Reproductive female medaka show larger amount of food intake.

(A) Light conditions for breeding and non-breeding status. (B) Food intake (10 mins) of female medaka in LD (orange; n = 7) and SD (blue; n = 7) conditions, normalized to the amount of artemia eaten by medaka in LD (breeding) condition (p = 0.02519). (C) Food intake (10 mins) of male medaka in LD (orange; n = 7) and SD (blue; n = 7) conditions, normalized to the amount of artemia eaten by medaka in LD (breeding) condition (p = 0.6540). Mann-Whitney U test, * p < 0.05. n.s., not significant.

agrp1-, agrp2-, and npyb-expressing neurons are distributed locally, while npya-expressing neurons are distributed more widely in the brain.

(A) Illustration of the lateral view of medaka brain and distributions of cell bodies expressing each agrp or npy gene. The oblique lines labeled with a-e indicate the level of the frontal sections in a-e. (a-e) Illustrations of frontal sections showing the distribution of neurons expressing each gene. The localization of neurons is indicated in the right half of the illustrations. dDm dorsal region of area dorsalis telencephali pars medialis, Dl area dorsalis telencephali pars lateralis, BO bulbus olfactorius, Vd area ventralis telencephali pars dorsalis, Vv area ventralis telencephali pars ventralis, Vs area ventralis telencephali pars supracommissuralis, lfb lateral forebrain bundle, Vl area ventralis telencephali pars lateralis, TO tectum opticum, NVT nucleus ventralis tuberis, PIT pituitary, NAT nucleus anterior tuberis, NRL nucleus recessus lateralis. (B) agrp1-expressing neurons are localized in NVT. Scale bar: 100 μm. (C) agrp2-expressing neurons are localized in Vv. Scale bar: 100 μm. (D) npya-expressing neurons are distributed in dDm, Dl, BO, Vs, Vl, lfb, NRL, NAT, NVT, and TO. Scale bar: 100 μm. (E) npyb-expressing neurons are localized in Vd. Scale bar: 50 μm. (F) agrp1 (magenta) and npya (green) are distributed in NVT, but the two genes are not co-expressed. (G-J) agrp and npy expressions in the whole brain of female medaka under normally fed condition (Fed; white; n = 7) or 2-week food restricted (FR; gray; n = 8). (G) agrp1 (p = 0.001243) (H) agrp2 (p = 0.9551) (I) npya (p = 0.2319) (J) npyb (p = 0.0003108) Mann-Whitney U test, ** p < 0.01, *** p < 0.001. n.s., not significant.

agrp1 and npyb show higher expression levels in LD than in SD female.

(A) agrp1 expression in the whole brain of LD (orange; n = 8) and SD (blue; n = 8) female medaka. (p = 0.001865) (B) ISH of agrp1-expressing neurons in LD and SD female medaka. Scale bar: 100 μm. (C) The number of neurons expressing agrp1 in LD (orange; n = 5) and SD (blue; n = 5). (p = 0.02828) (D) Time course of the number of neurons showing ISH signals for agrp1 at each 30-min time point from the beginning of the chromogenic reaction in LD (orange; n = 5) and SD (blue; n = 5). The positive neurons were counted from 0 min to 300 min after application of NBT/BCIP (see Material and Methods), and the reaction saturated at 300 min onward. (E) Ratio of cell number at 90 min divided by that at 300 min. (p = 0.2948) (F) npyb expression in the whole brain of LD (orange; n = 8) and SD (blue; n = 8) female medaka. (p = 0.0001554) (G) The number of neurons expressing npyb in LD (orange; n = 4) and SD (blue; n = 4). (p = 0.3429) The upper, middle, and lower bars show the third quartile, median, and the first quartile, respectively. Mann-Whitney U test, * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., not significant.

In juvenile female medaka, expression levels of neither agrp1 nor npyb shows significant change according to the day-length.

(A) agrp1 expression in the brain of juvenile female medaka. (p = 0.4179) (B) npyb expression in the brain of juvenile female medaka. (p = 0.3357) LD: orange, n = 8; SD: blue, n = 7. The upper, middle, and lower bars show the third quartile, median, and the first quartile, respectively. Mann-Whitney U test. n.s., not significant.

Estrogen, which is secreted from mature ovary, may affect the expression of agrp1.

(A) Relative expression of agrp1 in Sham (white; n = 6), OVX (Ovariectomized medaka, gray; n = 6), and OVX + E (OVX medaka kept in the tank containing E2, yellow; n = 7). Sham vs OVX: p = 0.04310, OVX vs OVX+E: p = 0.05790, Sham vs OVX+E: p = 0.2000. (B) Relative expression of npyb in Sham, OVX, and OVX + E. Sham vs OVX: p = 0.1386, OVX vs OVX+E: p = 0.9991, Sham vs OVX+E: p = 0.0812. The upper, middle, and lower bars show the third quartile, median, and the first quartile, respectively. Steel-Dwass test, * p < 0.05. n.s., not significant. (C) Photographs of brain slices after experiments of double in situ hybridization (agrp1 (green) and estrogen receptors (magenta)). (D) Expanded photograph of agrp1 and esr2a co-expressing neurons (white arrowhead). Scale bars: 50 µm.

agrp1−/− female medaka show decrease in food intake and the number of fertilized eggs.

(A) Lateral views of representative agrp1+/+ and agrp1−/− female medaka (Left), and body weight, body length, and abdominal height (right) of agrp1+/+ (white; n = 6) and agrp1−/− (gray; n = 6). All the fish are 4-month-old adult medaka. Scale bar: 1 cm. body weight: p = 0.04113; body length: p = 0.3939; abdominal length: p = 0.002165. (B) Food intake (10 min) of agrp1+/+ (white; n = 6) and agrp1−/− (gray; n = 6) female medaka. Each amount of food intake is normalized by the average number of that of agrp1+/+ medaka. (p = 0.004329) (C) Food intake (10 min) of LD agrp1−/− (white; n = 9) and SD agrp1−/− (gray; n = 7) female medaka. Each amount of food intake is normalized by the average number of that of LD agrp1−/− medaka. (p = 0.5953) (D) The number of eggs spawned by agrp1+/+ (white; n = 6) and agrp1−/− (gray; n = 5) female medaka in a day. Each female was paired with a wildtype male. (p = 0.008658). (E) Photograph of ovary in representative agrp1+/+ and agrp1−/− female (left) and the gonado-somatic index (GSI, right). n = 6 of each group. (p = 0.06494) Scale bar: 1 mm. (F) Expression of gonadotropin genes (lhb and fshb) in the pituitary of agrp1+/+ (white; n = 6) and agrp1−/− (gray; n = 6) female medaka. lhb: p = 0.008658); fshb (p = 0.02597). The upper, middle, and lower bars show the third quartile, median, and the first quartile, respectively. Mann-Whitney U test, * p < 0.05, ** p < 0.01. n.s., not significant.

Illustration of mechanisms of breeding season-dependent feeding behavior in medaka suggested by the present study

(A) Long day (LD) condition in breeding season induces ovarian maturation, which facilitates release of estrogen (E2) from the mature ovary. (B) High concentration of serum E2 increases agrp1 expression in the brain via the estrogen receptors, especially, esr2a. (C) Higher expression of AgRP1 is suggested to activate neural circuitry for feeding, which leads to an increase in food intake and egg spawning of female medaka in the breeding season.