Molecular Characterization, Developmental Expression and Immunolocalization of Clathrin Heavy Chain in the Ovary of the American Cockroach, Periplaneta Americana During Oogenesis

Clathrin is the principal protein involved in receptor mediate endocytosis and the main component of the coated vesicles. It is composed of three identical clathrin heavy chains (CHC), each with an attached light chain. We characterized the deduced amino acid sequence of the partial cDNA clone of the American cockroach, Periplaneta americana (Pam) CHC. The analysis showed that this sequence is represented as multiple alpha helical repeats occurred in the arm region of the CHC and displayed a high level of identity and similarity to mosquitoes and Drosophila melanogaster CHCs. This is the first report on CHC from a hemimetabolous insect. The amplified CHC probe could hybridize two CHC transcripts in the current preparations, 6.3 kb and 7.3 kb. The Northern blot analysis confirmed that a 6.3 kb transcript is specifically expressed in ovarian tissues at high levels throughout the ovarian development, especially in previtellogenic ovaries (Days 1-4) but dropped during the vitellogenic period (days 5-7) and ultimately no transcript was detected in fully vitellogenic ovaries (days 9-13). Immunoblot analysis detected an ovary specific CHC protein of ~175 kDa that was present in previtellogenic ovaries on the day of female emergence and after initiation of vitellogenesis and onset of Vg uptake. Immunocytochemistry localized CHC protein to germ-line derived cells, oocytes, and revealed that CHC translation begins very early during oocyte differentiation in the germarium. The present work suggested a possible role for clathrin in the early fluid phase endocytosis (pinocytosis) in addition to its role in receptor-mediated endocytosis

Molecular characterization, developmental expression and immunolocalization of clathrin heavy chain in the ovary of the American cockroach, Periplaneta americana during oogenesis

Clathrin is the principal protein involved in receptor mediate endocytosis and the main component of the coated vesicles. It is composed of three identical clathrin heavy chains (CHC), each with an attached light chain. We characterized the deduced amino acid sequence of the partial cDNA clone of the American cockroach, Periplaneta americana (Pam) CHC. The analysis showed that this sequence is represented as multiple alpha helical repeats occurred in the arm region of the CHC and displayed a high level of identity and similarity to mosquitoes and Drosophila melanogaster CHCs. This is the first report on CHC from a hemimetabolous insect. The amplified CHC probe could hybridize two CHC transcripts in the current preparations, 6.3 kb and 7.3 kb. The Northern blot analysis confirmed that a 6.3 kb transcript is specifically expressed in ovarian tissues at high levels throughout the ovarian development, especially in previtellogenic ovaries (Days 1-4) but dropped during the vitellogenic period (days 5-7) and ultimately no transcript was detected in fully vitellogenic ovaries (days 9-13). Immunoblot analysis detected an ovary specific CHC protein of ~175 kDa that was present in previtellogenic ovaries on the day of female emergence and after initiation of vitellogenesis and onset of Vg uptake. Immunocytochemistry localized CHC protein to germ-line derived cells, oocytes, and revealed that CHC translation begins very early during oocyte differentiation in the germarium. The present work suggested a possible role for clathrin in the early fluid phase endocytosis (pinocytosis) in addition to its role in receptor-mediated endocytosis

Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods

The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it Leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system

Serotonin Receptor B May Lock the Gate of PTTH Release/Synthesis in the Chinese Silk Moth, <i>Antheraea pernyi</i>; A Diapause Initiation/Maintenance Mechanism?

<div><p>The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, <i>Antheraea pernyi</i>. We have cloned cDNAs encoding two types of serotonin receptors (5HTR_{A and B}). 5HTR_A-, and 5HTR_B-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA^5HTRB responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTR_A was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA^5HTRB resulted in early diapause termination, whereas that of dsRNA^5HTRA did not affect the rate of diapause termination. The injection of dsRNA^5HTRB induced PTTH accumulation, indicating that 5HTR_B binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTR_B may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.</p> </div

Relative mRNA levels of 5HTRs upon long-day activation.

<p>Diapause pupae were exposed to LD 16:8 for 0, 5 and 10 cycles at 25°C and mRNA level of 5HTR_A (gray bar) and 5HTR_B (white bar) in the BR-SOG was determined by real time PCR. The results are presented as the mean ± S.E.M. from three independent experiments. Asterisks indicate significant difference from 0-day incubation by one-way ANOVA (Fisher's, LSD). <i>p<</i>0.05.</p

Pharmacological confirmation of RNAi effect targeting at 5HTR_B.

<p>Effect of injections of 5HT, 5,7-DHT and luzindole on photoperiodism. (A) Diapause pupae were injected either with 5 µl water and 5 µl DMSO (Mock injection) or 5 pmoles Luzindole plus 5 pmoles 5HT in the same volume of solvent and placed under LD: 16:8 at 25°C. Cumulatively 5% adults emerged in 40 days after injection. Cont.: untreated. M: injection with distilled water and DMSO. Luzindole +5HT: luzindole and 5HT co-injected. (B) Diapause pupae were injected with 5,7-DHT at three doses and thereafter the pupae were kept under LD 12:12 at 25°C. Cont.: untreated. M: mock injection with 10 µl distilled water. 5,7-DHT: injected with 5,7-DHT dissolved in the same volume as in the mock. 5 percentage points, 30 percentage points and 100 percentage points adult emergence was observed in 40 days. Asterisks indicate significant difference from control by Kaplan-Meier. ** <i>p</i><0.01.</p

Colocalization of 5HTR_A- and PTTH-ir in the adult BR-SOG of

<p><b><i>A. pernyi</i></b>. <i>Bm</i>PTTH-ir/<i>Ap</i>PTTH-ir in the BR-SOG of female adult with 24hrs after emergence was co-localized with <i>Ap</i>5HTR_A-ir. (A) The locations of detected cells. Lower-case letters correspond to the regions (capital letters) shown in the photographs (e.g., b to B). (B) Two large PTTH-ir neurons in the DL region. (C, D, H) Two large 5HTR_A-ir neurons in the DL region. (E) <i>Bm</i>PTTH-ir in the DL region. (F) Merged image of <i>Bm</i>PTTH- and 5HTR_A-ir in the DL region. (G) <i>Ap</i>PTTH-ir in the DL region. (I) Merged image of <i>Ap</i>PTTH and 5HTR_A in the DL region. Scale bar = 100 µm.</p

Schematic illustration of 5HTRs role on diapause induction/maintenance in pupal diapause of <i>A. pernyi</i>.

<p>The moth has two 5HTR subtypes, 5HTR_A and 5HTR_B.The former subtype shows no transcription rhythm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079381#B24" target="_blank">24</a>] and did not respond to photoperiodic activation by long day, while the latter showed rhythmic expression and responded to photoperiodic activation. Therefore, it may be regulated by circadian system. Via one type of arylalkylamine N-acetyltransferase, aaNAT. This engyme is encoded by <i>nat</i> is that a circadian-controlled gene (ccg) since dsRNA^CYC, and dsRNA^CLK suppressed <i>nat</i> transcription and dsRNA^NAT dysfunctioned photoperiodism. Transcription of 5HTR_A was not rhythmic. MT-binding closes the endocrine switch to PTTH release that finally terminates diapause, while 5HTR_B cuts this circuit to enforce or initiate diapause. Diapause of <i>A.penyi</i> is therefore under binary regulation and circadian system regulates at least two points in this system, <i>nat</i> transcription and 5HTR_B expression. aaNAT, arylalkylamine N-acetyltransferase. PER, Period protein, a negative regulator of transcription translation feedback. CYC/CLK, heterodimeric circadian transcription regulator. Mel, melatonin. MT, melatonin receptor. LD, long day. SD, short day. PTTH, prothoracicotropic hormone. PTG, prothoracic gland. E, ecdysone. 20E, 20 hydroxyecdysone.</p

Colocalization of 5HTR_B- and PTTH-ir in the erly pupal BR-SOG of

<p><b><i>A. pernyi</i></b>. <i>Bm</i>PTTH-ir/<i>Ap</i>PTTH-ir in the 5-day-old pupal brain of <i>A. pernyi</i> and its colocalization with <i>Ap</i>5HTR_B-ir (red filled circles). <i>Bm</i>EH-ir in the 5-day-old pupa brain and its colocalization with <i>Ap</i>5HTR_B-ir (blue filled circles) and unique distribution (open circles) of <i>Bm</i>EH-ir in other regions of the brain. (A) The loaction of detected cells. Lower-case letters correspond to the regions shown in the photographs (e.g., b to B). (B) One <i>Bm</i>EH-ir neuron in the PI. (C) <i>Bm</i>EH-ir in the DC region. (D, H) 5HTR_B-ir in the DL region. (E) <i>Bm</i>PTTH-ir in the DL region. (F) Merged image of <i>Bm</i>PTTH-ir and 5HTR_B-ir in the DL region. (G) <i>Ap</i>PTTH-ir in the DL region. (I) Merged image of <i>Ap</i>PTTH-ir and 5HTR_B-ir in the DL region. (J) 5HTR_B-ir in the DC region. (K) One EH-ir neuron in the DC. (L) Merged image of <i>Bm</i>EH-ir and 5HTR_B-ir in the DC region. Scale bar = 100 µm.</p

Colocalization of 5HTR_A- and PTTH-ir in the early pupal BR-SOG of

<p><b><i>A. pernyi</i></b>. <i>Bm</i>PTTH-ir/<i>Ap</i>PTTH-ir was co-localized with <i>Ap</i>5HTR_A-ir in the BR-SOG of 5-day-old pupa. (A) The location of detected cells. Lower-case letters correspond to the regions shown in the photographs (e.g., b to B). (B) Two large PTTH-ir neurons in the DL region. (C, D, H) Two large 5HTR_A-ir neurons in the DL region. (E) <i>Bm</i>PTTH-ir in the DL region. (F) Merged image of <i>Bm</i>PTTH- and 5HTR_A-ir in the DL region. (G) <i>Ap</i>PTTH-ir in the DL region. (I) Merged image of <i>Ap</i>PTTH-ir and 5HTR_A-ir in the DL region. Scale bar = 100 µm.</p