Angiotensin I-converting enzyme (ACE) activity of the tomato moth, Lacanobia oleracea: changes in levels of activity during development and after copulation suggest roles during metamorphosis and reproduction

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that removes C-terminal dipeptides from relatively short oligopeptides, usually smaller than 15 amino acids. In mammals, the enzyme has several important roles in the metabolism of vasoactive peptides, but its physiological role in insects is not fully understood. We now report the properties of an ACE in a lepidopteran species (the tomato moth, Lacanobia oleracea) and suggest new physiological roles for the enzyme in this insect. ACE activity increases four-fold during the last stadium and in early pupae, a rise which, in its timing, is similar to what has been observed previously in the transition of larva to pupa in Drosophila melanogaster. This suggests that the increase in ACE activity might be of general importance for peptide metabolism during metamorphosis in holometabolous insects. High levels of ACE activity were found in the haemolymph of sixth stadium larvae and adult insects, and in the reproductive tissues of both male and female adults. Almost all of the ACE activity in the reproductive tissues was found in the accessory glands of the male and the spermatheca and bursa copulatrix of the female. The decline in accessory gland ACE in mated males and the concomitant rise in ACE activity in the spermatheca and bursa copulatrix of the female suggested the transfer of ACE from the male to the female during copulation. Using several convenient peptides as substrates, we have shown that the spermatophore/bursa copulatrix taken from mated female insects possess an aminopeptidase, a carboxypeptidase and a dipeptidase, in addition to high levels of ACE. These peptidases might be involved in the breakdown of proteins to peptides and eventually to amino acids in the spermatophore. Evidence for such a proteolytic pathway and its role in providing substrates for the TCA cycle has been obtained previously in a study of reproduction in Bombyx mori.     

The structural mechanism of trypsin-induced intrinsic motility in Manduca sexta spermatozoa in vitro

Lepidopteran males produce eupyrene (nucleate) and apyrene (anucleate) spermatozoa, but in the female only eupyrene spermatozoa leave the spermatheca and fertilize the eggs. Both kinds of spermatozoa lack intrinsic motility in the male genital duct. They become motile in the spermatophore, in a process involving proteases from the male duct. In vitro, trypsin induces immotile spermatozoa to become motile. We studied the changes spermatozoa of Manduca sexta undergo during trypsin-induced motility and found that (a) they mimick rather closely those occurring in vivo during normal sperm maturation in genital ducts and (b) they are time- and dose-dependent. As in vivo, they comprise, successively, (a) disappearance of an extracellular matrix that maintains the integrity of eupyrene bundles in the seminal vesicle, (b) dispersion of the eupyrene bundles and intermingling of eupyrene and apyrene spermatozoa and (c) "hatching" of eupyrene spermatozoa from individual enclosing envelopes that are formed in the seminal vesicle. "Hatching" may not directly be related to motility since eupyrene spermatozoa become motile before "hatching" and motile apyrene spermatozoa never "hatch". Rather "hatching" may be related to the capacitation of eupyrene spermatozoa to either leave the spermatheca or fertilize the eggs, or both, as neither apyrene spermatozoa, nor those eupyrene spermatozoa that fail to "hatch", leave the spermatheca.     

Possible Removal of Rival Sperm by the Elongated Genitalia of the Earwig, Euborellia plebeja

Sperm displacement is a sperm competition avoidance mechanism that reduces the paternity of males that have already mated with the female. Direct anatomical sperm removal or sperm flushing is known to occur in four insect orders: Odonata, Orthoptera, Coleoptera and Hymenoptera. In a fifth order, Dermaptera (earwigs), I found that the virga (the elongated rod of the male genitalia) of Euborellia plebeja seems to be used to remove rival sperm from the spermatheca (a fine-tubed female sperm storage organ). In this species, copulation lasted on average 4.6 minutes, during which time the male inserted the virga deep into the spermatheca, and then extracted it ejaculating semen from the opening of the virgal tip. The extraction of virgae (with its brim-like tip) appeared to cause removal of stored sperm in the spermatheca. The virga was as long as the body length of males, and the spermatheca was twice the female body length. The long length of the spermatheca and the possible removal function of the virga may select for virgal elongation.     

Sperm numbers,their storage and usage in the fly Dryomyza anilis

In the fly Dryomyza anilis females have two kinds of sperm storage organs: one bursa copulatrix and three spermathecae (two spermathecae with a common duct form the doublet, and the third is a singlet spermathecal unit). At the beginning of a mating the male deposits his sperm in the bursa copulatrix. After sperm transfer the male taps the female''s abdomen with his claspers. This behaviour has been shown to increase the male''s fertilization success. After mating, the female discharges large quantities of sperm before oviposition. To find out where the sperm remaining in the female are stored, I counted the number of sperm in the droplet and in the female''s sperm storage organs after different types of mating. I carried out three mating experiments. In experiment 1, virgin females were mated with one male and the matings were interrupted either immediately after sperm transfer or after several tapping sequences. The results show that during male tapping more sperm moved into the singlet spermatheca. In addition, the total number of sperm correlated with sperm numbers in all sperm storage organs, and male size was positively related to the number of sperm remaining in the bursa. In experiment 2, females mated with several males. The number of sperm increased with increasing number of matings only in the doublet spermatheca. No increase in the number of sperm in the singlet spermatheca during consecutive matings suggests that sperm were replaced or did not reach this sperm storage organ. In experiment 3, virgin females were mated with a single male and half of them were allowed to lay eggs. The experiment showed that during egglaying, females primarily used sperm from their singlet spermatheca. The results from the three experiments suggest that sperm stored in the singlet spermatheca is central for male fertilization success and male tapping is related to sperm storage in the singlet spermatheca. The different female''s sperm storage organs in D. anilis may have separate functions during sperm storage as well as during sperm usage.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1 complex in the poly(ADP-ribosyl)ation pathway after DNA damage

A defect in the Werner syndrome protein (WRN) leads to the premature aging disease Werner syndrome (WS). Hallmark features of cells derived from WS patients include genomic instability and hypersensitivity to certain DNA-damaging agents. WRN contains a highly conserved region, the RecQ conserved domain, that plays a central role in protein interactions. We searched for proteins that bound to this region, and the most prominent direct interaction was with poly(ADP-ribose) polymerase 1 (PARP-1), a nuclear enzyme that protects the genome by responding to DNA damage and facilitating DNA repair. In pursuit of a functional interaction between WRN and PARP-1, we found that WS cells are deficient in the poly(ADP-ribosyl)ation pathway after they are treated with the DNA-damaging agents H2O2 and methyl methanesulfonate. After cellular stress, PARP-1 itself becomes activated, but the poly(ADP-ribosyl)ation of other cellular proteins is severely impaired in WS cells. Overexpression of the PARP-1 binding domain of WRN strongly inhibits the poly(ADP-ribosyl)ation activity in H2O2-treated control cell lines. These results indicate that the WRN/PARP-1 complex plays a key role in the cellular response to oxidative stress and alkylating agents, suggesting a role for these proteins in the base excision DNA repair pathway.     

平疣桑椹石磺生殖系统结构及精子储存场所

雌雄同体贝类精子的储存和利用规律一直是国内外贝类生物学研究的难点之一,本文利用活体解剖、显微观察、组织切片和扫描电镜技术,综合研究了平疣桑椹石磺(Platevindex mortoni)的生殖系统及精子储存场所。结果显示,其生殖系统包括生殖器本部、雌性生殖部分和雄性生殖部分。生殖器本部由两性腺、两性输送管、蛋白腺、黏液腺、支囊组成;雌性生殖部分包括输卵管、受精囊、阴道,位于身体中后方体腔内;雄性生殖部分包括输精管、刺激器、阴茎、阴茎鞘和阴茎牵引肌,位于身体前端右侧体腔内;其阴茎有阴茎鞘,阴茎表面布满倒刺。平疣桑椹石磺阴茎为直线状,无雄性附属腺。未交配的性成熟个体支囊内充满细长精子,受精囊内无精子;而交配后充当雌性个体的支囊内均为细长的自体精子,受精囊内有大量活力较强的粗短精子,其支囊为自体精子的存储场所,而受精囊为异体精子的存储场所。其精子储运情况为:两性腺内精子成熟后暂存于支囊,交配时通过输精管运输至阴茎,由阴茎输送精子至对方的阴道,异体精子进入受精囊内存储待用。     

Evolution of specialized spermatheca morphology in ant queens: Insight from comparative developmental biology between ants and polistine wasps

In many ant species, the queens can keep spermatozoa alive in their spermatheca for several years, which goes along with unique morphological characteristics of the queen's spermatheca. The relative spermatheca size in ant queens is prominently larger than that in social wasps. Furthermore, the epithelium lining the spermatheca reservoir of ants consists of columnar cells in the hilar region and squamous cells in the distal region, whereas it is formed by columnar cells only in social wasps. To study the evolution of the unique spermatheca morphology in ant queens, we compared the various processes during spermatheca development between two ponerine ant species of the genus Pachycondyla (= Brachyponera) and three polistine wasp species of the genus Polistes. From histological observations, we can define four developmental events in the ant queens: (1) invagination of the spermatheca primordium, (2) the reservoir wall thickness becomes unequal, (3) the reservoir diameter doubles as the lining epithelial cells become flattened except for the hilar region, and (4) the increase in thickness of the reservoir epithelium is limited to the hilar region which doubles in thickness. In polistine wasps, the second and the third developmental events are absent and the entire epithelium of the spermatheca wall becomes thick in the final step. We therefore conclude that for ant queens the second and third steps are crucial for the enlargement of the spermatheca size, and that the second to the fourth steps are crucial for the specialization of the reservoir wall structure.     

Sperm storage by spermatodoses in the spermatheca of Trioza alacris (Flor, 1861) hemiptera,psylloidea, triozidae: A structural and ultrastructural study

Female insects generally store sperm received during mating in specific organs of their reproductive tract, i.e., the spermathecae, which keep the sperm alive for a long time until fertilization occurs. We investigated spermatheca morphology and ultrastructure in the psylloidean insect Trioza alacris (Flor, 1861 ) in which spheroidal sperm packets that we refer to as ‘spermatodoses’ are found after mating. The ectoderm‐derived epithelium of the sac‐shaped spermatheca that has a proximal neck, consists of large secretory and flat cuticle‐forming cells. Secretory cells are characterized by a wide extracellular cavity, bordered by microvilli, in which electron‐dense secretion accumulates before discharge into the spermathecal lumen. The cuticle‐forming cells produce the cuticular intima of the organ and a peculiar specialized apical structure, through which secretion flows into the lumen. At mating, the male transfers bundles of sperm cells embedded in seminal fluid into the spermathecal neck. Sperm cells proceed towards the spermathecal sac lumen, where they are progressively compacted and surrounded with an envelope that also encloses secretions of both male and female origin. We describe the formation of these sperm containing structures and document the contribution of the female secretion to spermatodose or female‐determined spermatophore construction. We also discuss the choice of the term ‘spermatodose’ for T. alacris and suggest it be used to refer to sperm masses constructed in the female reproductive organs, at least when they involve the contribution of female secretion. © 2011 Wiley Periodicals, Inc.     

Sperm utilisation by Melipona quadrifasciata Lepeletier (Hymenoptera, Apidae) queens subjected to multiple mating

Summary In most Hymenoptera species the queen mates once but in a small number of species, multiple matings can occur normally. So, in this study, physogastric M. quadrifasciata queens were mated with a second male to investigate how these queens, naturally inseminated and laying eggs, use spermatozoa stored in their spermatheca, when they are mated with a second male. Results demonstrate that spermatozoa of different males mix in the spermatheca of M. quadrifasciata queens and that there is a gradual increase in the utilisation of spermatozoa of the second male, which could be explained by a competition among spermatozoa of different drones over the way in which spermatozoa are stored in the spermatheca.     

Membrane specializations in the spermatozoa of collembolan insects

Here we describe the membrane specializations displayed by "dormant" collembolan spermatozoa, during the period when they are temporarily stored in the male genital ducts before being laid in the soil as a spermatophore, which is then picked up by the female. In the male deferent ducts and in the spermatophore, these "dormant" spermatozoa are rolled up into flattened ellipsoids that surround a central extracellular cavity filled with dense material. In this condition, the plasma membranes that surround the central extracellular cavity contain two types of membrane specializations. One consists of parallel rows of intramembrane particles on the E-face of the membrane facing the outside of the spermatozoa. The other consists of small orthogonal plaques of intramembrane particles intermingled with these rows. These plaques associate with the P-face of the membrane, and are therefore likely to represent preferential sites for receiving and transmuting environmental signals, especially whatever signal(s) induce these spermatozoa to transform into filiform and motile cells upon reaching the female spermatheca.     

Post-mating effects in the grasshopper, Gomphocerus rufus L. mediated by the spermatheca

In the female grasshopper Gomphocerus rufus mating replaces copulatory readiness with immediate and long-lasting `secondary defense', during which further mating attempts are efficiently repelled. The behavioral change is caused by secretions from the male accessory glands' white secretory tubule 1 which is injected with the spermatophore material into the female's spermathecal duct. A bristle field of contact chemoreceptors at the entry of the spermathecal duct into the endbulb is assumed to be stimulated by the secretion. Ablation of the bristle field, interruption of the nervous pathway between the spermatheca and the ventral nervecord, or severance of the latter sustains sexual receptivity after mating. Both the secretion from white secretary tubule 1 and the spermatophore contained in the spermatheca of a mated female are digested by proteolytic enzymes from spermathecal gland cells. Dissolved material is resorbed by similar glandular-like cells. The intersexual conflicts of interest and their evolutionary consequences are discussed. Accepted: 4 December 1998     

Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of a rat carcinoma cell line

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.     

The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans

The ancestry of the cells in the hermaphrodite and male gonadal somatic structures of C. elegans has been traced from the two gonadal somatic progenitor cells (Z1 and Z4) that are present in the newly hatched larvae of both sexes. The lineages of Z1 and Z4 are essentially invariant. In hermaphrodites, they give rise to a symmetrical group of structures consisting of 143 cells, and in males, they give rise to an asymmetrical group of structures consisting of 56 cells. The male gonad can be distinguished from the hermaphrodite gonad soon after the first division of Z1 and Z4. However, the development of Z1 and Z4 in hermaphrodites shares several features in common with their development in males suggesting that the two programs are controlled by similar mechanisms. In the hermaphrodite lineage, a variability in the positions of two cells is correlated with a variability in the lineages of four cells. This variability suggests that cell-cell interaction may play a more significant role in organisms that develop by invariant lineages than has hitherto been considered. None of the somatic structures (e.g., uterus, spermatheca, vas deferens) develops as a clone of a single cell. Instead, cells that arise early in the Z1–Z4 lineage generally contribute descendants to more than one structure, and individual structures consist of descendants of more than one lineage.     

Physiological basis for sterilizing effects of constant light in Lymantria dispar

ABSTRACT Females of gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), mated to males kept in constant light (LL) as pharate adults fail to oviposit. In males, a rhythm of sperm release from the testis that occurs in light-dark (LD) cycles is abolished in LL, and the total amount of sperm released from the testis is approximately half of that of LD males. Moreover, any sperm that may be released from the testis of LL males tend to remain in the vasa deferentia instead of moving into the duplex as in LD males. Consequently, in LL very few sperm bundles are transferred to the bursa copulatrix during mating; furthermore, these bundles fail to disperse into spermatozoa and sperm do not reach the spermatheca. The presence of a spermatheca filled with sperm must play an important role in controlling oviposition because their removal from mated females prevents egg-laying. Our results indicate that the rhythm of sperm release from the testis is essential for the ability of sperm to migrate in male and female reproductive tracts. The rhythms may help to synchronize final stages of sperm development with the activity of phagocytic and secretory cells lining the reproductive tract.     

Copulation patterns in the golden orb-web spider Nephila madagascariensis

A consequence of multiple mating by females can be that the sperm of two or more males directly compete for the fertilisation of ova inside the female reproductive tract. Selection through sperm-competition favours males that protect their sperm against that of rivals and strategically allocate their sperm, e.g., according to the mating status of the female and the morphology of the spermatheca. In the majority of spiders, we encounter the otherwise unusual situation that females possess two independent insemination ducts, both ending in their own sperm storage organ, the spermatheca. Males have paired mating organs, but generally can only fill one spermatheca at a time. We investigated whether males of the African golden orb-web spider Nephila madagascariensis can prevent rival males from mating into the same spermatheca and whether the mating status of the female and/or the spermatheca causes differences in male mating behaviour. There was no significant difference in the duration of copulations into unused spermathecae of virgin and mated females. We found that copulations into previously inseminated spermathecae were generally possible, but shorter than copulations into the unused side of mated females or with virgins. Thus, male N. madagascariensis may have an advantage when they mate with virgins, but cannot prevent future males from mating. However, in rare instances, parts of the male genitals can completely obstruct a female genital opening.     

Coevolution of male and female reproductive traits in a simultaneously hermaphroditic land snail

Inter- and intraspecific studies in gonochoristic animals reveal a covariation between sperm characteristics and the size of the female reproductive tract, indicating a rapid evolutionary divergence, which is consistent with the theory of post-copulatory sexual selection. Simultaneous hermaphrodites differ from species with separate sexes (gonochorists) in that they possess both functional male and female reproductive organs at the same time. We investigated whether in hermaphroditic animals intraspecific variation in reproductive traits results from divergent coevolution, by quantifying the variation in male and female traits among six natural populations of the snail Arianta arbustorum and examining the covariation in interacting traits. There was a significant among-population variation in spermatophore volume, number of sperm transferred and sperm length, as well as in volume of the sperm storage organ (spermatheca) and number of tubules, but not in spermatheca length. We found a positive association between sperm number transferred and spermatheca volume. This result suggests that the same post-copulatory mechanisms as in gonochorists drive the correlated evolution of reproductive characters in hermaphrodites.     

Dynamics of eupyrene and apyrene sperm storage in ovipositing females of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

A male swallowtail butterfly, Papilio xuthus, transfers both eupyrene and apyrene sperm during copulation, both of which migrate to the spermatheca via the spermatophore in the bursa copulatrix of the female. Because the spermatheca seems to remain constant in size during the female lifespan, the excess sperm migration may cause the spermatheca to overflow. Approximately 9000 eupyrene and 265 000 apyrene spermatozoa were transferred during a single copulation, and approximately 1000 eupyrene and 1100 apyrene spermatozoa successfully arrived in the spermatheca. The number of both types of spermatozoon decreased in the spermatheca after the onset of oviposition, and no eupyrene spermatozoa were found by 7 days after copulation, partly due to insemination. The spermathecal gland leading from the distal end of the spermatheca was gradually filled by eupyrene spermatozoa. Although the function of the gland remains unclear, the final destination of the sperm is likely to be the gland.     

The spermathecal complex in Ips typographus (L.): Differentiation of the spermathecal gland related to age and reproductive state

The spermathecal complex of the bark beetle, Ips typographus, comprises the following elements: spermathecal duct, spermatheca and spermathecal gland. The spermathecal duct connects the vagina and the spermatheca and consists of a cuticular tube surrounded by an epithelial layer and circular muscles. The spermatheca is bottle-shaped and has a cuticle-lined lumen. Muscles are attached to both ends of the spermatheca. The spermathecal gland which is connected to the spermatheca possesses three cell types: glandular, hypodermal, and ductule. The glandular cells have different structural characteristics depending on the age and reproductive state of the females. After the emergence of the brood, two different kinds of secretory material are present in the glandular cells. There is evidence that one type of secretion is emitted during the first few days after brood emergence, while the other type accumulates to be secreted during later stages.