), and arylalkylamine-N-acetyltransferase had previously been mapped in the brain-subesophageal ganglion (SOG) complex. CYC-ir and CLK-ir occurred predominantly in the cytoplasm of the neurons distributed mainly in the central brain, SOG, and corpora cardiaca. Double-labeling experiments showed that CYC-ir and CLK-ir were co-localized only in the mandibular and maxillary neuromeres of the SOG. The neuronal processes in the dorsolateral region of the protocerebrum partially shared the immunoreactivities, whereas most of the other immunoreactivities were unique. The optic lobe showed reactivity to anti-CYC at small proximal frontodorsal cells and to anti-CLK at small proximal frontoventral cells. The frontal ganglion exhibited CYC-ir in the cell bodies that lacked CLK-ir. No difference in their number, distribution, or staining intensity was found between sampling under light:dark regimes of 16:8 and 12:12. The levels of both CYC-ir and CLK-ir showed no oscillation throughout a 24-h period. The co-localization pattern suggests that the midline cells of the SOG share most of the circadian-related immunoreactivities, thus constituting the heart of the circadian clock in A. allardi.

Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure.

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.