Crosstalk among Indoleamines, Neuropeptides and JH/20E in Regulation of Reproduction in the American Cockroach, Periplaneta americana

Abstract

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 (dsRNA^aaNAT) 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 dsRNA^aaNAT 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.

Keywords: 20-hydroxyecdysone; Periplaneta americana; aaNAT; biogenic amines; juvenile hormone; neuropeptides; oocytes; vitellogenesis.

Figure 1

Aralkylamine N-acetyltransferase (aaNAT) acts in different aspects of vitellogenesis. (A) The relative expression of aaNAT mRNA in female brain and fat body compared to actin standard in adult females of P. americana 4 days post-injection with dsRNA^aaNAT. (B) Oocyte length of adult females of P. americana 4 days post-injection with dsRNA^aaNAT. (C) Expression of vitellogenin 1 and 2 (Vg1 and Vg2) and vitellogenin receptor (VgR) mRNAs in female fat body of P. americana 4 days after dsRNA^aaNAT injection. (D) Expression of genes encoding JH acid methyltransferase (JHAMT) and farnesoate O-methyltransferases (FAMeT) in the brain of adult females of P. americana 4 days post-injection with dsRNA^aaNAT. Data are presented as mean ± SEM. The significant differences are indicated by * (p < 0.05; B) or ** (p < 0.01; A, C, D), as compared to the controls, using Student’s t-test.

Figure 2

Concentrations of hemolymph crustacean cardioactive peptide (CCAP) (A) and short neuropeptide F (sNPF) (B) 4- and 7-days post-injection of P. americana adult females with dsRNA^aaNAT. Data is displayed as mean ± SEM, (n = 25 individuals/condition). n.s., no statistically significant differences were observed between the tested groups using the Student’s t-test.

Figure 3

Effect of CCAP injection on oocyte growth and 20E titers in hemolymph. (A) Effect of CCAP injection on oocyte size in adult females. Adult females were injected daily with 10 pmol CCAP (dissolved in 5 μL of PBS), from day 1 to day 15 of the adult stage. Controls were injected with 5 μL PBS. (B) Effect of CCAP injections on ecdysteroid concentrations in the hemolymph. A total amount of 10 p moles of CCAP (in 5 μL of PBS) was injected daily into the hemocoel of female adults from day 1 to day 15. Control insects were injected with 5 μL of PBS. Ecdysteroid level was measured by the enzyme immunoassay (EIA). Data are expressed as mean ± SEM (n = 25 individuals/condition). ***: represent significant difference (p < 0.001) as compared to the control group, using Tukey’s test.

Figure 4

Effect of sNPF treatments on oocyte size and 20E titers in hemolymph. (A) Effect of sNPF injection on oocyte size in adult females. Adult females were injected daily with 10 pmol sNPF (dissolved in 5 μL PBS), from day 1 to day 15 of the adult stage. Control insects were injected with 5 μL PBS into the hemolymph. (B) Effect of sNPF injections on ecdysteroid concentrations in the hemolymph. A total 10 pmol of sNPF (in 5 μL of PBS) was injected daily into the hemocoel of female adults from day 1 to day 15. Control insects were injected with 5 μL of PBS. Ecdysteroid level was measured by the EIA. Values are displayed as mean ± SEM (n = 25 individual/condition). ***: significantly different (p < 0.001) compared to the controls, using Tukey’s test.

Figure 5

Vg1, Vg2, and VgR expression in the fat body of normally fed adult females of P. americana 4 days post-injection with CCAP and sNPF. Data are presented as mean ± SEM. Means followed by different letters are significantly different (p < 0.05), using Duncan’s multiple range test.

Figure 6

JHAMT and FAMeT expression in the fat body 4 days after CCAP and sNPF injection into normally fed P. americana. Data are presented as mean ± SEM. Means followed by unlike letters are significantly different (p < 0.05), using Duncan’s test.

Figure 7

Immunocytochemical detection of VgR in P. americana ovaries from virgin female by immunofluorescence. Control (A), CCAP injected (B) and sNPF injected (C) of normally fed insect 4 days after injection. Scale bar = 200 μm.

Figure 8

Dose response for induction by 20E. Expression of JHAMT and FAMeT in the brain of adult P. americana females 4 days after injection with 10 µL of the different given concentrations of 20E. Control roaches were injected with 10 µL of ca. 0.1% EtOH solvent. Data are presented as mean ± SEM. Columns with symbols (*: JHAMT; #: FAMeT) are significantly different at p < 0.05, compared to 0 concentration, using Tukey’s test.

Figure 9

Dose response for reduction by 20E. Expression of Vg1, Vg2, and VgR mRNA in P. americana female fat body 4 days after injection with 10 µL of the different doses of 20E. Control roaches were injected with 10 µL of ca. 0.1% EtOH solvent. Data are shown as mean ± SEM. Columns with symbols (*: Vg1; #: Vg2; □: VgR) are significantly different at p < 0.05, compared to 0 concentration, using Tukey’s test.

Figure 10

The “physiological wormhole model” for control of insect reproduction. Three distinct routes of neuroendocrine pathways, running in parallel or interactive or linearly connected. Probably non-endocrine regulatory pathways operate to regulate reproductive timing, associative behaviors, metabolic supports, JH/20E, CCAP/sNPF and monoamines as well as the biological clock, longevity, and cell cycle machinery.