Journal of Insect Physiology 56 (2010) 1576–1586

Serotonin- and two putative serotonin receptors-like immunohistochemicalreactivities in the ground crickets Dianemobius nigrofasciatus andAllonemobius allardi

Qi-Miao Shao a,b, Maged Mohamed Ali Fouda a, Makio Takeda a,*a Graduate School of Agriculture Science, Kobe University, 1-1 Rokkodai-cho, Kobe 567-8501, Japanb Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

A R T I C L E I N F O

Article history:

Received 26 February 2010

Received in revised form 18 May 2010

Accepted 18 May 2010

Keywords:

Serotonin (5-hydroxytryptamine

5-HT)

5-HT1A

5-HT1B

Immunohistochemistry

A B S T R A C T

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.

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1. Introduction

Serotonin (5-hydroxytryptamine; 5-HT) is a monoamine neuro-transmitter that regulates a wide range of behaviors including mood,cognition, appetite, sleep, memory, and sex in mammalian systems(Lucki, 1998). In insects, 5-HT functions as a neurotransmitter,neuromodulator, and neurohormone. 5-HT is responsible for themodulation of various behaviors in insects, such as short-termmemory, sensitivity to olfactory stimuli (Mercer and Menzel, 1982;Menzel et al., 1999) and foraging behavior in honeybees (Schulzet al., 2002). Lines of evidence show that 5-HT may be involveddirectly in the circadian rhythm of many different insects. In thecrickets, Gryllus bimaculatus, serotonergic neurons are distributedacross almost the entire area of the lamina and medulla neuropil ofthe optic lobe, and the serotonin content in the optic lobe fluctuatesdepending on the time of day, in synchrony with circadian changes

* Corresponding author.

E-mail address: mtakeda@kobe-u.ac.jp (M. Takeda).

0022-1910/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2010.05.015

in the sensitivity of visual interneurons (Tomioka et al., 1993). In G.

bimaculatus, pigment dispersing factor (PDF) enhances and 5-HTsuppresses the photoresponsiveness of the medulla bilateralneurons, which are the most likely candidate neurons forsynchronization of the pacemakers of the two hemispheres(Saifullah and Tomioka, 2002, 2003). In the cockroach Leucophaea

maderae, injections of 5-HT induced phase shifts in circadianlocomotor activity (Page, 1987). In the house fly, Musca domestica,some monopolar neurons in the lamina change their axon diametersin a circadian manner, and this can be influenced antagonistically byinjections of 5-HT and PDF (Pyza and Meinertzhagen, 1996;Meinertzhagen and Pyza, 1999). 5-HT-expressing neurons innervatethe larval optic neuropil that overlaps with the dendritic arboriza-tion of the ventral lateral neuron, which may suggest that the larvalpacemaker neurons of Drosophila may be directly modulated by 5-HT-producing neurons (Rodriguez Moncalvo and Campos, 2005).Injection of the specific neurotoxin 5,7-DHT which causes selectivedegeneration of serotonergic neurons, modified the level oflocomotor activity and the period of circadian rhythmicity in theblowfly (Cymborowski, 2003).

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–1586 1577

The effects of 5-HT are primarily mediated through interactionswith a large number of guanine nucleotide binding and regulatoryproteins (G-protein)-coupled receptor proteins that activatemultiple effectors pathways (Raymond et al., 2001). In mammals,there are at least 30 types of 5-HT receptors. Most of the receptorsbelong to a large family of proteins that are coupled with G-proteins. 5-HT receptors have been conventionally divided intoseven subfamilies. All but one of the 5-HT receptor groups arecoupled to G-proteins, while only 5-HT3 receptors are 5-HT gatedion channels (Raymond et al., 2001). Drosophila is known toexpress four 5-HT receptor subtypes that are predicted to beorthologs of the mammalian 5-HT1A, 5-HT1B, 5-HT2, and 5-HT7receptors. These are designated 5-HT1ADro, 5-HT1BDro, 5-HT2Dro, and 5-HT7Dro, respectively (Nichols, 2006). Recentreports have indicated that the 5-HT1ADro and 5-HT1BDroreceptors are involved in both circadian processes and sleep inthe fly (Yuan et al., 2005, 2006). In Drosophila, the cholinergicinputs, which act on nicotinic receptors on the s-LNvs, are likely tobe derived primarily from the larval photoreceptors of the Bolwigorgan so that they possibly subserve light entrainment in the larva(Wegener et al., 2004). These inputs may also mediate light inputsfor rapid behavioral responses such as photo-aversion (Mazzoniet al., 2005).

Recent studies on 5-HT receptors have advanced the field ofresearch considerably in insects other than Drosophila. A 5-HTreceptor was cloned from both the antennae of Bombyx mori andHeliothis virescens (B150Bom and K15Hel; von Nickisch-Rosenegket al., 1996). Another 5-HT receptor was cloned from Papilio xuthus L.(Px5HT) (Ono and Yoshikawa, 2004). Dacks et al. (2006) cloned twosubtypes of 5-HT from Manduca sexta (Ms5HT1A and Ms5HT1B) andrevealed that both subtypes existed in the antennae, thoracicganglion, and abdominal ganglia of adults or pharate adults. Twoputative 5-HT receptors (Ap5-HT1A and Ap5-HT1B) were clonedfrom the brain of the Chinese oak silkmoth, Antheraea peryni

(Hiragaki et al., 2008). However, no information about serotoninreceptors in hemimetabolous insects has been reported. The presentstudy investigated the expression of 5-HT-like and two 5-HTreceptors-like immunoreactivities in the two ground cricketsDianemobius nigrofasciatus and Allonemobius allardi, with specialinterest on their relationship with the circadian clock. The twocricket species serve as good model animals to study the centralnervous system because of their large and easily detectable neurons.The expression pattern of many clock related proteins has beenmapped in these two crickets by immunohistochemistry (IHC) (Shaoet al., 2006, 2008a,b; Sehadova et al., 2007).

2. Materials and methods

2.1. Animals

Both male and female adults of D. nigrofasciatus (Gryllidae:Nemobiinae) and A. allardi (Gryllidae: Nemobiinae) were used forimmunohistochemisty. D. nigrofasciatus was collected in Kako-gawa, Japan (34.78N, 134.88E), and A. allardi were collected fromSouth Carolina, USA (348N, 828W; See Shao et al., 2006). They werekept at 25 8C under LD 16:8 or LD 12:12 and fed with a commercialanimal diet (MF mix, Oriental Yeast Corp., Tokyo, Japan).

2.2. SDS–PAGE and Western blotting analysis

Western analysis was performed on head samples eachconsisting of 4 heads of D. nigrofasciatus collected at 6 h intervalsunder LD 12:12 with the antennae, mouth parts, and scales removed.The samples were homogenized in 200 ml sample buffer (25% 0.5 MTris–HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol,0.8 mg/ml bromophenol blue, 37% Millipore water). The homoge-

nate was centrifuged (10,000 � g, 5 min at 4 8C) to eliminate thecuticle and cell debris, and the supernatant was collected anddenatured at 95 8C for 10 min before storage at �20 8C until use. A5 ml sample was loaded per lane on a 10% SDS–polyacrylamide gel,and Broad Range Prestained protein marker 6–175 kDa (BioLabs,New England) was used to estimate the molecular size of separatedproteins. Protein samples were transferred electrophoretically ontoa nitrocellulose membrane (Sigma-Aldrich) using a semidry blottingsystem (ATTO Corporation AE-6675; 40 mV for 450). After washingwith phosphate-buffered saline, the membrane was treated for 1 hat rT with 5% non-fat dry milk in PBS supplemented with 0.1%Tween-20 (PBS-Tw) to block unspecific IgG binding sites and thenincubated overnight at 4 8C under gentle agitation in primaryantibody solution containing 5% bovine serum albumin in PBS-Tw(1:40,000 dilution). After washing with PBS-Tw, the membrane wasincubated for 1 h at rT with the goat anti-rabbit IgG conjugated withhorse radish peroxidase (HRP) (Amersham Biosciences, UK) dilutedat 1:2000 in PBS-Tw, and washed with PBS-Tw. The immunoreactionwas visualized using a ECL system.

2.3. Immunohistochemistry

Antibodies: anti-5-HT was produced and the specificity waschecked as described previously in Nishiitsutsuji-Uwo et al.(1984). A peptide corresponding to the 18 amino acids from 447to 464 of the deduced sequence of A. pernyi 5-HT1A and anotherpeptide corresponding to 20 amino acids from 429 to 448 of thededuced sequence of A. pernyi 5-HT1B were synthesized andconjugated with KLH from Genmed Co. (Texas, USA). Because thecDNA sequence of 5-HT1A and 5-HT1B were not known at presentin crickets, we compare the peptides sequence between hemime-tabolous insects and A. peryni. About half of the amino acids are thesame, indicating high possibility of catching the same receptors incrickets. Polyclonal antibodies against each peptide were raised intwo rabbits and both two rabbits gave us similar reactivities.Antibody specificity was verified both by preabsorption tests withthe provided peptide-KLH antigen and Western blotting.

Heads were harvested in the sterilized Ringer’ solution andfixed overnight at 4 8C in Bouin fluid. Standard techniques wereused for tissue dehydration, embedding in paraffin, sectioning(8 mm), deparaffinization, and rehydration as described by Shaoet al. (2006). The sections were blocked with 1.5% normal goatserum diluted in PBS for 30 min at rT. Subsequent overnightincubation with the primary antibodies diluted with the blockingserum was conducted in a humidified chamber at 4 8C. Primaryantibodies diluted 1: 500 were replaced with normal serum in thecontrol staining. Bound IgG was detected with the rabbit IgG-Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA)following the procedures described by Shao et al. (2006). Theactivity of the horseradish peroxidase (HRP) was visualized usinghydrogen peroxide (0.005%) and 3,30-diaminobenzidine tetrahy-drochloride (DAB, 0.25 mM in 0.1 M Tris–HCl, pH 7.5). Stainedsections were mounted in Biolet medium (Kouken Rika, Osaka,Japan) and visualized using a BX50F4 microscope (Olympus,Japan).

CLK/5HT1A double-labeling and CLK/5HT1B double-labelingwere conducted using fluorescent dye-conjugated secondaryantibodies. GST-CLK fusion protein was produced and this wasused as an antigen. The specificity was checked and describedpreviously (Shao et al., 2008a,b). CLK antibody was diluted 1:500,and incubated overnight at 4 8C. After rinsing with PBS, the slideswere incubated with Cy3-conjugated (red) goat anti-rabbit IgG for90 min at rT. After rinsing with PBS, goat normal serum (3% dilutedin PBS) was added to remove the primary antibody. Subsequently,goat polyclonal antibody to rabbit IgG H&L-Fab fragment (Rhoda-mine) (Abcam) was added to retrieve the extra-binding sites of the

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–15861578

first secondary antibody. After these retrieval steps, the slides wereincubated with anti-5-HT1A or anti-5-HT1B (1:500) overnight at4 8C. After washing with PBS, Alexa488-conjugated (green) goatanti-rabbit IgG was added and incubated for 90 min at rT. Finally,the slides were rinsed with PBS and mounted in Aqua Polymountand visualized using a BX50F4 microscope (Olympus, Japan).

3. Results

3.1. 5-HT-ir in the cephalic ganglia of D. nigrofasciatus and A. allardi

5-HT-ir showed only slight differences in the numbers andintensity of the immunoreactive cells in two crickets. 5-HT-ir wasintense in the pars intercerebalis, central body, optic lobe,dorsolateral region of the protocerebrum, posterior cortex of theprotocerebrum, frontal ganglion, deutocerebrum, and SOG(Table 1, Figs. 1 and 2). No sexual differences were detected andthe signal was found only in the cytoplasm. Axonal arborizationwas found in the midline of the brain and the calyes of themushroom body. Strongly stained axonal process was foundextending to the posterior region of the protocerebrum (Fig. 2b,arrow). Furthermore, one axonal process extended from theprotocerebrum to the deutocerebrum region (Fig. 2c, arrow).Intensively immunoreactive fibers were found in all eightsegments of the fan-shaped body of the upper region of thecentral body (Figs. 1c and 2d). The posterior optic commissure wasstrongly stained in both crickets (Figs. 1b and 2e, arrow). Around28 (in D. nigrofasciatus) and 24 (in A. allardi) small, strongly stainedcells were observed in the posterior cortex region of theprotocerebrum (Figs. 1d and 2f). Strong neuronal arborizationwas distributed in the lamina of the optic lobe and moderatesignals were also found in the medulla region of the optic lobe(Figs. 1e and 2k). Around 10 small cell bodies that appeared to beamacrine cells were located in the boundary between the medullaand the lobula (Figs. 1f and 2i). Seven to eight strongly stainedneurons in D. nigrofasciatus (Fig. 1f, arrow) and 7–8 stronglystained and 5–6 weakly stained neurons in A. allardi with obvious

Table 1The number and intensity of immunoreactive cells in the cephalic ganglia of D. nigrofa

D. nigrofasciatus

5-HT 5-HT1A

OL aMe 7–8++++ 1++

Pro PI* 2–3++++� 6++++

DL 4+++ 1+++

Optic tract � 3+++

Posteriolateral cortex 28++++ 2++

De 2+++ �Tr � �FG* 6++++ �

SOG Mdb-Vm �Mxl-Vm � 2+

Mdb-Lm 2++++ �Mxl-Lm 4+++

Mdb-L 4++++ 1++

CC* Cortex arborization � +++

NCA I ++ ++

Oscillation No No

Immunoreactivity was graded as absent (�), weak (+), moderate (++), considerable (++

numbers are shown either for the entire ganglion (*) or for half of it, e.g., one brain hemis

1B; aMe, accessory medulla; Pr, protocerebrum; PI, pars intercerebralis; De, deutoce

subesophageal ganglion; Mdb-Vm, Mxl-Vm, and Lb-Vm, ventromedial cell clusters in the

Mxl-Lm, lateral medial cells in the mandibular, maxillary, respectively; Mdb-L, the latera

nervi corporis allati I.

neuronal processes were observed in the accessory medulla regionof the optic lobe (Fig. 2i). Four small cells in D. nigrofasciatus

(Fig. 1g) and two large cells in A. allardi (Fig. 2j) in the dorsolateralregion of the protocerebrum were moderately stained. Two cellbodies were located in the deutocerebrum: one cell near theantennal lobe (Figs. 1h and 2g) and one cell body close to theanterolateral region of the antennal lobe (Fig. 1i and 2h). Intenselystained neuronal arborization was observed in the antennal lobeglomeruli of both crickets. Six large neurons in D. nigrofasciatus

(Fig. 1j) and eight large neurons showing granular staining in A.

allardi (Fig. 2l) with axonal processes extending to the cortex of thefrontal ganglion were found in the dorsal region of the frontalganglion. Neuronal arborization distributed all over the frontalganglion and neuronal processes projected toward the tritocer-ebrum through the frontal connectives. Five groups of neuronswere strongly stained in the SOG in both crickets. Two large motorneurons were located in the fronto-median region of the SOG(Figs. 1l and 2n), two large neurons in the ventrolateral region ofthe mandibular neuromere (Fig. 1M) and four large neurons in theventrolateral region of the maxillary neuromere were stronglystained (Figs. 1n and 2o). Four large neurons were intensely stainedin the lateral region of the mandibular neuromere (Figs. 1o and 2p).Two large neurons were located in the posterior region of the SOG(picture not shown). Intense arborization was observed all over theSOG and strong axonal processes was found extending from themedian region of the SOG to the lateral region although theirneuronal process was difficult to follow (Fig. 2q).

3.2. 5-HT1A-like immunoreactivity the cephalic ganglia of D.

nigrofasciatus and A. allardi

5-HT1A-ir shared by D. nigrofasciatus and A. allardi was found inthe pars intercerebralis, dorsolateral region of the protocerebrum,posterior cortex of protocerebrum, optic tract, deutocerebrum, andcorpora cardiaca, while 5-HT1A-ir in the SOG was quite different inthe two crickets. The immunoreactivity was always located in thecytoplasm and no intensive neuronal fibrication was detected all

sciatus and A. allardi.

A. allardi

5-HT1B 5-HT 5-HT1A 5-HT1B

13++ 7–8++++

5–6++

– 12–14+++

4++++ 4++++

3++

2++

2++++

2++ 2++++ 1++

2+++

2++ 24++ 2–3++ 2+++

1+ 2++++ 1++

4–5++ 4–5++++ 4–5++++

8++++

4++ 4++ 4+++

8–10+ 8–10++ 8–10+++

2++++ � �4++ 2+++

2++ 4++++ 1++++ 12+++

+ � ++ +

+ +++ +++ +

No No No No

+), or strong (++++). Evaluation scales were set for each antibody separately. The

phere or one optic lobe. 5-HT1A, serotonin receptor 1A; 5-HT1B, serotonin receptor

rebrum; DL, dorsolateral region of the protocerebrum; Tr, tritocerebrum; SOG,

mandibular, maxillary neuromere and labial neuromere, respectively; Mdb-Lm and

l cells in the mandibular neuromere; FG, frontal ganglion; CC, corpora cardiac; NCAI,

[(Fig._1)TD$FIG]

Fig. 1. 5-HT-ir in the brain–SOG complex of D. nigrofasciatus. (a) Representation of the numbers and topography of 5-HT-ir cells brain–SOG. (b) 5-HT-ir in the neuronal

processes of the anterior region of the central body. (c) Strong staining of the fan-shaped upper division of the central body, and the posterior optic commissure (POC) was also

strongly stained by the 5-HT antibody (arrow). (d) Several strongly stained cell bodies located in the posterior cortex of the protocerebrum. (e) Intensive staining of the

neuronal arborization in the lamina of the optic lobe. (f) Representative cell bodies that were found between the medulla and the lobular and some neurons located in the

accessory medulla of the optic lobe (arrow). (g) One cell body found in the dorsolateral region of the protocerebrum but much near to the mushroom body (arrow) and three

other small cell bodies clustered together in the dorsolateral region of the protocerebrum closer to the optic lobe (black star). (h) Strongly stained neuronal process distributed

throughout the deutocerebrum and one neuron located much closer to the antennal lobe (arrow). (i) One neuron observed much nearer to the outer region of the

deutocerebrum. (j) One representative, large and very intensely stained neuron located in the upper division of the frontal ganglion and strongly stained neuronal

arborization in the middle region of the frontal ganglion. (k) Two strongly stained neurons located in upper region of the frontal ganglion. (l) Two large, strongly stained

neurons located at the very front of the SOG. (m) Two large neurons in the ventrolateral mandibular region. (n) Three large neurons in the ventrolateral maxillary region. (o)

Two representative cells in the lateral mandibular region. Scale bar = 100 mm.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–1586 1579

over the brain and SOG like what has been detected with 5-HTantibody in both crickets. Six strongly stained cell bodies in D.

nigrofasciatus (Fig. 3b) and four strongly stained cells and threeweakly stained cell bodies in A. allardi (Fig. 4b and c) were found inthe pars intercerebralis region. The central body was free fromstaining in both crickets, but the nodulus below the central bodywas found to be intensely stained in D. nigrofasciatus. No stainingwas observed here in A. allardi (Fig. 3c). One moderately stainedmedium-sized cell body was observed in the dorsolateral region ofthe protocerebrum in D. nigrofasciatus but not in A. allardi (Fig. 3d,arrow). Two weakly stained cell bodies in D. nigrofasciatus and twomoderately stained cell bodies in A. allardi were found in the

posterior cortex of the protocerebrum (Figs. 3e and 4d). There wasonly one cell body located in the ventral medulla of the optic lobein D. nigrofasciatus, while no cell was found in A. allardi (Fig. 3f,arrow). Three large cell bodies were located in the optic tractregion of D. nigrofasciatus and two large cell bodies in A. allardi

(Figs. 3g and 4e). 5-HT1A-ir was found in the border of the corporacardiac and nervi corporis allati I (NCA I), but not in the corporaallata in both crickets (Fig. 3h, 4h and 4i).

Immunoreactivities in the tritocerebrum and SOG were grosslydifferent between the two crickets. The tritocerebrum was freefrom staining in D. nigrofasciatus but four to five small grouped cellbodies were found in A. allardi (Fig. 4g). In D. nigrofasciatus, only

[(Fig._2)TD$FIG]

Fig. 2. 5-HT-ir in the brain–SOG complex of A. allardi. (a) Representation of the numbers and topography of 5-HT-ir cells in the brain–SOG. (b) Neuronal process starting from

the pars intercerebralis region extending to the ventral protocerebrum region (arrow). (c) The strongly stained neuronal processes extending from the ventral protocerebrum

to the deutocerebrum (arrow). (d) Intense staining in the fan-shaped upper division of the central body. (e) Neuronal process in the POC. (F) Several cell bodies distributed in

the posterior cortex of the protocerebrum. (g) A neuron close to the antennal lobe was located in the deutocerebrum. (h) A neuron near the lateral region of the

deutocerebrum. (i) Intense neuronal arborization in the medulla region and several intensely stained neurons with axonal processes in the accessory medulla region of the

optic lobe and several cell bodies located between the medulla and the lobula. (j) One representative large cell body was intensely stained in the DL. (k) Neuronal arborization

in the lamina and medulla, and an axonal process from the optic lobe to the ventral protocerebrum (arrow). (l) Four representative large neurons in the frontal ganglion. (m)

Neuronal process in the nervi corporis allati I (NCA I). (n) Two strongly stained neurons located in the very frontal region of the SOG. (o) Two large motorneurons in the

ventrolateral region of the maxillary neuromere. (p) Cell bodies in the lateral mandibular region. (q) Axonal process extending from the median region to the lateral region of

the SOG. Scale bar = 100 mm.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–15861580

[(Fig._3)TD$FIG]

Fig. 3. 5-HT1A-ir in the brain–SOG complex of D. nigrofasciatus. (a) Map showing the numbers and topography of 5-HT1A-ir cells in the brain–SOG and possible routes of their

nerve processes. (b) Two strongly stained neurons located in the pars intercerebralis. (c) A strongly stained nodulus belonging to the central complex. (d) One small cell body

located in the dorsolateral region of the protocerebrum. (e) Four moderately stained cell bodies in the posterior cortex of the protocerebrum. (f) The only one cell found in the

ventral of the inner optic chiasma. (g) Two intensely stained large neurons in the optic tract. (h) Cortex staining in the corpora cardiac (CC). (i) Two weakly stained cell bodies

located in the mandibular neuromere of the SOG. Scale bar = 100 mm.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–1586 1581

two weakly stained neurons in the mandibular neruomere of theSOG were detected and three poorly stained cells were located inthe dorsal region of the SOG (Fig. 3i). However, in A. allardi, anintensely stained frontal commissure was found in the dorsalregion of the SOG (Fig. 4j). A group of four large neurons withaxonal processes extending backward to the dorsal region werefound in the mandibular neuromere (Fig. 4k, arrow), and 8–10large neurons clustered together at maxillary neuromere were alsofound (Fig. 4l, arrow). One cell body in the lateral mandibularneuromere was observed (Fig. 4m, arrow). The cell bodies locatedin the tritocerebrum and SOG also showed CLK-ir when double-labeling was conducted by fluorescence IHC (Fig. 4n–v).

Adult females and males reared under long-day and short-dayconditions were compared with respect to the distribution andstaining intensity of 5-HT1A-ir. No differences in the stainingpattern were found between sexes. Furthermore, no regularvariation in immunostaining was detected among samplescollected at 6 h intervals throughout a day. The Western blottingresults verified the IHC results in the head extracts of D.

nigrofasciatus. A single band with a size around 49 kDa wasdetected but without daily fluctuation under LD 12:12 (Fig. 7a).

3.3. 5-HT1B-like immunoreactivity in the cephalic ganglion of D.

nigrofasciatus and A. allardi

5-HT1B-ir in both crickets resembled each other but had muchhigher intensity in A. allardi. 5-HT1B-ir was always found in thecytoplasm. Faint signals were detected in the dorsolateral region of

the protocerebrum, posterior cortex of the protocerebrum, opticlobe, deutocerebrum, tritocerebrum, and the SOG in D. nigrofas-

ciatus, while strong reactivity was found in all these regions andpars intercerebralis in A. allardi. Two strongly stained cell bodiesand two weakly stained cell bodies were observed in the parsintercerebralis in A. allardi (Fig. 6b). Two moderately stained cellbodies were located in the posterior cortex of the protocerebrum inboth crickets (Figs. 5c and 6c). Two weakly stained cell bodies in D.

nigrofasciatus and 1 strongly stained cell body in A. allardi weredetected in the dorsolateral region of the protocerebrum (Figs. 5band 6d). Around 10–12 slightly stained cell bodies were observedin the accessory medulla region of the optic lobe in both crickets(Figs. 5d, arrow and 6f, arrow). Three to four weakly stained cellswere found in the dorsal part of the medulla in both crickets(Fig. 5d, arrowhead). A cluster of four to five weakly stained cellbodies was located in the tritocerebrum in both crickets (Figs. 5eand 6j). Three groups of neurons in the SOG were detected atdifferent intensities in both crickets, weakly in D. nigrofasciatus andstrongly in A. allardi. Four large cell bodies were located in themandibular neuromere (Figs. 5f and 6k) and 8–10 in the maxillaryneuromere in both crickets (Figs. 5g and 6l). Two neurons, with oneshowing an axonal process reaching to the dorsolateral maxillaryregion, were detected in the lateral labial neuromere of D.

nigrofasciatus (Fig. 5h). Two cell bodies were located in the lateralmandibular neuromere in both crickets (Figs. 5i and 6i). Double-labeling with anti-CLK and 5-HT1B sera showed that 5-HT1B-ir andCLK-ir were co-localized in the tritocerebrum and SOG in A. allardi

(Fig. 6m–t).

[(Fig._4)TD$FIG]

Fig. 4. 5-HT1A-ir in the brain–SOG complex of A. allardi. (a) Map showing the numbers and topography of 5-HT1A-ir cells in the brain–SOG and possible route of their nerve

processes. (b) and (c) Several neurons located in the pars intercerebralis region. (d) A representative cell body in the posterior cortex of the protocerebrum. (e) One of the two

large neurons situated in the optic tract (arrow). (f) A neuron in the lateral region of the deutocerebrum. (g) A cluster of small intensely stained cell bodies in the

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–15861582

[(Fig._5)TD$FIG]

Fig. 5. 5-HT1B-ir in the brain–SOG complex of D. nigrofasciatus. (a) Map of the numbers and topography of 5-HT1B-ir cells in the brain–SOG and possible routes of their nerve

processes. (b) Two faintly stained neurons in the dorsolateral region of the protocerebrum. (c) A representative cell body in the posterior cortex of the protocerebrum. (d)

Several slightly stained cell bodies located in the accessory medulla region of the optic lobe (arrow) and in the dorsal part of the medulla (star). (e) A cluster of small cell bodies

located in the tritocerebrum. (f) Two moderately stained neurons in the mandibular neuromere. (g) A group of slightly stained neurons in the maxillary neuromere. (h) A

neuron in the lateral labial neuromere with a long axonal process extending to the dorsal region of the lateral mandibular neuromere. (i) A moderately stained neuron in the

lateral mandibular neuromere. Scale bar = 100 mm.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–1586 1583

Adult females and males reared under LD 16:8 and LD 12:12conditions were compared with respect to the distribution andstaining intensity of 5-HT1B-ir. No differences in the stainingpattern were found between two sexes or between thesephotoperiods. Furthermore, no oscillation of 5-HT1B-ir wasdetected among the samples fixed at 6 h intervals throughout aday. The Western blotting results verified the IHC results in theheads extracts of D. nigrofasciatus. A single band around 29 kDawas detected but without daily fluctuation under LD 12:12(Fig. 7b).

4. Discussion

This study investigated the immunoreactivities against 5-HTand two 5-HT receptors (5-HT1A and 5-HT1B) in two groundcrickets, D. nigrofasciatus and A. allardi. 5-HT-ir has beeninvestigated in many insects, including Schistocerca gregaria

(Wurden and Homberg, 1995), Leucophaea madearae (Petri et al.,1995), Rhodnius prolixus (Miksys and Orchard, 1994), Periplaneta

americana (Bishop and O’Shea, 1983; Nishiitsutsuji-Uwo et al.,

tritocerebrum. (h) The CC was free from staining. (i) High intensity staining was observed

the dorsomedian region of the SOG. (k) Four large neurons with strongly stained axonal

maxillary neuromere. (m) A neuron situated in the lateral mandibula neuromere (arrow)

CLK-ir in the tritocerebrum. (o) 5-HT1A-ir in the tritocerebrum. (p) Merged image of CLK

SOG. (r) 5-HT1A-ir in the mandibular neuromere. (s) Merged image of CLK-ir and 5-HT1A

(u) 5-HT1A-ir in the maxillary neuromere. (v) Merged image of CLK-ir and 5-HT1A-ir in t

color in this figure legend, the reader is referred to the web version of the article.)

1984), M. sexta (Homberg and Hildebrand, 1989), Apis mellifera

(Schurmann and Klemm, 1984), Epitheca sp. and Pachydiplax

longipennis (Longley and Longley, 1986), and Aedes aegypti andAnopheles gambiae (Siju et al., 2008). However, this reportexamined the distributions of 5-HT-ir and two 5-HT receptors-irs in the same species and their potential relationship withcircadian clock.

5-HT-ir was widely and intensely expressed in the cephalicganglia of the two crickets and other species described above. Bothcrickets showed intense 5-HT-ir in the optic lobe. For example, 30small immunoreactive somata were found between the medullaand lamina in Triatoma infestans. Strong 5-HT-ir neurons found inthe accessory medulla region of the optic lobe of both cricketsresembled those found at the medial edge of the medulla in T.

infestans (Settembrini and Villar, 2004), and in the anterior medullaof the praying mantis optic lobe (Leitinger et al., 1999). Theaccessory medulla region of the optic lobe serves as a putativecentral pacemaker in D. nigrofasciatus, where PERIOD andDOUBLETIME were proved to be located (Shao et al., 2006).Furthermore, the optic lobe could be an output center from the

in the NCA I, but not in corpora allta (CA). (j) The strongly stained neuronal process in

processes in the mandibular neuromere. (l) A group of large neurons located in the

. (n–v) Double-labeling using anti-CLK (red) and anti-5-HT1A (green) antibodies. (n)

-ir and 5-HT1A-ir in the tritocerebrum. (q) CLK-ir the mandibular neuromere of the

-ir in the mandibular neuromere. (t) CLK-ir in the maxillary neuromere of the SOG.

he maxillary neuromere. Scale bar = 100 mm. (For interpretation of the references to

[(Fig._6)TD$FIG]

Fig. 6. 5-HT1B-ir in the brain–SOG complex of A. allardi. (a) Map of the numbers and topography of 5-HT1B-ir cells in the brain–SOG and possible routes of their nerve

processes. (b) Two strongly stained small cell bodies located in the pars intercerebralis. (c) One of the 5-HT1B-ir cells located in the posterior cortex of the protocerebrum. (d)

A neuron situated in the dorsolateral region of the protocerebrum. (e) The antennal lobe was strongly stained. (f) Several moderately stained cell bodies in the accessory

medulla region of the optic lobe. (g) Weakly stained neuronal arborization in the frontal ganglion. (h) The CC was moderately stained while the CA was free from staining. (i)

Two strongly stained neurons in the lateral mandibular neuromere. (j) A group of small weakly stained cell bodies in the tritocerebrum. (k) A representative large neuron in

the mandibular neuromere with a strongly stained axonal process extending to the dorsal SOG. (l) A group of several large neurons located in the maxillary neuromere. (m–t)

Double-labeling with anti-CLK and anti-5HT1B in the tritocerebrum and SOG. (m) CLK-ir in the tritocerebrum. (n) 5-HT1B-ir in the tritocerebrum. (o) Merged image of CLK-ir

and 5-HT1B-ir in the tritocerebrum. (p) CLK-ir in the mandibular neuromere. (q) 5-HT1B-ir in the mandibular neuromere. (r) Merged image of CLK-ir and 5-HT1B-ir in the

mandibular neuromere. (s) CLK-ir in the maxillary neuromere. (t) 5-HT1B-ir in the maxillary neuromere. (u) Merged image of CLK-ir and 5-HT1B-ir in the maxillary

neuromere. Scale bar = 100 mm.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–15861584

circadian pacemaker in both crickets, because PDF—a putativeoutput signal of the circadian rhythm in Drosophila, is located inthis region in both crickets (Shao, unpublished observation).Meinertzhagen and Pyza (1996) and Cymborowski (1998)implicated 5-HT as a neurotransmitter in the visual system of

insects. The extensive network of 5-HT-ir in the lamina, medulla,and the strongly stained neurons in the optic lobe may indicate 5-HT as a neurotransmitter in the regulation of visual sensitivity forboth crickets. The intensive staining of 5-HT-ir neuropiles in theantennal lobe of the deutocerebrum suggest serotonin may work

[(Fig._7)TD$FIG]

Fig. 7. 5-HT1A and 5-HT1B immunoreactivities on the western blots of proteins

extracted from the heads of D. nigrofasciatus. Samples were collected at 6-h intervals

under LD 12:12 Zeitgeber time 0 (ZT lanes) is designated the time of light-on. (a) A

single band around 49 kDa was detected with the 5-HT1A antibody, but no obvious

daily oscillation was observed. (b) A single band around 29 kDa was detected with

the 5-HT1B antibody, but no obvious daily oscillation was observed.

Q.-M. Shao et al. / Journal of Insect Physiology 56 (2010) 1576–1586 1585

as olfactory receptor or tactile sense receptor in the two cricketsalthough no direct proof available. Changes in the volume of the 5-HT-ir varicosities in the antennal lobe throughout a 24 h periodwas detected in mosquitoes (Siju et al., 2008). However, such achange was not clearly observed in the present study. A largedeutocerebral 5-HT-ir neuron arborized in the glomeruli of theantennal lobes found in cockroach (Salecker and Distler, 1990) andthe honey bee (Schurmann and Klemm, 1984) was also detected inthese crickets. 5-HT content in the OL shows a daily fluctuation tosynchronize the sensitivity among the vision interneurons in thecricket, G. bimaculatus (Tomioka et al., 1993), while present studyshowed no daily oscillation of 5HT-ir in both crickets. The fanshaped upper division of the central body showed intense 5-HT-irbut the lower division was devoid of staining, as found in otherspecies such as S. gregaria, S. americana (Homberg, 1991), and T.

infestans (Settembrini and Villar, 2004). However, unlike the 64 5-HT-ir columnar neurons connecting the protocerebal bridge of S.

gregaria (Homberg, 2002), and in Periplaneta where the proto-cerebral bridge was most strongly stained (Nishiitsutsuji-Uwoet al., 1984), the present study showed no neuronal arborization inthe protocerebral bridge. The large neurons near the root of themandibular nerve, maxillary nerve, and labial nerve in the SOGshown in the two crickets also occurred in the cockroach, P.

americana (Davis, 1987; Nishiitsutsuji-Uwo et al., 1984), T.

infestans (Settembrini and Villar, 2004), and two mosquitoes, A.

aegypti and A. gambiae (Siju et al., 2008). The efferent neuronsstained strongly in the frontal ganglion in both crickets were alsodetected in M. sexta (Homberg and Hildebrand, 1989) and P.

americana (Nishiitsutsuji-Uwo et al., 1984). The intense and widedistribution of the 5-HT-ir in the brain–SOG system of both cricketsindicates that 5-HT may serve as a very strong neuroactivesubstance in the two species.

To date, very little is known about the function and distributionof 5-HT receptors in the central nervous system of insects. In D.

melanogastor, 5-HT1A loss of function mutant flies had short andfragmental sleep, which could be rescued by expressing thereceptor in the adult mushroom bodies (Yuan et al., 2006). In D.

melanogastor, the 5-HT2 receptor was expressed in the proto-cerebrum and ellipsoid body, which are believed to regulate higherorder behaviors, including learning, locomotion, and sensoryperception (Nichols, 2007). In G. bimaculatus, the 5HT7-likereceptor mediates the serotonergic suppression of the photo-responsiveness of the medulla bilateral neurons (Saifullah andTomioka, 2003). The 5-HT1A receptor has been identified at bothpresynaptic and postsynaptic locations (Miquel et al., 1992; Radjaet al., 1992). This study showed distinct 5-HT-ir and two receptor-like-ir pattern in the brain–SOG system. This suggests that thesereceptors are most likely postsynaptic receptors rather thanrelease-modulating autoreceptors. Neither receptor was detectedin the central body or frontal ganglion, where a strong 5-HT signalwas detected. And, only one small weak 5-HT1A-ir cell body in D.

nigrofasciatus and no 5-HT1A-ir signal in A. allardi were detected inthe optic lobe where intensive 5-HT-ir neuronal staining wasobserved, indicating that some other receptor may exist in theseregions. Although both 5-HT-ir and receptor-ir were detected inthe SOG, 5-HT occurred in completely different locations from thetwo receptors: 5-HT was located in the lateral region of the SOG,while the two receptors were located in the midline of the SOG.This means that both receptors are postsynaptic receptors, notautoreceptors in SOG region. 5-HT1A-ir and 5-HT1B-ir co-localizedwith CLK-ir in the mandibular and maxillary neuromere region inA. allardi, while in D. nigrofasciatus only 5-HT1B-ir was co-localizedwith CLK-ir. Previous results demonstrated that PER-ir and CKIa-irare co-localized in the accessory medulla region of the optic lobe inD. nigrofasciatus, while they may be coexpressed in the tritocer-ebrum and the maxillary and mandibular neuromeres of the SOGin A. allardi (Shao et al., 2006). Our previous study also showed thatimmunoreactivity against some neuropeptides, such as crustaceancardioactive peptide (CCAP), corazonin, and diapause hormone(DH), were co-localized in the tritocerebrum and the maxillary andmandibular neuromeres of the SOG in both species (Sehadovaet al., 2007). It was also found previously that CYC-ir and CLK-ir areco-localized in these groups of neurons in both crickets (Shao et al.,2008a,b). 5-HT1A-ir in the SOG differed between the two crickets.As reported previously PER-ir and casein kinase Ia (CKIa)-ir (Shaoet al., 2006), an interspecific difference was found between thesetwo crickets. Similarly, weak 5-HT1A-ir was found in two neuronsin the SOG in D. nigrofasciatus, while strong reactivity appeared inthe tritocerebrum and SOG in A. allardi. These results suggest that5-HT may have some relationship with the circadian clock in thesecrickets although no functional analysis was done in this study.This is also in agreement with earlier reports in other cricketspecies and Drosophila. For example, in G. bimaculatus, injectionwith 5,7-dihydroxytryptamine (5,7-DHT) into the optic loberesulted in enhanced masking activity at lights-on that occurredafter a 6 h phase advance (Germ and Tomioka, 1998). Furthermore,in G. bimaculatus, an increase in the cerebral 5-HT level occurredalmost simultaneously with rhythm reversal from diurnal tonocturnal, suggesting that 5-HT plays a role in the post-embryonicchange in the locomotor rhythm (Nishinokubi and Tomioka, 2000).In the cricket, Acheta domesticus, an injection of p-CPA (para-chlorophenylanalnin), an inhibitor of serotonin biosynthesis,resulted in arrhythmicity followed by a reversed phase activityrhythm (Renucci et al., 1989). In Drosophila, d5-HT1B wasexpressed in the clock network and affected circadian lightsensitivity by decreasing the activity of GSK3, which, in turn,produced increased stability of TIMELESS (Yuan et al., 2005).

Acknowledgements

This work was supported by JSPS Postdoctoral Fellowship forForeign Researchers (IP No. P07428) and a JSPS grand-in-aid forscientific research (B 18380042).

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