Susan M. Rankin

Cockroach allatostatin-immunoreactive neurons and effects of cockroach allatostatin in earwigs

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to demonstrate the presence of allatostatin-immunoreactive cells and fiber tracts in the neuroendocrine system of the earwig Euborellia annulipes. The corpora cardiaca cells were not immunoreactive, nor were the neurosecretory endings of fiber tracts from the brain to the corpora cardiaca. No immunoreactive material was detected in the corpus allatum, although the corpus allatum contained neurosecretory endings, and some cells of the brain, including medial and lateral protocerebral cells, showed immunoreactivity. In addition, the recurrent and esophageal nerves were allatostatin-positive. The last abdominal ganglion contained immunoreactive somata, and immunoreactive axons of the proctodeal nerve innervated the rectum, anterior intestine, and posterior midgut. We did not detect reactive endocrine cells in the midgut. Allatostatin I at concentrations of 10(-5) and 10(-7) M did not inhibit juvenile hormone biosynthesis by E. annulipes corpora allata in vitro. This was true for glands of low activity from 2-day females and brooding females, as well as for relatively high activity glands from 10-day females. In contrast, 10(-7) M allatostatin I significantly and reversibly decreased hindgut motility. Motility was decreased in hindguts of high endogenous motility from 2-day females and in those of relatively low activity from brooding females. These results support the notion that a primary function of allatostatin might be to reduce gut motility.

Juvenile hormone in earwigs: Roles in oogenesis, mating, and maternal behaviors

The role of juvenile hormone (JH) in courtship, mating, maternal behavior, and the ovarian cycle was studied in the ring-legged earwig, Euborellia annulipes (Lucas). The single, median corpus allatum makes and secretes JH III. JH III production was low in newly eclosed adult females, increasing as oocytes developed, maximal at about the time of oviposition, and low again in brooding females. Application of 35 or 122 μg JH III to newly eclosed females hastened the onset of courtship behavior, but had no effect on the age at which females first mated nor on the duration of mating, though the trend is toward advanced onset. Hormone treatment advanced the age of first oviposition and reduced clutch size and the proportion of eggs hatching but did not affect the interval from oviposition of the first clutch to oviposition of the second clutch, nor the size and proportion hatching of the second clutch. Acetone treatment and treatment with 6 μg JH III did not affect these parameters. Application of 50 μg JH III to females on the day of oviposition shortened the duration of maternal care and advanced the onset of the second gonadotropic cycle, compared with that of acetone-treated and precocene II-treated females. The duration of maternal care was positively correlated with the proportion of eggs hatching. JH titer analysis confirmed JH III to be the predominant hormone in this species and clearly demonstrated the absence of other homologues. This work also confirmed our hypothesis that intermediate to high levels of JH are associated with oocyte growth, mating, and cessation of maternal care; low levels of JH are associated with the period of maternal behavior and slow ovarian development. We are currently investigating factors which might regulate corpus allatum activity during the reproductive cycle and the subsequent period of maternal care. Arch. Insect Biochem. Physiol. 35:427–442, 1997.

Maternal behavior and clutch manipulation in the ring-legged earwig (Dermaptera: Carcinophoridae).

The expression and maintenance of maternal behavior in the earwig,Euborellia annulipes, was examined through manipulation of clutch size, age, and species and through observations of interactions between brooding females. Females underwent discrete gonadotrophic cycles culminating in oviposition of first clutches that were highly variable in size. Neither the head capsule width nor the age of the mother was correlated with clutch size. Maternal care extended through embryogenesis and for the week following hatching. Clutch removal significantly shortened the interclutch interval, indicating that the presence of brood inhibited the onset of the second gonadotrophic cycle. Brooding females readily accepted replacement clutches of the same age. Thus, mothers did not appear to distinguish their own eggs from those of other females. Experimental doubling of clutch size did not significantly reduce the proportion hatching or fledging. In contrast, reducing clutch size diminished the percentage successfully fledging. Manipulation of clutch age resulted in reduced hatching/fledging success. Placing two females, each with newly laid clutches, in the same cage usually resulted in egg transfer from the nest of one female to that of the other within 12 h. Nests of females with larger forceps were significantly more likely to contain both clutches. When mothers with first clutches were paired with mothers with third clutches, eggs were more likely to be transferred to the nest of the older female.E. annulipes females with newly laid clutches appeared to accept as replacement clutches eggs of the earwigDoru taeniatum. Alien clutches were maintained for the typical duration of embryogenesis; however, noD. taeniatum hatchlings were observed.

Physiological correlates to courtship, mating, ovarian development and maternal behaviour in the ring‐legged earwig

Abstract. The involvement of the endocrine system in courtship, mating, maternal behaviour and the gonadotrophic cycle was studied in the earwig, Euborellia annulipes, after application of JH III and, alternatively, precocene II.

Biosynthesis and release of juvenile hormone during the reproductive cycle of the ring-legged earwig

Corpora allata of adult female Euborellia annulipes, incubated in medium containing 3H-methionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 ± 1.6% of the total juvenile hormone III synthesized was released and 96.5% ± 0.3 in medium supplemented with 60 μM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 ± 0.004 mm), hormone production was only 0.59 ± 0.13 fmol hr−/corpus allatum; production increased to 1.52 ± 0.25 fmol hr−1/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 ± 0.13 fmol hr−1/corpus allatum. The addition of 60 μM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of l-methionine in the medium also enhanced hormone biosynthesis. Maximal hormone production was 32.8 ± 10.9 fmol hr−1/corpus allatum, at an l-methionine concentration of 51 μM.

Partial characterization and isolation of earwig `allatostatins': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroach, Diploptera punctata, that inhibit juvenile hormone production in that species (but do not do so in earwigs), and inhibit hindgut muscle contractions in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adult female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatostatin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. Material from each peak at 10 nM Dip-AST equivalents inhibited juvenile hormone biosynthesis in vitro by corpora allata of 2-day virgin D. punctata cockroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) purified extracts of brains of earwigs containing 1 nM Dip-AST equivalents failed to suppress hindgut contractions in vitro of 2-day earwigs and of brooding female earwigs. In contrast, 1 nM cockroach allostatin 1 (Dip-AST 7) reversibly inhibited hindgut contractions in vitro. These results suggested the presence of another brain factor, such as proctolin, that counteracts the inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at 1 nM; the effects of equal concentrations of allatostatin and proctolin varied with the stage of the female. Furthermore, HPLC-separated fractions that co-eluted with cockroach allatostatin and were immunoreactive with antibodies to Dip-AST suppressed hindgut contractions in vitro of 2-day female earwigs. Finally, crude brain extracts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro in glands of low, but not in glands of high, activity. Thus, earwig brain extract after HPLC separation has Dip-AST-like material that inhibits cockroach corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extracted earwig brain material, however, does not inhibit earwig gut contraction. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain extract that does have this action.

Effects of selected neuropeptides, mating status and castration on male reproductive tract movements and immunolocalization of neuropeptides in earwigs.

In earwigs, the male reproductive system is complex, comprising accessory glands and long dual intromittent organs for transfer of materials to the female and for removal of rival sperm. We investigated potential factors altering contractions of the male reproductive tracts in vitro. Tracts from 0-day (newly emerged) males displayed relatively little motility in vitro; however, those from 5-day (intermediate stage of sexual maturity) and 8-day (fully mature) males pulsed vigorously. Both 1 and 100 nM proctolin (RYLPT-OH) stimulated the rate of contraction of reproductive tracts from both 5-day and 8-day males. In contrast, 1 nM and 100 nM FGLa AST (cockroach allatostatin) did not affect pulsations. However, 10 microM FGLa AST decreased activity of reproductive tracts. Mating decreased motility of tracts from 5-day old males, but did not alter motility of tracts from 8-day old males. Castration of larvae significantly suppressed reproductive tract motility in subsequent 8-day old adults compared with those of intact or sham-operated adults. Castration also suppressed seminal vesicle size. Lastly, we assessed the presence and distribution of proctolin-like and allatostatin-like immunoreactivity in tissues. Immunoreactivity to FGLa AST and proctolin was widespread, occurring in the brain and ventral ganglia. Surprisingly, we did not detect immunoreactivity to either FGLa AST or proctolin within the reproductive system; however, proctolin immunoreactivity was evident in nerves extending from the terminal ganglion of 8-day, but not 0-day, males. Collectively, these experiments demonstrate that the male earwig reproductive system is an appropriate model for use in addressing sexual maturation and activities in male insects.

Juvenile hormone esterase activity during the reproductive cycle of the ring-legged earwig☆

Corpora allata of the ring-legged earwig, Euborellia annulipes, produce and secrete juvenile hormone III (JH III). The primary degradation product of JH III in vitro by diluted hemolymph was juvenile hormone acid, as assessed by thin-layer and high-performance liquid chromatography. This result was confirmed by derivatization of the degradation product using diazomethane. Conversion of JH III to JH acid by E. annulipes hemolymph in vitro occurred at a constant rate for at least 4 hr. The addition of 10−6 M O-ethyl-S-phenyl phosphoamidothiolate or, alternatively, 3-octyl thio-1,1,1-trifluoropropan-2-one inhibited hemolymph esterase activity; O,O-diisopropylphosphorofluoridate and ethanol alone had no effect on the conversion of JH III to JH acid. At 10−4 M, each of the selected inhibitors reduced hemolymph esterase activity relative to ethanol-treated and untreated controls. JH esterase activity of diluted hemolymph was relatively high in females with basal follicles < 0.2 mm and low in those with larger basal follicles. The change in JH esterase activity of the hemolymph was not correlated with changes in hemolymph protein content; hemolymph protein content was generally 50–100 μg/μ1 and did not change significantly with either age or basal follicle length.

Effects of diet and mating status upon corpus allatum activity, oocyte growth, and salivary gland size in the ring‐legged earwig

The effect of diet on mating behavior and the subsequent effects of diet and mating status on the biosynthesis of juvenile hormone III, basal follicle length, salivary gland size and total body weight were assessed in the ring‐legged earwig, Euborellia annulipes (Lucas) (Family Carcinophoridae; subfamily Carcinophorinae) during the first 15 days of adult life (the first gonadotrophic cycle of those fed presumably near‐optimal diets of catfood) and again on day 25 (late vitellogenesis of the second gonadotrophic cycle of those fed catfood). Diets of catfood, honey, fructose and total starvation, respectively, imposed on 0‐day adult females did not affect sexual receptivity, mating success or duration of mating as assessed on day 7. With the addition of a group of virgin, catfood fed females, we noted that only those females maintained on catfood oviposited within 25 days; enforced virginity virtually abolished oviposition. Total food deprivation of females as well as diets of honey or fructose abolished the cycles in total body weight, basal follicle length, salivary gland size and juvenile hormone production. Thus, starvation decreased the reproductive success of these insects, and carbohydrates only (fructose) or in combination with trace amounts of nutrients and protein (honey) were not sufficient to promote reproduction and associated cycles in this insect. Furthermore, virgins failed to undergo the decreases in salivary gland size that were characteristic of mated females. Among mated, catfood‐fed females, the second cycle in juvenile hormone production appeared to be smaller than the first.

The effect of ovariectomy on reproductive behaviour and physiology of adult female ring‐legged earwigs, Euborellia annulipes

The effect of ovariectomy on feeding, mating, Juvenile Hormone (JH) production, and maternal behaviour was assessed in female ring‐legged earwigs, Euborellia annulipes (Lucas) (Dermaptera: Carcinophoridae), during the first 16 days of adult life (the first gonadotrophic cycle and early brooding).