Ann-Shyn Chiang

Hormonal control of sexual receptivity in cockroaches

Many animals exhibit specific behaviors associated with sexual receptivity only when they are reproductively competent. In insects with gonadal maturation cycles, these behaviors usually coincide with ovarian matruation. In the cockroachBlattella germanica, juvenile hormone (JH), produced by the corpora allata (CA), regulates female reproductive physiology. Various experimental manipulations, including ablation of the CA, therapy with JH analogs, CA denervation, ovariectomy, and changing nutrient quality, coupled with time-lapse video recording, support the hypothesis that JH also controls female sexual receptivity. A re-examination of the role of the CA in the maturation of male sexual readiness shows that, while sexual behavior develops in the absence of JH in bothB. germanica andSupella longipalpa, JH accelerates the expression of sexual readiness.

Improved conditions for culture of biosynthetically active cockroach corpora allata

SummaryCurrently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.

Control of Cell Proliferation in the Corpora Allata During the Reproductive Cycle of the Cockroach Diploptera punctata

Unlike in Blettelta gcrmanica and Supella longipalpa, the corpora allata (CAI of Diploptera punctafa exhibited cyclic changes in cell number during the reproductive cycle. In mated females, a wave of DNA synthesis followed by mitosis resulted in a significant increase in CA cell number from about 9,000 cells on day 0 to 12,000 cells at ovulation on day 8. Subsequently, the number of cells per CA underwent a gradual decline to about 10,000 cells by day 64. During this long period of gestation, mitotic activity was undetectable (by colchicine arrest) and pycnotic nuclei were frequently observed by transmission electron microscopy. Just before parturition on day 72 another mitotic wave was detected and CA cell number increased again. The early wave of CA cell proliferation could be postponed by delaying mating or abolished by maintaining females as virgins. Neural disconnection of the CA from the brain mimicked the effect of mating, suggesting that enhanced cell proliferation is permitted by the removal of inhibitory signals from cerebral neurosecretory cells. The proliferative activities after mating were neither abolished by ovariectomy, which suppressed the normal increase in JH synthesis, nor elevated by unilateral allatectomy, which doubled the rates of JH synthesis in the remaining CA. These data corroborate previous results (Szibbo and Tobe, 1981 a; Tobe et al., 1984; Johnson et at., 1993) and suggest that waves of cell proliferation and JH synthesis, though simultaneous, are regulated independently by inhibitory signals from cerebral neurosecretory cells. D 1996 Witey-Liss, Inc.

Neural and hormonal regulation of growth of corpora allata in the cockroach, Diploptera punctata

DNA synthesis and mitosis in the corpora allata (CA) of adult Diploptera punctata males were investigated with total cell count after 5-bromo-2-deoxyuridine immunodetection and colchicine arrestment both in vivo and in vitro. The CA exhibited a single wave of DNA synthesis followed by cell division during the first 4 days after the imaginal ecdysis. A second mitotic wave was experimentally induced after the nervous connections between the CA and the brain were severed on day 4. Spontaneous mitosis was abolished in cockroaches treated with a juvenile hormone (JH) analog. This inhibitory regulation in vivo appeared to act through brain neurosecretory cells since in the denervated CA mitotic activity was unaffected by JH treatment. An in vitro system supporting growth of the corpus allatum was established to study direct hormonal effects. By using continuous bromodeoxyuridine labeling in vitro for 6 days, we showed that DNA synthesis of corpus allatum cells was unaffected by direct contact with JH. In contrast, 20-hydroxyecdysone exerted direct mitogenic action on allatal cells. These and previous results suggest that CA cells alternate between JH synthesis and a proliferative state in which they divide in a self-renewing fashion to yield differentiated progeny. We propose that in newly enclosed adult Diploptera punctata males, low JH titer and high ecdysteroid titer promote mitosis in CA cells. As the ecdysteroid titer declines, JH produced by the CA acts on brain neurosecretory cells which dispatch inhibitory signals through nerves to prevent continuous proliferation of CA cells.

Allatostatin inhibition and farnesol stimulation of corpus allatum activity in embryos of the viviparous cockroach, Diploptera punctata

Juvenile hormone (JH) biosynthesis by corpora allata (CAI from embryos of the cockroach Diploptera punctata was measured at four stages during the latter half of embryogenesis. Individual glands from 32-day-old embryos that had completed 49% of embryonic development synthesized 0.3 pmol JH Ill h-. By day 46 (70% development) gland activity rose to 1.1 pmol JH h-, but on subsequent days JH synthetic rates declined, measuring only 0.8 pmol h- on day 56 (86% development) and 0.5 pmol h- on day 60 (92% development). Differences in JH biosynthesis by CA from different-aged embryos were more evident when gland activity was corrected for either corpus allatum cell number, which increased progressively from fewer than 200 cells per gland on day 32 to almost 700 cells per gland on day 60, or embryo mass, which increased from 1.6 mg per embryo on day 32 to 10.8 mg per embryo on day 60. JH biosynthetic rates were significantly inhibited in a medium containing M Dip-allatostatin 7 which suppressed CA activity by 68, 83, 76, and 51°% on days 32, 46, 56, and 60, respectively. In all embryonic stages JH production was significantly stimulated by incubation of glands with 200 pM farnesol, a late precursor in the JH biosynthetic pathway. D 1~96 Wcy-Liss, Inc.

Neural control of cell size in the corpora allata during the reproductive cycle of the cockroach Diploptera punctata (Dictyoptera: Blaberidae)

Summary Rising and subsequent falling rates of juvenile hormone (JH) synthesis occurred concurrently with synchronous growth and atrophy of CA cells during the ovarian cycle in mated adult females of Diploptera punctata. Ultrastructural observations revealed that growth of CA cells resulted from synchronous proliferation of cellular machinery required for JH synthesis. Cell growth was suppressed in CA of virgin females, in which rates of JH synthesis remained low, but was stimulated by mating or by severance of nerves leading from the brain to the CA. Atrophy of CA cells during declining rates of JH synthesis was due to synchronous autophagy of cellular organelles. While the mechanism initiating autophagy is unclear, it is independent of nervous connections between the CA and brain. We propose that under normal physiological conditions the quantity of JH synthesized by a corpus allatum is determined largely by the total amount of cellular machinery available for JH production. Therefore, the cycle of JH s...

Juvenile hormone synthesis in relation to corpus allatum development in embryos of the viviparous cockroach Diploptera punctata

Summary Few studies have addressed endocrinology of the corpora allata (CA) in insect embryos. We now report on development and biosynthetic activity of CA in embryos of a viviparous cockroach, Diploptera punctata. When newly-eclosed adult females of D. punctata were mated, they oviposited and gave birth, respectively, about 8 and 73 days later; thus, gestation and corresponding embryogenesis lasted approximately 65 days. Dorsal closure, which coincides with differentiation of the CA, was concluded when embryos were about 13 days old and had completed 20% of embryogenesis. Reverse phase-high performance liquid chromatography revealed that embryonic CA released predominantly juvenile hormone III (JH) in vitro. Furthermore, an in vitro radiochemical assay showed that between day 28 of embryogenesis (43% of embryonic development completed) and hatch rates of JH synthesis rose, plateaued and then fell. When CA activity was increasing or was high, from day 28 to 54 (83% development), mitosis occurred at low an...

Development‐activity relationships in nymphal corpora allata of the cockroach, Diploptera punctata

Abstract. In females of Diploptera punctata the corpora allata undergo a gradual increase in volume during most of the second nymphal stadium. In the first half of the stadium, steady growth of the glands results from a progressive increase in the size of constituent cells. Late in the stadium, cell size declines but the volume of the glands continues to rise due to an increase in cell number. Changes in cell size during the stadium displayed a distinct pattern in relation to Juvenile Hormone (JH) synthesis. Both cell size and activity increased during the first two‐thirds of the stadium, peaked early in the last third of the stadium, and decreased before the moult. The rise in cell numbers late in the stadium corresponded to a wave of cellular mitosis and occurred after a steep decline in the rate of JH biosynthesis. Exposure of late second instars to fenoxycarb. a JH analogue, depressed mitosis significantly, suggesting autocrine regulation of cell proliferation in the corpora allata. Possible mechanisms modulating sequential cycles of growth and atrophy of cells and cell proliferation in these glands are discussed in relation to temporal patterns of JH and ecdysteroid titres in nymphs.

IN VITRO GROWTH OF CORPORA ALLATA FROM DIPLOPTERA PUNCTATA

SummaryAn in vitro organ culture system was established to support growth of corpora allata from the cockroachDiploptera punctata. During a 1-wk incubation in L-15B medium supplemented with 10% fetal bovine serum (FBS) and 10% cockroach hemolymph, adult male corpora allata exhibited a cycle of de novo DNA synthesis followed by cell division. The number of S-phase cells and metaphase cells per corpus allatum were counted from whole-mount monolayers after labeling in vitro with 5′-bromo-2′-deoxyuridine and exposure to colchicine, respectively. While both FBS and cockroach hemolymph were essential for proliferation of allatal cells, the growth-promoting effect of insect hemolymph was not species-specific and adult female hemolymph was more potent than hemolymph from adult males. Furthermore, DNA synthesis of corpus allatum cells was stimulated in vitro by 20-hydroxyecdysone. This sensitive assay system will be of immense utility in the search for allatal growth factors.