Quantification of cockroach allatostatin-like peptide and its myotropic effects in males of the earwig Euborellia annulipes

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to establish a competitive enzyme‐linked immunosorbent assay for quantification of allatostatin‐like peptides in the hindgut of the adult male earwig, Euborellia annulipes. Hindguts of 0‐day males contained significantly more allatostatin‐positive material than those of 8‐day males fed on catfood. However, males starved for the first 8 days of adult life had significantly higher levels of allatostatin‐positive material than those of either 0‐day or of 8‐day fed males. Hindguts from 0‐day old males exhibited lower spontaneous motility in vitro than those from 8‐day males. Hindguts from males at both ages responded to allostatin with reversible, dosage‐dependent decreases in hindgut motility, and responded to proctolin with reversible, dosage‐dependent increases in hindgut motility. When both allatostatin and proctolin were applied to hindgut preparations simultaneously and in equal concentrations, the response varied with the stage of the male. Starvation enhanced hindgut motility and abolished the response to allatostatin, but not to proctolin. These results indicate the presence of material similar to cockroach allatostatins in male earwigs, and that the levels change with age and physiological stage. Furthermore, such peptides may indeed be regulatory neuropeptides and could modulate hindgut contraction. There was an increase in sensitivity to exogenous allatostatin in the hindgut during development from day 0 to day 8 in feeding males, but a loss in sensitivity in response to starvation; sensitivity to exogenous proctolin also increased with age, but such responsiveness was not diminished by starvation.     

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.     

Effect of aging on male reproduction in zebrafish (Danio rerio)

The study was designed to test the hypothesis that male aging is associated with a change in reproductive function in the zebrafish. Young (290 ± 37 d) and older (911 ± 48 d) males were combined with females (604 ± 24 d) to test the effect of male age on the number and fertility of eggs laid by their mates. 48% of breeding trials with young males and 25% of the trails with older males resulted in egg deposition. Although young males were associated with significantly more successful breeding attempts than older males, number of eggs laid per clutch, number and percent of fertilized eggs and the number and percent living embryos were not statistically different between young and older males. These data suggest that male aging is associated with altered reproductive behavior and/or female response but not in sperm quality per se. Consistent with this interpretation were the findings that percent motility and sperm motility characteristics did not differ between sperm from young and older males as assessed by computer-assisted sperm analysis. However, older males contained higher quantities of extractable sperm than did young males, perhaps associated with fewer successful breeding attempts. Age-related effects on male reproductive in the zebrafish may therefore be a consequence of behavioral or morphological features that play a role in female mate choice and/ or male sexual response.     

Sperm stock and mating of males in a parasitoid wasp

Hymenoptera are haplodiploid insects, consequently sex ratio depends on female's sperm management which itself arises from the reproductive capacity of neighbouring males. To study the influence of ageing on male reproductive potential, laboratory experiments were conducted on Dinarmus basalis (Hymenoptera, Pteromalidae) males, a tropical wasp in which sperm counts are known to constrain sex ratio. Two groups of virgin males were compared: 1-day and 30-days old. Parameters recorded were sperm quantity and viability in seminal vesicles, shape of testis, mating ability in both individual and competitive situations and sperm stored by females after male multiple mating. Older males had twice as much sperm as young males, but their reproductive capacities did not differ. They were able to copulate with 20 successive virgin females in a short period. Sperm stored in spermathecae decreased with female mating order. In competition, old and young males had the same access to females. The difference between old and young males was visible at the level of reproductive tract: young males have functional testis and old males have empty non-functional testis. Spermatozoa are kept viable in male seminal vesicles for long periods. In this species, the reproductive potential of males is not altered by ageing. At the population level this may represent an adaptation for maintaining continuous reserves of sperm at the disposal of females.     

Determinants of Mating Frequency in the Spiny Orbweaving Spider,Micrathena gracilis (Araneae: Araneidae)

Male Micrathena gracilis require two copulations, separated by a dismount, in order to inseminate both reproductive tracts of the female. We examined several factors that might influence a male's copulatory success. Web structure influenced male courtship and dismount tactics, but not copulatory frequency. The presence of another male reduced the likelihood of a given male copulating with both tracts, a limitation mediated by sexual responsiveness of the female. Mating status of the female did influence copulatory frequency; males were less likely to copulate a second time with nonvirgin females. In summary, males modify mating activities to reduce predation by females, to reduce intermale competition, and to avoid expending gametes when there is little chance of fertilization. Females influence males by predatory activities, mediated through web structure, and enhancing sperm competition among males.     

Are non-reproductive male highveld mole-rats, Cryptomys hottentotus pretoriae physiologically suppressed while in the confines of the natal colony?

The highveld mole-rat, Cryptomys hottentous pretoriae is a social, subterranean, co-operative breeder with a high reproductive skew. The relationship between reproductive status and reproductive physiology in male highveld mole-rats was investigated. Testicular morphometrics, histological parameters, plasma testosterone concentrations and sperm motility parameters were studied in 31 males (14 reproductive and 17 non-reproductive males). Reproductive males were significantly larger than non-reproductive males, with testicular mass and volume corrected for body mass considerably larger for the reproductive males. Circulating plasma testosterone concentrations of reproductive males were not significantly higher than non-reproductive males (reproductive males 10.3±1.8 nmol l⁻¹ vs non-reproductive males 7.3±1.74 nmol l⁻¹). Sperm motility parameters were measured, but no significant differences were found between reproductive and non-reproductive males. All 14 reproductive males had motile sperm, whereas 13 of the 17 (76%) of non-reproductive males possessed motile sperm. A typical ejaculate of a reproductive male contains 48.3% motile sperm characterized by a high percentage of flagellar defects, whereas the non-reproductive male has an ejaculate containing 45.6% motile sperm with a high percentage of head defects. It is apparent that non-reproductive males are not physiologically suppressed from reproducing. Moreover, the non-reproductive males are excluded from incestuous matings as a result of being the offspring of the reproductive female.     

Reproductive potential of male silver foxes Vulpes vulpes after long selection for the domesticated behavior type

Age peculiarities of reproductive activity and fertility during the reproductive season as well as the hormonal and the testis spermatogenic function were studied in silver foxes Vulpes vulpes from the population selected for a long time for domesticated behavior. It has been established that during the first reproductive season the male sexually activity (the coupled females’ number) was reduced as compared with subsequent seasons, and about one third of the males were not coupled at all with females; however, the young (one-year old) males did not differ from adults by the testis weight, ejaculate spermatozoid number, and the fraction of abnormal spermatozoids. Comparison of indexes of spermatogenesis and the testicular hormonal function in the young males with a decreased and “normal” sexual activity did not reveal any statistically significant differences. It is suggested that the decreased (or absent) sexual activity in young silver fox males can be a response of the population to the human-created environment.     

Experimental evolution exposes female and male responses to sexual selection and conflict in Tribolium castaneum

Between-individual variance in potential reproductive rate theoretically creates a load in reproducing populations by driving sexual selection of male traits for winning competitions, and female traits for resisting the costs of multiple mating. Here, using replicated experimental evolution under divergent operational sex ratios (OSR, 9:1 or 1:6 ♀:♂) we empirically identified the parallel reproductive fitness consequences for females and males in the promiscuous flour beetle Tribolium castaneum. Our results revealed clear evidence that sexual conflict resides within the T. castaneum mating system. After 20 generations of selection, females from female-biased OSRs became vulnerable to multiple mating, and showed a steep decrease in reproductive fitness with an increasing number of control males. In contrast, females from male-biased OSRs showed no change in reproductive fitness, irrespective of male numbers. The divergence in reproductive output was not explained by variation in female mortality. Parallel assays revealed that males also responded to experimental evolution: individuals from male-biased OSRs obtained 27% greater reproductive success across 7-day competition for females with a control male rival, compared to males from the female-biased lines. Subsequent assays suggest that these differences were not due to postcopulatory sperm competitiveness, but to precopulatory/copulatory competitive male mating behavior.     

Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.     

IDENTIFICATION OF THE FGL‐AMIDE ALLATOSTATIN GENE OF THE PRIMITIVE TERMITE Mastotermes darwiniensis AND THE WOODROACH Cryptocercus darwini

Allatostatins with the C‐terminal ending Tyr/Phe‐Xaa‐Phe‐Gly‐Leu/Ile‐amide (FGLa/ASTs) are widespread neuropeptides with multiple functions. The gene encoding the FGLa/AST polypeptide precursor was first isolated from cockroaches and since then could be identified in many insects and crustaceans. With its strictly conserved regions in combination with variable regions the gene seems to be a good candidate for phylogenetic analyses between closely and distantly related species. Here, the structure of the FGLa/AST gene of the most primitive termite, the giant northern termite Mastotermes darwiniensis Froggatt, was identified. The FGLa/AST gene of the woodroach Cryptocercus darwini was also determined. Precursor sequences of both species possess the general organization of dictyopteran FGLa/AST precursors containing 14 putative FGLa/AST peptides. In M. darwiniensis, only 11 out of the 14 FGLa/AST‐like peptides possess the C‐terminal conserved region Y/FXFGL/I/V/M and four of the putative peptide structures are not followed by a Gly residue that would lead to nonamidated peptides. Phylogenetic analyses show the high degree of similarity of dictyopteran FGLa/AST sequences. The position of termites, nested within the Blattaria, confirms that termites have evolved from primitive cockroaches.     

Age‐specific oxidative status and the expression of pre‐ and postcopulatory sexually selected traits in male red junglefowl, Gallus gallus

Oxidative stress is emerging as a key factor underpinning life history and the expression of sexually selected traits. Resolving the role of oxidative stress in life history and sexual selection requires a pluralistic approach, which investigates how age affects the relationship between oxidative status (i.e., antioxidants and oxidative damage) and the multiple traits contributing to variation in reproductive success. Here, we investigate the relationship between oxidative status and the expression of multiple sexually selected traits in two‐age classes of male red junglefowl, Gallus gallus, a species which displays marked male reproductive senescence. We found that, irrespective of male age, both male social status and comb size were strongly associated with plasma oxidative status, and there was a nonsignificant tendency for sperm motility to be associated with seminal oxidative status. Importantly, however, patterns of plasma and seminal antioxidant levels differed markedly in young and old males. While seminal antioxidants increased with plasma antioxidants in young males, the level of seminal antioxidants remained low and was independent of plasma levels in old males. In addition, old males also accumulated more oxidative damage in their sperm DNA. These results suggest that antioxidant allocation across different reproductive traits and somatic maintenance might change drastically as males age, leading to age‐specific patterns of antioxidant investment.     

Reproduction in the male sub-Antarctic fur seal Arctocephalus tropicalis

The reproductive tracts of male sub-Antarctic fur seals Arctocephalus tropicalis ( n = 123), taken at Gough Island (40° 20'S, 9° 54'W) between November 1977 and October 1978, were examined. The presence of spermatozoa in the epididymal tubules showed that all males ≥4 years old had reached puberty, and the marked slower increase in mean testis weight and baculum length suggested that sexual (social) maturity was approached by 8-year-olds. Full adulthood was attained at 10–11 years of age based on the peak in mean testis and prostate weights, and mean baculum length. Secondary sexual characteristics were only fully developed in males ≥9 years old. A significant decline in mean testosterone concentration, and in testis, epididymis and prostate weights showed that adult males were reproductively quiescent during winter from May to July when seminiferous and epididymal tubules had lowest mean diameters with no spermatozoa present. Both the mean plasma testosterone concentration and mean testis weight peaked twice during the austral summer. The first peak coincided with the breeding season, and the second peak with the moulting period when adult males were impotent. Photoperiodic cueing might explain this seasonal trend.     

Characterization of male reproductive anatomy of the endangered Arabian oryx (Oryx leucoryx)

Reproductive tracts of four male Arabian oryx (Oryx leucoryx) from Shaumari Nature Reserve in Jordan were examined to characterize their reproductive anatomy. Animals were allocated into two groups based on their age: Group 1 (n = 2, males were 12 and 14 mo old) and Group 2 (n = 2, males were 7 and 9 yrs old). Observations regarding the morphology, position and orientation of different reproductive organs were made. The external and internal genital organs of male oryx were similar to other domestic ruminant species with minor differences.     

Androgens in relation to prenatal development and postnatal inversion of the gubernacula in rats.

Exposure of male rats to the anti-androgen flutamide during fetal life, from day 10 after conception to the day of birth, allowed quantitatively unaltered development of the gubernacula. Apparently, androgens play no important role or no role at all in their growth. Castration of newborn male rats did not interfere with the inversion during further postnatal life of the gubernacula to create the muscular parts of the scrotum (cremaster muscles). Prenatal exposure to flutamide, followed by castration immediately after birth, also allowed gubernacular inversion and cremaster muscle growth. Neonatal administration of testosterone, after castration at birth, did not enhance gubernacular inversion or promote cremaster muscle growth in infancy or during adulthood. Apparently, postnatal gubernacular inversion and cremaster muscle growth are independent not only of androgens, but also of all testis hormones. Neonatal administration of the potent androgen 5 alpha-dihydrotestosterone propionate suppressed gonadotrophin secretion and, in intact males, inhibited testicular growth. Administration from the day of birth to day 33 delayed testicular descent and enhanced growth of the genital apparatus, but did not affect the size of the cremaster muscles. These experiments indicate that androgens are not involved in the processes that create the cavities into which testes descend to acquire their full reproductive potential.     

Metabolism of exogenous androgens in rams and wethers: differential clearance rates

The effect of the presence of the testes on the clearance rates of 5 and 50 mg of testosterone and androstenedione was examined in 8-month old rams and wethers. Castration did not affect (P greater than 0.10) the clearance of androstenedione or testosterone at the 5 mg dosage. Castration had an effect (P less than 0.05) on the clearance of 50 mg of androstenedione and testosterone. These data indicate that reproductive status has a differential effect on the metabolism of exogenously administered steroids.     

Role of septum and preoptic area in regulating masculine and feminine sexual behavior in male rats

The effects of septal or preoptic lesions on both masculine and feminine sexual behaviors were examined in castrated adult male rats. Three weeks after brain surgery, animals were implanted with Silastic tubes containing testosterone (T) and observations of masculine sexual behavior were carried out four times every 5 days. T tubes were removed immediately after the end of the masculine behavioral tests. Two weeks later, animals implanted with Silastic tubes containing estradiol-17 beta(E2) were subjected to three feminine sexual behavioral tests at 5-day intervals. The bilateral lateral septal lesion (LSL) and the medial preoptic lesion (MPOL) effectively suppressed the performance of mounts, intromissions, and ejaculations, whereas the medial septal lesion (MSL), the dorsolateral preoptic lesion (DPOL), and the sham operation did not show any significant suppression of these behaviors. In the feminine sexual behavioral tests, intact and sham-operated control males showed only a low lordotic activity. However, the performance of the lordosis reflex was markedly facilitated by LSL or DPOL, while the lordotic activity of MSL and MPOL males was not significantly different from that of control males. These results suggest that the lateral septum exerts not only a facilitatory influence on masculine sexual behavior but also an inhibitory influence on feminine sexual behavior in male rats. On the other hand, the medial preoptic area may play a critical role in regulating masculine sexual behavior in male rats.     

Subordinate male cichlids retain reproductive competence during social suppression

Subordinate males, which are excluded from reproduction often save energy by reducing their investment in sperm production. However, if their position in a dominance hierarchy changes suddenly they should also rapidly attain fertilization capability. Here, we asked how social suppression and ascension to dominance influences sperm quality, spermatogenesis and reproductive competence in the cichlid Astatotilapia burtoni, where reproduction is tightly coupled to social status. Dominant territorial (T) males are reproductively active while subordinate non-territorial (NT) males are suppressed, but given the opportunity, NT males will perform dominance behaviours within minutes and attain T male testes size within days. Using the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) to label germ cell proliferation, we found that the spermatogenic cycle takes approximately 11-12 days, and social status had no effect on proliferation, suggesting that spermatogenesis continues during reproductive suppression. Although sperm velocity did not differ among social states, NT males had reduced sperm motility. Remarkably, males ascending in status showed sperm motility equivalent to T males within 24 h. Males also successfully reproduced within hours of social opportunity, despite four to five weeks of suppression and reduced testis size. Our data suggest that NT males maintain reproductive potential during suppression possibly as a strategy to rapidly improve reproductive fitness upon social opportunity.     

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. Arch. Insect Biochem. Physiol. 38:155–165, 1998. © 1998 Wiley-Liss, Inc.     

Scent-marking behaviour by male prairie voles,Microtus ochrogaster,in response to the scent of opposite- and same-sex conspecifics

We conducted an experiment using the prairie vole (Microtus ochrogaster) to test predictions associated with the proposed functions of scent marking as a sexual attractant, in reproductive competition, and as a self-advertisement. We allowed an oestrous female, an anoestrous female, and an adult male to scent mark three portions of a clean substrate and then exposed a second male to this substrate for secondary marking. We did not support a sexual attraction hypothesis in that males did not place more scent marks in response to oestrous than anoestrous females. Similarly, we did not support a reproductive competition hypothesis in that males did not place more scent marks in response to marks of males than to those of females or bare substrate. Males did not overmark the scent of males or females and thus we did not support a scent-masking or scent-blending hypothesis. In that males deposited scent similarly in response to males, females, and on bare substrate, our results suggest that the frequency and placement of scent marks by males function primarily to advertise individual identity in an area.