Vitellogenins (Vg) genes code for the major egg yolk protein precursor in insects and many other oviparous species. In insects, the Vg gene is expressed extra-ovarially in the fat body in sex-, tissue-and stage-specific manners. During the reproductive phase, the Vg mRNA is expressed in large quantities, which is then translated, secreted into hemolymph and ultimately taken up by the developing oocytes through receptormediated endocytosis. Once sequestered, the Vgs are stored as vitellin (Vn), the main nutritional reserve for the developing embryo. The regulation of Vg genes is directly under the control of hormones at the transcriptional level. Hormones involved in Vg gene transcription are juvenile hormone (JH), ecdysteroids and some neuropeptides. The overall understanding that has emerged is that the insects can be classified, based on the system of hormonal regulation of Vg gene transcription, into three groups: (i) insects (like most of hemipterans) that use only JH for Vg gene transcription; (ii) insects (like dipterans) that need both JH and ecdysteroids for Vg regulation; and (iii) insects like lepidopterans that require JH, ecdysteroids and additional hormones to regulate their reproductive biology. However, why insect species diverge in using different hormones to govern their reproductive physiology remains unclear. The present contribution focuses on the current status of knowledge regarding the regulation of Vg genes in insects. Besides a brief information on biochemical and molecular features, the role of Vg genes as a target of endocrine disruptors will be addressed. Also, the molecular mechanism of Vg gene regulation will be discussed.

Although the regulation of vitellogenesis in insects has been mainly discussed in terms of ‘classical’ lipid hormones, juvenile hormone (JH), and 20-hydroxyecdysone (20E), recent data support the notion that this process must be adjusted in harmony with a nutritional input/reservoir and involvement of certain indoleamines and neuropeptides in regulation of such process. This study focuses on crosstalks among these axes, lipid hormones, monoamines, and neuropeptides in regulation of vitellogenesis in the American cockroach Periplaneta americana with novel aspects in the roles of arylalkylamine N-acetyltransferase (aaNAT), a key enzyme in indoleamine metabolism, and the enteroendocrine peptides; crustacean cardioactive peptide (CCAP) and short neuropeptide F (sNPF). Double-stranded RNA against aaNAT (dsRNAaaNAT) was injected into designated-aged females and the effects were monitored including the expressions of aaNAT itself, vitellogenin 1 and 2 (Vg1 and Vg2) and the vitellogenin receptor (VgR) mRNAs, oocyte maturation and changes in the hemolymph peptide concentrations. Effects of peptides application and 20E were also investigated. Injection of dsRNAaaNAT strongly suppressed oocyte maturation, transcription of Vg1, Vg2, VgR, and genes encoding JH acid- and farnesoate O-methyltransferases (JHAMT and FAMeT, respectively) acting in the JH biosynthetic pathway. However, it did not affect hemolymph concentrations of CCAP and sNPF. Injection of CCAP stimulated, while sNPF suppressed oocyte maturation and Vgs/VgR transcription, i.e., acting as allatomedins. Injection of CCAP promoted, while sNPF repressed ecdysteroid (20E) synthesis, particularly at the second step of Vg uptake. 20E also affected the JH biosynthetic pathway and Vg/VgR synthesis. The results revealed that on the course of vitellogenesis, JH- and 20E-mediated regulation occurs downstream to indoleamines- and peptides-mediated regulations. Intricate mutual interactions of these regulatory routes must orchestrate reproduction in this species at the highest potency.

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.

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Melatonin (MEL) orchestrates daily and seasonal rhythms (eg, locomotion, sleep/ wake cycles, and migration among other rhythms) in diverse organisms. We investigated the effects of pharmacological doses (0.03-1 mM) of exogenous MEL intake in the cockroach, Periplaneta americana, on locomotor activity. As per os MEL concentration increased, cockroach locomotor rhythm in light-dark (LD) cycles became more synchronized. The ratio of night activity to 24-h activity increased and the acrophase (peak) slightly advanced. MEL application also influenced total activity bouts in the free-running rhythm. Since MEL slightly influenced τ in the freerunning rhythms, it is not a central element of the circadian pacemaker but must influence mutual coupling of multi-oscillatory system components. Arylalkylamine N-acetyltransferase (aaNAT) regulates enzymatic production of MEL. aaNAT activities vary in circadian rhythms, and the immunoreactive aaNAT (aaNAT-ir) is colocalized with the key clock proteins cycle (CYC)-ir and pigment-dispersing factor (PDF)-ir These are elements of the central pacemaker and its output pathway as well as other circadian landmarks such as the anterior and posterior optic commissures (AOC and POC, respectively). It also partially shares immunohistochemical reactivity with PER-ir and DBT-ir neurons. We analyzed the role of Pamericana aaNAT1 (PaaaNAT1) (AB106562.1) by injecting dsRNA aaNAT1 . qPCR showed a decrease in accumulations of mRNAs encoding PaaaNAT1. The injections led to arrhythmicity in LD cycles and the arrhythmicity persisted in constant dark (DD). Continuous administration of MEL resynchronized the rhythm after arrhythmicity was induced by dsRNA aaNAT1 injection, suggesting that PaaaNAT is the key regulator of the circadian system in the cockroach via MEL production. PaaaNAT1 contains putative E−box regions which may explain its tight circadian control. The receptor that mediates MEL 2 of 19 | KAMRUZZAMAN et Al How to cite this article: Kamruzzaman ASM, Hiragaki S, Watari Y, et al. Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach, Periplaneta americana, via melatonin/MT2-like receptor.