Spermathecae Of Queens Research Articles

A new gene, SRP16, differentially expressed in the spermathecae of honeybee queens (Apis mellifera) related with reproduction status

Honey bee queens have the ability to store sperm in spermathecae for fertilizing eggs throughout their life. To investigate mechanisms for sperm storage in Apis mellifera, we employed suppression subtractive hybridization (SSH) to find differentially expressed fragments in spermathecae between virgin queens and newly mated queens. A new gene, named SRP16, was obtained by joining the SSH products with 5'-RACE and 3'-RACE. SRP16 is predicted to encode a 41 kDa protein with 363 amino acid residues. Its expression was found in the spermathecae dominantly in honey bee queens but not in honey bee workers, with the highest expression found in spermathecae of virgin and newly mated queens. SRP16 expression was weak in other tissues of queens other than in the spermathecae and showed no obvious change with reproductive status of queens. The results suggest that SRP16 may play important roles in sperm storage and honey bee reproduction.

The viability of sperm in lateral oviducts and spermathecae of instrumentally inseminated and naturally mated honey bee (Apis mellifera L.) queens

SummaryInstrumentally inseminated queens (IIQ) were compared with naturally mated ones (NMQ) to detect the effect of the instrumental insemination procedure on sperm viability in the reproductive organs. The viability of sperm in the reproductive organs of IIQ and NMQ was determined at 4 h and 24 h after they were inseminated or they mated. No significant difference was found between IIQ (88.6%) and NMQ (87.6%) in viability of sperm in the lateral oviducts at 4 h. There was also no difference between IIQ (98.3%) and NMQ (97.8%) in viability of sperm in spermathecae at 4 h. The mean viability of sperm in spermathecae of IIQ (97.5%) did not differ from the mean viability of sperm in spermathecae of NMQ (96.8%) after 24 h. These results showed that the insemination procedure had no negative effect on viability of sperm in reproductive organs of queens. The difference in sperm viability between lateral oviducts (88.1%) and spermathecae (98.1%) of queens at 4 h was found to be statistically significant (P < 0.001). Nevertheless, no significant difference was found between viability of sperm in spermatheca at 4 h (98.1%) and 24 h (97.2%). The possible reasons for the differences in viability of sperm in reproductive organs are discussed.

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.

Visible and near-infrared spectroscopy detects queen honey bee insemination

The abdomens of honey bee queens and semen from drone bees were analyzed by visible and near-infrared spectroscopy. Mated honey bee queens could be distinguished from virgin queens by their absorption spectra with 100% accuracy. Spectra of semen showed that classifications of queens were likely influenced by the presence or absence of semen in the queen spermathecae. However, physiological or morphological changes that occur in the queens after mating probably influenced the classifications also.

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Under complementary sex determination (CSD), females of Hymenoptera arise from diploid, fertilized eggs and males from haploid, unfertilized eggs. Incidentally, fertilized eggs that inherit two identical alleles at the CSD locus will develop into diploid males. Diploid males are usually unviable or sterile. In a few species, however, they produce diploid sperm and father a triploid female progeny. Diploid males have been reported in a number of social Hymenoptera, but the occurrence of triploid females has hardly ever been documented. Here, we report the presence of triploid females, diploid males, and diploid sperm (produced by diploid males and stored in queen spermathecae) in the ant Tapinoma erraticum. Moreover, we show variations in the frequency of triploids among female castes: Triploid females are more frequent among workers than virgin queens; they are absent among mated, reproductive queens. The frequency of triploid workers also varies between populations and between nests within populations.

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.

Reproductive disturbance of Japanese bumblebees by the introduced European bumblebee Bombus terrestris

The European bumblebee, Bombus terrestris, is an invasive eusocial species whose distribution is expanding greatly beyond its native range because numerous colonies are imported to or locally produced in non-native countries for pollination of agricultural crops. Closely related species exist in Japan where the unrestricted import and use of B. terrestris has resulted in the establishment of wild colonies. Laboratory studies previously showed that B. terrestris and Japanese native species can copulate and produce fertilized eggs. Although these eggs do not hatch, the interspecific mating can cause a serious reproductive disturbance to native bumblebees. In this study, we determined the frequencies of interspecies mating between B. terrestris males and native bumblebee queens in the wild on the islands of Hokkaido and Honshu by analyzing the DNA sequences of spermatozoa stored in spermathecae of native queens. We found that 20.2% of B. hypocrita hypocrita queens and 30.2% of B. hypocrita sapporoensis queens had spermatozoa of B. terrestris males in their spermathecae. Given that a Bombus queen generally mates only once in her life, such high frequencies of interspecific mating with B. terrestris pose serious threats to the populations of native bumblebees in Japan.

Dominant Lethal Mutations in the Honeybee: A Perspective 50 Years Later

Dominant Lethal Mutations in the Honeybee: A Perspective 50 Years Later

Testing of the isolation of the Rog-Ponikve mating station for Carniolan (Apis mellifera carnica) honey bee queens

SummaryMating nuclei populated with virgin queens and worker bees were sited at three mating stations in order to test and compare their isolation from drones. After 22 days, the nuclei were examined for the presence of brood, the spermatozoa stored in the queens' spermathecae were counted, and the bodies and ovaries of the queens were weighed. The queens and 10 worker bees from each nucleus were also examined for Nosema spores. No queen produced brood at the Rog-Ponikve station, whilst 50% and 81% queens produced brood at the ČremoČnJice and Semic stations, respectively. Spermathecae of queens from Rog-Ponikve station did not contain sperm. The average body weight of the queens from Rog-Ponikve, ČremošnJice and SemiČ, were 169.25, 200.25 and 203.61 mg, respectively and their average ovary weights were 21.00, 51.57 and 57.31 mg, respectively. Thus egg laying queens from the ČremošnJice and SemiČ mating stations were 1.2 × heavier and their ovaries were 2.7 and 2.4 × heavier respectively, than those of the non ovipositing queens from the Rog-Ponikve station (P<0.001). No significant differences were found in the average number of Nosema spores (P>0.05) in the worker bees from the 3 stations and no spores were detected in any of the queens. The experiment showed that the Rog-Ponikve mating station has a secure level of drone isolation. The importance of testing the isolation of mating stations is discussed in this study.

MONITORING THE REPRODUCTIVE INDIVIDUALS IN THE ORIENTAL HORNET Vespa orientalis

Individuals of the oriental hornet, Vespa orien-talis Fab. were monitored at the apiary of Faculty of Agriculture .Ain Shams University throughout the year of 2006. The individual which was first detected in early spring at the first week of Febru-ary was the mated queens, later during the last week of April workers were appeared. At the first week of September individuals large in size ap-peared which were workers with developed ovary and not a new queen as believed previously. New queens started to appear at the second week of November after about three weeks from appear-ance the drones which appear at the last week of October. The numbers of ovarioles varied from 7 to 8 for each ovary in queens either in spring or in autumn. On the other hand the numbers of ovari-oles were only 7 for each ovary in workers either normal or with developed ovary. The measure-ments of the spermatheca showed no significant differences between all female individuals. The average numbers of spermatozoa in the queen's spermatheca of spring queen hornet were 1.486± 0.370 million spermatozoa and in the new queens who appear in the end of the year, the numbers of spermatozoa recorded 1.343±0.332 million. The counts of spermatozoa in the seminal vesicles of drone hornet recorded 2.336 ± 0.408 million sperms per drone.

Mating biology of the leaf-cutting antsAtta colombica andA. cephalotes

Copulation behavior has often been shaped by sexually selected sperm competition or cryptic female choice. However, manipulation of previously deposited ejaculates is unknown in the social Hymenoptera and the degree to which sperm competes after insemination or is actively selected by females has remained ambiguous. We studied the mating process in the leaf-cutting ants Atta colombica and A. cephalotes, which belong to one of the few derived social insect lineages where obligate multiple mating has evolved. As copulations often occur at night and in remote places, direct observations were impossible, so we had to reconstruct the sequential copulation events by morphological analysis of the male and female genitalia and by tracking the process of sperm transfer and sperm storage. We show that Atta male genitalia have two external rows of spiny teeth, which fit into a specialized pouch organ in the female sexual tract. Reconstruction of the sperm storage process indicated that sperm is transferred to the spermatheca during or immediately after ejaculation and without being mixed with sperm and seminal fluids from other males. A convergent mechanism of direct sperm transfer to the spermatheca of queens is known from two species of dwarf honeybees. Direct sperm transfer may restrict female control over the sperm storage process and the number of males that contribute to the stored sperm.

Insemination of honey bee, Apis mellifera, queens with non-frozen stored semen: sperm concentration measured with a spectrophotometer

SUMMARYNon-frozen storage of honey bee semen is one possible method to preserve this species' germplasm, as honey bee queens naturally store sperm for years at brood nest temperatures. In the past, most work has used cell counting chambers and small repeated samples to assess the number of sperm stored in a queen's spermatheca. To calibrate a spectrophotometer to measure sperm concentration using the entire contents of the spermatheca, five groups of queens—virgins, naturally mated, or inseminated with one, four or eight μl of semen—were assayed. Absorbance at 260 nm and 550 nm gave measures that were significantly correlated with direct cell counts using a counting chamber. Although the viability (percentage of live sperm) was high for semen stored at room temperature for eight weeks, a second population of queens inseminated with semen stored for four weeks or longer had reduced concentrations of sperm in the spermatheca. The area and quality (drones in worker cells) of brood from queens inseminated with fresh and two-week old semen were similar, but there was a trend toward reduced brood area and increased drone laying in queens receiving semen stored over four weeks.

Functional characterisation of semen in honeybee queen (A.m.ligustica S.) spermatheca and efficiency of the diluted semen technique in instrumental insemination

Differences over time in the quality of semen present in the honey bee (Apis mellifera ligustica) queen spermatheca were studied. An increase in the non-vital spermatozoa was shown to be evident (P>0.05) between the 12th and 24th month. The study of semen viability demonstrated that the passage of the semen to the spermatheca is due to sperm motility. In the queen inseminated with non-viable spermatozoa, no semen was detected in the spermatheca. Queens inseminated twice with a Hyes solution/semen mixture (1:1) stored as many spermatozoa in their spermatheca as those inseminated once with the classic technique. Queen replacement, oviposition and other functional characteristics were similar to those observed in the classic insemination procedure.

Sperm length, sperm storage and mating system characteristics in bumblebees

Multiple insemination induces sperm competition and may select for longer, faster moving sperm in species where sperm is short-lived and egg fertilization takes place almost immediately after ejaculation. Here we report the first detailed analysis of sperm length in social insects with long-term storage of sperm, using three bumblebee species with different mating systems as models. We show that individual males produce only one size-class of sperm, but that sperm length is highly variable among brothers, among unrelated conspecific males, and among males of different species. Males of Bombus hypnorum, a species with multiple-mating queens, have longer sperm than males of B. terrestris and B. lucorum whose queens are single mated. Although the sample size on the species level was too small to perform a phylogenetic analysis, this finding supports the hypothesis that, all other things being equal, multiple mating may select for longer sperm. Sperm length was positively correlated with male body size in B. terrestris and possibly in B. hypnorum, but not in B. lucorum. The variance of sperm length within single B. terrestris males before mating was consistently higher than the variance of ejaculated sperm that was stored in a queen's spermatheca. Both longer sperm and shorter sperm could be preferentially stored, depending on the colony in which the males and queens were born and raised. These results indicate that the genotype of males may affect sperm length and that cryptic female choice of sperm length may have a genetic component as well.

Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus.

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.

Biased sperm use by polyandrous queens of the ant Proformica longiseta

The occurrence and genetic effects of polyandry were studied in the ant Proformica longiseta using three microsatellite markers. The average queen mating frequency (QMF) estimated from the sperm dissected from the spermathecae of 61 queens was 2.4 with 69% of the queens being multiply mated. QMF estimated from worker offspring in a subsample of eight monogynous colonies was 3.5, but the effective paternity (m e,p) was only 1.23. The difference between these values reflected unequal sperm use by the queens. Most colonies of P. longiseta were polygynous and the average relatedness among workers was 0.35. Polyandry thus added only marginally to the genetic diversity of colonies, and our results gave little support to the genetic-variability hypothesis for explaining polyandry. Diploid male load was low, as only 1% of males were diploid. A large majority (92%) of nests produced one sex only, with males produced in colonies that had higher than average worker relatedness. This contradicted the predictions derived from worker control of sex ratios. Males produced enough sperm to fill the spermathecae of several queens. Thus, the results indicated that diploid male load, sperm limitation and sex ratio conflict are also unlikely explanations of polyandry. Plausible hypotheses for polyandry include mating by convenience, as the sex ratio is male biased and the mating costs to a female can be low because the females are wingless and have no mating flight. The observed unequal sperm use furthermore points to sperm choice and sperm competition as important factors in the evolution of polyandry.

The importance of the spermathecal duct in bumblebees

The elongated spermathecal duct of bumblebees has been studied in hibernating queens, queens shortly after hibernation, mature egg-laying queens and uninseminated queens captured during summer, and workers. Only rather small size differences are found when comparing spermathecae of queens and workers. Clear differences between bumblebee queens and workers are found when comparing the histochemistry of the spermathecal ducts. Adult queens, regardless of age and reproductive status have spermathecal ducts that contain PAS positive material, whereas workers do not. It is suggested that the polysaccharides in the spermathecal ducts of queens are necessary as a source of energy for the rapid activation of spermatozoa passing through the duct prior to oocyte fertilization. An ultrastructural investigation revealed the presence of high glycogen content in the cells lining the duct of queens. Assuming that sperm cells are kept in a rather inactive state in the reservoir, the carbohydrate (glycogen) probably serves as an energy source for the sperm. The comparatively increased spermathecal duct length of bumblebees may increase the retention time of sperm inside the lumen.

Relationship between semen quality and performance of instrumentally inseminated honey bee queens

Techniques to effectively store honey bee semen must meet some minimally acceptable level of spermatozoa survival. To determine this level, honey bee queens were inseminated using var- ious mixes of fresh and freeze-killed semen, and were allowed to lay eggs in small colonies for three weeks. The queens receiving all freeze-killed spermatozoa (0% fresh) had no spermatozoa in their sper- mathecae, and produced only drone pupae (unfertilized eggs). The proportions of live and dead sper- matozoa (determined by dual fluorescent staining) in the spermathecae of queens receiving 25 to 100% fresh semen were not significantly different at 27 days post-insemination. Queens receiving 50% fresh semen or more produced only worker pupae (all eggs were fertilized). Therefore, a program to improve storage of semen should only have to reach survival levels of 50% of the spermatozoa to have functional semen.

Mating frequency of ant queens with alternative dispersal strategies, as revealed by microsatellite analysis of sperm

In social Hymenoptera (ants, bees, and wasps), the number of males that mate with the same queen affects social and genetic organization of the colony. However, the selective forces leading to single mating in certain conditions and multiple mating in others remain enigmatic. In this study, I investigated whether queens of the wood ant Formica paralugubris adopting different dispersal strategies varied in their mating frequency (the number of males with whom they mated). The frequency of multiple mating was determined by using microsatellite markers to genotype the sperm stored in the spermatheca of queens, and the validity of this method was confirmed by analysing mother–offspring combinations obtained from experimental single‐queen colonies. Dispersing queens, which may found new colonies, did not mate with more males than queens that stayed within polygynous colonies, where the presence of numerous reproductive individuals ensured a high level of genetic diversity. Hence, this study provides no support to the hypotheses that multiple mating is beneficial because it increases genetic variability within colonies. Most of the F. paralugubris queens mated with a single male, whatever their dispersal strategy and life history. Moreover, multiple mating had little effect on colony genetic structure: the effective mating frequency was 1.11 when calculated from within‐brood relatedness, and 1.13 when calculated from the number of mates detected in the sperm. Hence, occasional multiple mating by F. paralugubris queens may have no adaptive significance.

Sperm usage in honey bees

Sperm usage was investigated in a naturally mated honey bee queen. We collected worker progeny arising from eggs that were laid sequentially during three sampling periods. Paternity was determined by analysis of three polymorphic microsatellite loci, leading to the conclusion that the queen had mated with seven males. Direct analysis of the sperm from the spermatheca revealed no evidence that sperm from additional males was present inside the spermatheca. Frequencies of different subfamilies differed significantly and ranged from 3.8% to 27.3%. In the short term, the frequencies of subfamilies among the eggs laid did not change over time. The frequency of eggs of a particular subfamily was statistically independent of the previous egg's subfamily. Thus, there is no evidence for non-random fine-scale sperm usage, and we estimate the effect of sperm clumping to be less than 6%. We conclude that the sperm is mixed completely inside the queen's spermatheca. Our results suggest that taking brood samples from comb cells next to each other is a statistically correct way of independent sampling of subfamilies at a given time in honey bee colonies. Furthermore, any bias in subfamily frequencies in offspring queens due to sperm usage can be excluded. However, the analyses of progeny samples taken 12 months apart do not allow us to exclude moderate fluctuations of subfamily frequencies in the long-term.