Spermathecal Contents Research Articles

Genomic DNA extraction from honey bee (Apis mellifera) queen spermathecal content

Genetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.

HRM Analysis of Spermathecal Contents to Determine the Origin of Drones that Inseminated Honey Bee Queens

Abstract Europe, Africa and the Middle East have several original subspecies of the western honey bee (Apis mellifera L.), each with distinctive characteristics. These subspecies are the product of natural selection in their native range. Nevertheless, anthropogenic impacts such as migratory beekeeping and use of non-native queens result in an admixture of these subspecies and their ecotypes. I aimed to develop a SNP-based method to detect whether queen honey bees were mated with drones from foreign subspecies. For this purpose, Caucasian and Italian queens and drones were reared. Each queen was instrumentally inseminated with mixed semen collected from Caucasian (4 μl) and Italian drones (4 μl). The spermathecae of queens were dissected out after the onset of oviposition. The DNA was extracted from each spermatheca and from the thoraces of Caucasian and Italian drones. Seven regions on mtDNA that were isolated from drones were sequenced to determine the SNPs, enabling the discrimination of Caucasian sperm from Italian in spermathecal contents. Based on one SNP (11606. bp, T/C) residing on the Cytb gene, a specific primer was designed to be used in High Resolution Melting (HRM) analysis. HRM analysis indicated that heteroduplex peak profiles were present in all spermathecal contents of instrumentally inseminated queens. The results provide proof of the concept that the presence of likely non-native mitochondrial lineages can be detected by HRM analysis based on the SNP genotyping of spermathecal contents.

Ecological disjunction across the sexes in an alloparasitoid: Host requirements, sex ratios and mate localization (Hymenoptera: Aphelinidae)

AbstractThe larval females of Coccophagus sp. nr gurneyi Compere are primary parasitoids of lantana mealybugs, whereas males develop hyperparasitically through other parasitoids (never their own females), so the species is alloparasitic. Males are seldom even reared from lantana mealybugs (<0.3%, n = 4,212), and have not yet been reared from any other host. Adults were sampled in the field to establish that this species is sexual (by assessing female spermathecal content), and to quantify relative abundance of the sexes around host infestations. Adult males were scarce above hosts (3%, n = 314), but were attracted in relatively high numbers to caged virgin females within those infestations. Caged females outside infestations did not attract males, suggesting that mate attraction requires environmental signals other than those from females. Most females collected in the field above host infestations had sperm in their spermathecal capsules. They presumably had mated with males that developed elsewhere (so mate localization might involve searching across substantial distances). Virgin females were present only early in the day and evidently mate soon after eclosion. Evidence of sperm depletion in mated females was not found. The spatial scale of male and female movements needs to be quantified, but the ongoing movement of individuals (as a consequence of their sex‐related host relationships) seems to be a regular aspect of their ecology. The spatial and temporal dynamics across the sexes illustrates that their abilities to localize one another for mating leaves the sexes free to diverge ecologically, and their sex ratios to vary.

Fertilization and fertility in the female of Varroa destructor, a key point for the parasite population dynamics

In Varroa destructor, the spermathecal content allows the female mite to carry out several successive reproductive cycles after a single mating event. The present study aimed to identify the main factors influencing the sperm content. Mite adult females were sampled at different bee colony phenological phases and on different host stages/sexes. Spermatozoa was characterized and counted. Spermatozoa maturity was acquired sufficiently fast (less than 5 days after the imaginal moult), their viability (ca. 91.5 %) and the fertilization success (ca. 1 spermatozoa spent per oocyte) were sufficiently high so that none of these parameters were limiting factors in the dynamics of mite populations. Moreover, no modulation was detected in the spermathecal content according to the brood status and the seasonal activity of the colony.

Reproductive interference between honeybee species in artificial sympatry

Reproductive isolation between closely related species is often incomplete. The Western honeybee, Apis mellifera, and the Eastern hive bee, Apis cerana, have been allopatric for millions of years, but are nonetheless similar in morphology and behaviour. During the last century, the two species were brought into contact anthropogenically, providing potential opportunities for interspecific matings. Hybrids between A.mellifera and A.cerana are inviable, so natural interspecific matings are of concern because they may reduce the viability of A.cerana and A.mellifera populations - two of the world's most important pollinators. We examined the mating behaviour of A.mellifera and A.cerana queens and drones from Caoba Basin, China and Cairns, Australia. Drone mating flight times overlap in both areas. Analysis of the spermathecal contents of queens with species-specific genetic markers indicated that in Caoba Basin, 14% of A.mellifera queens mated with at least one A.cerana male, but we detected no A.cerana queens that had mated with A.mellifera males. Similarly, in Cairns, no A.cerana queens carried A.mellifera sperm, but one-third of A.mellifera queens had mated with at least one A.cerana male. No hybrid embryos were detected in eggs laid by interspecifically mated A.mellifera queens in either location. However, A.mellifera queens artificially inseminated with A.cerana sperm produced inviable hybrid eggs or unfertilized drones. This suggests that reproductive interference will impact the viability of honeybee populations wherever A.cerana and A.mellifera are in contact.

Correlation of queen size and spermathecal contents and effects of miticide exposure during development

Normal mating of honey bee (Apis mellifera L.) queens and drones results in extreme polyandry. Larger queens are reported to produce more brood, but do they also store more semen? Measurements of queen weight, spermatheca weight and volume, and numbers of sperm in the spermatheca were made on normally reared queens and queens exposed to miticide from early larval stage to emergence. In the normally reared group, larger queens had more sperm in spermathecae of greater volume and weight. The presence of miticide during queen development skewed the relationship of queen weight to spermatheca size and the number of sperm stored such that they were not correlated. This sublethal effect of miticide in the colony has an unknown impact on queen performance.

An adaptive view of caste differentiation in the neotropical wasp Polybia (Trichothorax) sericea Olivier (Hymenoptera: Vespidae)

The tribe Epiponini comprehends the swarm-founding Neotropical wasps, with several species endemic to Brazil, which are extremely important in studies of social evolution of wasps. The Epiponini diverge in several ways from the definitions of high eusociality, since caste syndromes range from species without morphological caste differentiation to those with complete caste dimorphism, and all species are polygynous. Frequently, indirect studies based on morphometry and physiology are the only solutions to collect data regarding the natural history and caste system in this tribe, since most species are extremely aggressive and build enveloped nests, usually in places of difficult access. We analyzed morphological parameters in seven colonies of the Epiponini species Polybia (Trichothorax) sericea Olivier in different phases of colonial development. Nine body variables were taken and females were classified according to their ovary development and spermathecal contents. The results showed that caste differences in this species are based on a contrast among variables: queens have larger mesosoma and abdomen, but are smaller in head width and wing length. These results suggest that morphological caste differentiation in this species is based mainly on body shape. We considered this combination of characters as being adaptive. We also showed that caste differences varied according to the colony cycle, with more conspicuous differences when queen number is reduced.

Caste differences in Pseudopolybia compressa (Hymenoptera: Vespidae: Polistinae)

Studies of morphological caste syndromes in the tribe Epiponini help us to understand the evolution of social behavior in insects. Few studies have integrated morphological syndromes of various species from the same or distinct genera. We studied morphological differences between castes from three colonies of Pseudopolybia compressa de Saussure, 1854, in order to relate their caste syndrome with those of other species in the genus. We took eight morphometric measures and females were classified in relation to ovarian development and spermathecal contents. Females of Colony 1 (C1) present a higher distinction between castes than females of Colonies 2 and 3 (C2 and C3), and, in general, queens of the three colonies are larger than workers in some measures but smaller in others. In Pseudopolybia Dalla Torre, 1894, shape rather than size is a more important trait in caste divergence.

Characteristics of the spermathecal contents of old and young honeybee queens

Sperm are often stored, for a long time after mating, in females of various animal species. In case of the queen honeybee ( Apis mellifera), sperm remain fertile for several years in the spermatheca. Little information is available regarding the effect of long-term storage of sperm on its fertility. To evaluate this, enzymes and/or sperm have been analysed from the spermatheca of 75 queens of various ages (0 year Y0, n = 14; one year Y1, n = 14; two years Y2, n = 7; virgin queen VQ, n = 40) and semen samples have been taken from 46 drones. The sperm from the spermatheca of older queens move more slowly ( F = 11.45, P < 0.0001) and show different movement patterns (Chi 2 = 90.0, P < 0.0001) from those of the other groups. The spermatheca content of differently aged mated queens differ significantly with respect to the activities of lactate dehydrogenase ( F = 3.37, P < 0.05), citrate synthase ( F = 6.24, P < 0.005) and arginine kinase ( F = 9.44, P < 0.0006). Glyceraldehyde 3-phosphate dehydrogenase ( F = 0.10, P = 0.91) does not differ significantly. The results suggest considerable changes in the energy metabolic profile of the spermatheca tissue, of the sperm or of both during sperm storage.

Rapid determination of sperm number in ant queens by flow cytometry

In social Hymenoptera, queens receive a given amount of sperm during a single or multiple inseminations once and for all. The amount of sperm stored at mating determines the maximum number of fertilized eggs queens can produce for the rest of their reproductive life. We propose flow cytometry (FCM) as a method to estimate the concentration of sperm cells, as well as their ploidy level, in queens’ spermathecae. Our data, obtained from 5 ant species, show that FCM is precise, repeatable, easy to conduct and rapid. Estimates of variation of spermathecal content always remain below 10%, and samples can be analysed in less than 5 minutes. Flow cytometry appears as an excellent method for comparative analyses of sperm number within and between ant species.

No Behavioral Control over Mating Frequency in Queen Honey Bees (Apis melliferaL.): Implications for the Evolution of Extreme Polyandry

Ever since the seminal papers of Hamilton (1964), a common focus of sociobiology has been the genetic relationships among individuals of insect societies. At issue are agents that reduce the average relatedness among colony members since they are difficult to interpret in light of the evolution of sociality by kin selection. One such factor is polyandry or female multiple mating. Low levels of polyandry are common among the social Hymenoptera, occurring across a broad range of taxa and social systems (Page and Metcalf 1982; Page 1986; Crozier and Pamilo 1996). In spite of that, high mating frequencies (‚2 mates/ female) are relatively rare among the highly eusocial Hymenoptera, limited to only one or a few genera in each of the ants, bees, and wasps (Boomsma and Ratnieks 1996). These extraordinary levels of polyandry have been of particular interest among students of social insect research since their adaptive significance is still in question and highly debated (Kraus and Page 1998; Sherman et al. 1998). The genus Apis has been studied in depth for both queen reproductive behavior and mating frequency, facts that allow insight into the evolution of extreme polyandry in these species. The reproductive behavior of honey bee queens (Apis mellifera) occurs early in their lifetimes. Virgin queens initiate mating behavior when they are about 1 wk old by briefly exiting the colony and orienting to local landmarks (Ruttner 1956). After such orientation * To whom correspondence should be addressed; e-mail: drtarpy@

Reproductive abnormalities in tsetse flies in Zimbabwe

Between November 1988 and July 1995 five technicians carried out ovarian dissections on 16,013 Glossina morsitans morsitans Westwood and 123,848 G. pallidipes Austen captured at Rekomitjie Research Station, Zambezi Valley, Zimbabwe. The ovarian age and uterine content were recorded, as were the lengths (l1, l2 and lu) of the largest and second largest oocyte, and of any uterine inclusion. Major abnormalities and abnormal spermathecal contents were found in <0.1% of all flies dissected. Apparent abortions rates varied significantly between dissectors and occurred at frequencies of 0.8–4.5% in G. m. morsitans and 0.3–2.8% in G. pallidipes. The lowest estimates give the best picture of the field situation. Abortion rates were higher in flies caught on electric nets than in trapped flies where the rate was only 0.15%, indicating that reproductive losses are negligible for most of the year at Rekomitjie. The rates did, however, increase to >2% when mean temperatures exceeded 27 °C and flies were captured in artificial refuges. There was little effect of ovarian age on the abortion rate, but the frequency of empty uteri declined markedly with age – with a suggestion, however, that it might increase again in the oldest flies. A knowledge of the rates of reproductive loss is important for the construction of realistic models of the dynamics of tsetse populations.

Sperm competition and paternity assurance during the first breeding period of female snow crab (Chionoecetes opilio) (Brachyura: Majidae)

Two highly polymorphic microsatellite DNA loci were used to determine paternity of larvae of primiparous Chionoecetes opilio females. In addition, female spermathecal contents were genotyped at the two loci and histological analysis of the organs was carried out to elucidate patterns of sperm competition. Females carrying their first brood from controlled laboratory matings and from matings in the wild were examined. Spermathecal contents from wild-caught females were cut into several cross sections and each section genotyped individually. Both wild and laboratory females commonly mated with several males whose ejaculates were stored and stratified in the spermathecae. Genetic typing of the offspring of laboratory-mated females revealed single paternity and indicated that the last mate to inseminate a female before oviposition gained paternity of the clutch. The predominant mechanism ensuring single paternity appeared to be sperm stratification. In wild-caught females, the microsatellite typing of the offspring also revealed single paternity, but larvae appeared to be sired by males whose genotypes were found in the spermathecal cross sections towards the dorsal end (blind end) of the spermathecae. This suggested that they were the first males to mate with females that they guarded until oviposition, and females remated with other males thereafter.

The relative roles of predation and sperm competition on the duration of the post-copulatory association between the sexes in the blue crab, Callinectes sapidus

In many species, post-copulatory mate guarding prevents other males from mating with the guarded female. In crabs, males stay with their mates to protect the female from predators because, in some species, mating occurs when she is soft and vulnerable after molting. I tested the relative roles of sperm competition and predation on the duration of the post-copulatory association in the blue crab, Callinectes sapidus. Unpaired females suffered greater predation mortality than paired females and males stayed with the female longer in the presence of predators than in their absence, suggesting that the post-copulatory association protects females during their vulnerable period. However, the association may also occur in blue crabs because of sperm competition since spermathecal contents of females in the field indicate that 12.4% mated twice. Females experimentally mated with two males contained both males ejaculates and each ejaculate had access to the unfertilized eggs, suggesting that the size of a male's ejaculate influences his fertilization rate in a multiply-mated female. Males stayed longest in response to a high risk of sperm competition. Longer post-copulatory associations allowed the first male's ejaculate to harden into a type of sperm plug, which limited the size of a second inseminator's ejaculate in a non-virgin female as compared with a virgin. Males passed larger ejaculates in the presence of rivals and when previous ejaculates were in the female spermathecae, another response to sperm competition. Larger ejaculates may need longer post-copulatory associations before a more effective sperm plug forms. Large males stayed with the female longer, which is consistent with their ability to pass larger ejaculates than small males and suggests that there may be costs to minimizing the duration of the post-copulatory association. In the field, associations last long enough to protect the female during her vulnerable phase and may ensure that the guarding male fertilizes the most eggs in the female, even if she remates. Thus, the post-copulatory association protects female blue crabs from additional inseminators as well as from predators.

ASPECTS OF SPERM METABOLISM IN THE SPERMATHECA OF THE BRACHYURAN CRAB METOPOGRAPSUS MESSOR (FORSKAL)

ABSTRACT Mated female brachyuran crabs store the spermatozoa within the spermatheca, pending internal fertilization. The present study reports on the metabolism of sperm stored in the spermatheca of the estuarine crab Metopograpsus messor. Results of enzyme assays indicate that lactate oxidation to pyruvate predominates over pyruvate reduction to lactate within the spermatheca of M. messor. The spermathecal contents also show considerable succinate dehydrogenase (SDH) activity which is greater than the fumarate reductase (FR) activity, the SDH/FR ratio being 5.7:1. Furthermore, manometric assays demonstrate that the sperm stored within the spermathecal lumen consume ambient oxygen, the rate of oxygen uptake per 1.000 x 108 spermatozoa being 0.108 ± 0.004 µl X h-1. The prevalence of lactate oxidation over pyruvate reduction and the occurrence of a high SDH/FR ratio within the spermatheca, together with the spermatozoal oxygen uptake, strongly suggest that, while stored in the spermatheca, the spermatozoa of M. messor rely on aerobic metabolism. The occurrence of high lipid and the relative sparseness in carbohydrates (with near absence of free glucose) within the spermathecal lumen further reinforce our suggestion for aerobic respiration by the sperm stored in the female reproductive tract.

Reproductive Cycle and Fecundity of Primiparous and Multiparous Female Snow Crab, Chionoecetes opilio, in the Northwest Gulf of Saint Lawrence

A total of 1 691 mature female Chionoecetes opilio was collected by beam trawl in Baie Sainte-Marguerite on seven occasions from April 1991 to May 1992. Quantitative analyses of ovaries, brood, eggs, and spermathecal contents were performed on a subsample of 318 females. Females moulted to maturity in March and April. Development of ovaries and brood were phased and lasted 24–27 mo. Eggs hatched mainly from April to June. Spermathecae contained zero to three ejaculates, and the mean blotted weight of freshly deposited ejaculates was significantly greater in primiparous (0.513 g) than in multiparous (0.249 g) females. The weight of stored ejaculates decreased over time independently of spawning events. Fecundity was correlated positively with carapace width and negatively with mean egg diameter. Eggs of primiparous females were on average 1.4–2.7% larger, but 16.4–22.7% fewer per brood, than those of multiparous females. Some large multiparous females had small broods, due possibly to lesser fertility. Females probably hatch only two broods in a lifetime and produce a total of 81 630 to 83 143 larvae at 57.4 mm carapace width. Primiparous females apparently contribute &gt;40% of larvae produced by the population.

Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female

Biochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm-specific fraction, LDHx, the M-type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm-agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constituents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.

FUNCTIONAL ANATOMY OF THE MALE REPRODUCTIVE SYSTEM AND THE FEMALE SPERMATHECA IN THE SNOW CRAB CHIONOECETES OPILIO (0. FABRICIUS) (DECAPODA: MAJIDAE) AND A HYPOTHESIS FOR FERTILIZATION

ABSTRACT To help elucidate the reproductive characteristics of the Atlantic snow crab Chionoecetes opilio, the functional anatomy of the male reproductive system and the female spermatheca was investigated using histological methods, transmission and scanning electron microscopic techniques, and microscopic observation of fresh material. Several fundamental corrections and additions to earlier descriptions of spermatozoan structure in C. opilio were made: radial arms are present, while a chromatin ring is not; the acrosome protrudes only slightly and spermatozoa are not mushroom-shaped. The spermatozoa and the matrix of the anterior vas deferens are packed into spermatophores and surrounded by a pellicle which appears to be secreted by the cells lining the anterior vas deferens proximal to the testis. The highly folded configuration of this pellicle may act as a safeguard against dehiscence induced by contact with sea water during copulation. The posterior vas deferens contains two distinct secretions which are probably ejaculated along with the spermatophores and the matrix of the more anterior vas deferens. Anatomical and in vitro observations suggest that fertilization is initiated by exposure of the spermatophores to a hypotonic medium. Such a medium may be generated within the spermathecae by dilution of the seminal fluids/spermatophore storage matrix with sea water prior to egg mass extrusion. Devagination of the liberated spermatozoa may be facilitated by the same mechanism. Most of the females processed for histology had distended spermathecae devoid of spermatophores or spermatozoa. It is thus impossible to deduce successful copulation and spermatophore storage without direct observation of spermathecal contents.

Manipulation of prostaglandin-mediated oviposition after mating inTeleogryllus commodus

Summary In T. commodus, mating causes increased egg release by PGE2-action, whereas virgins lay comparatively few eggs. Removal of the spermatheca, the locus of prostaglandin biosynthesis, or severance of the spermathecal duct after mating cancels the egg flood and the egg-laying rate continues at a level similar to that of virgins. Deprivation of the spermathecal content from mated females has the same effect, whereas implantation of crushed spermathecae from mated into the haemocoel of unmated crickets causes a threefold increase in the number of eggs laid. Since PGE2 occurs also in the haemolymph of mated females, it is suggested that spermathecal prostaglandins enter the haemocoel via the genital chamber and the haemolymph transports these lipids to as yet unknown receptor sites in the central nervous system.

Effect of Continuous Light on the Potency of Plodia interpunctella Males (Lepidoptera: Phycitidae)

Recent studies showed that the egg production of females of the Indian-meal moth, Plodia interpunctella (Hubner), that were mated to males reared under 12-hr light :12-hr dark (LD) cycles was always higher than that of females mated to males reared under continuous light (LL) (Lum and Flaherty 1969). Even though males from either light regime copulated readily and transferred spermatopheres, the percent of females that oviposited was greater in cultures mated with LD males than in cultures mated with LL males. Oviposition in the Indian-meal moth is, therefore, stimulated by neither the physical act of mating nor the presence of a spermatophore in the bursa copulatrix. Because it was previously suspected that the quantity of spermatozoa deposited in some female insects may be the stimulus to oviposition (Norris 1933), the spermathecal contents of females mated to LD males, and females mated to LL males were compared to find the causes leading to differences in egg production and percent females ovipositing.