Spermathecal Fluid Research Articles

Proteomic analysis of spermathecal fluid reveals factors related to long‐term sperm storage in ant queens

AbstractAnt queens can maintain a large number of sperm cells for over a decade after mating at the beginning of their adult lives until they die. This sperm storage ability is prominent; however, the cellular mechanisms involved remain unclear. Sperm cells are maintained in the female sperm storage organ—the spermatheca—which supplies a suitable environment for sperm cells. To reveal the molecular basis of the long‐term sperm storage mechanisms in ant queens, protein profiles enriched in the spermathecal fluid relative to the hemolymph were identified in Lasius japonicus using data‐independent acquisition‐based quantitative proteomics technology. Proteins related to the extracellular matrix, antioxidants, metabolic pathways, proteases, chaperones, and with uncharacterized functions were especially abundant with higher log ratio values in the spermathecal fluid relative to the hemolymph. These enriched proteins were shared with highly expressed genes previously detected by transcriptome analyses of the spermatheca in queens of Crematogaster osakensis that belong to a different subfamily than L. japonicus. It is likely that the ability for long‐term sperm storage evolved early in the ant lineage. Therefore, the common proteins identified in these two ant species are possibly crucial for this ability.

Near-anoxia induces immobilization and sustains viability of sperm stored in ant queens

After copulation, insect females store sperm in a spermatheca for some duration until fertilization. At the beginning of their adult lives, ant queens can preserve numerous viable sperm cells from copulation for over ten years. However, the key factors influencing long-term sperm storage have not been identified. Here we show that the spermathecal environment is nearly anoxic, which induces sperm immobilization. Furthermore, mitochondrial respiratory inhibitors suppress sperm motility, suggesting that sperm immobilization may be caused by a shortage of ATP generated from only glycolysis under near-anoxic conditions. Sperm immobilization is not induced by acidification via glycolytic metabolism because the spermathecal fluid is not acidic. Finally, we show that artificial anoxic conditions rather than aerobic conditions sustain viable sperm cells. Therefore, near-anoxia is a key factor influencing long-term sperm storage in ant queens. The viability of sperm cells under artificial anoxia, however, is lower than that of those dissected immediately from queens. Moreover, the immotile sperm cells under more than 4 h of anoxia do not begin swimming after aerobic exposure, unlike those under anoxic conditions for less than 2 h. This finding indicates that factors other than anoxia are also necessary for long-term sperm preservation.

Queen honey bees exhibit variable resilience to temperature stress.

Extreme temperature exposure can reduce stored sperm viability within queen honey bees; however, little is known about how thermal stress may directly impact queen performance or other maternal quality metrics. Here, in a blind field trial, we recorded laying pattern, queen mass, and average callow worker mass before and after exposing queens to a cold temperature (4°C, 2 h), hot temperature (42°C, 2 h), and hive temperature (33°C, control). We measured sperm viability at experiment termination, and investigated potential vertical effects of maternal temperature stress on embryos using proteomics. We found that cold stress, but not heat stress, reduced stored sperm viability; however, we found no significant effect of temperature stress on any other recorded metrics (queen mass, average callow worker mass, laying patterns, the egg proteome, and queen spermathecal fluid proteome). Previously determined candidate heat and cold stress biomarkers were not differentially expressed in stressed queens, indicating that these markers only have short-term post-stress diagnostic utility. Combined with variable sperm viability responses to temperature stress reported in different studies, these data also suggest that there is substantial variation in temperature tolerance, with respect to impacts on fertility, amongst queens. Future research should aim to quantify the variation and heritability of temperature tolerance, particularly heat, in different populations of queens in an effort to promote queen resilience.

Upregulation of Transferrin and Major Royal Jelly Proteins in the Spermathecal Fluid of Mated Honeybee (Apis mellifera) Queens.

Simple SummaryTo understand the mechanisms underlying long-term storage and survival of sperm in honeybee Apis mellifera queens, previous studies have elucidated the components of honeybee spermathecal fluid. However, the expression profiles of transferrin (Tf) and major royal jelly proteins 1–9 (MRJPs 1–9) in the spermatheca and spermathecal fluid of mated honeybee queens have still not been characterized. In this study, we confirmed upregulation of Tf and MRJPs in the spermatheca and spermathecal fluid of mated honeybee queens by using RNA sequencing, reverse transcription-polymerase chain reaction, and Western blot analyses. The levels of Tf and antioxidant enzymes were elevated in the spermathecal fluid of the mated queens, paralleling the levels of reactive oxygen species, H2O2, and iron. The increased levels of MRJPs, especially MRJP1, MRJP4, and MRJP6, in the spermathecal fluid of mated queens may be responsible for energy provision during sperm storage in honeybee queens. Overall, our findings indicate that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens, providing a novel insight into antioxidant defense and energy metabolism for stored sperm in honeybee queens.Sperm storage in the spermathecae of honeybee (Apis mellifera) queens is vital for reproduction of honeybees. However, the molecular mechanisms whereby queens store sperm in a viable state over prolonged periods in the spermatheca are not fully understood. Here, we conducted RNA sequencing analysis of the spermathecae in both virgin and mated A. mellifera queens 24 h after mating and observed that the genes encoding transferrin (Tf) and major royal jelly proteins (MRJPs) were differentially expressed in the spermathecae of mated queens. The concentrations of Tf and antioxidant proteins such as superoxide dismutase 1, catalase, and glutathione peroxidase as well as the levels of reactive oxygen species, H2O2, and iron were higher in the spermathecal fluid of the mated queens than in virgin queens. Tf upregulation is likely to perform a protective role against the Fenton reaction occurring between iron and H2O2 in the antioxidant pathway in the mated queen’s spermathecal fluid. Furthermore, MRJPs—especially MRJP1, MRJP4, and MRJP6—were upregulated in the mated queen’s spermathecal fluid, indicating that they may serve as antimicrobial and antioxidant agents as well as an energy source for stored sperm in the spermathecal fluid of honeybee queens. Together, our findings show that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens.

Honey bee queen health is unaffected by contact exposure to pesticides commonly found in beeswax

Honey bee queen health is crucial for colony health and productivity, and pesticides have been previously associated with queen loss and premature supersedure. Prior research has investigated the effects of indirect pesticide exposure on queens via workers, as well as direct effects on queens during development. However, as adults, queens are in constant contact with wax as they walk on comb and lay eggs; therefore, direct pesticide contact with adult queens is a relevant but seldom investigated exposure route. Here, we conducted laboratory and field experiments to investigate the impacts of topical pesticide exposure on adult queens. We tested six pesticides commonly found in wax: coumaphos, tau-fluvalinate, atrazine, 2,4-DMPF, chlorpyriphos, chlorothalonil, and a cocktail of all six, each administered at 1, 4, 8, 16, and 32 times the concentrations typically found in wax. We found no effect of any treatment on queen mass, sperm viability, or fat body protein expression. In a field trial testing queen topical exposure of a pesticide cocktail, we found no impact on egg-laying pattern, queen mass, emergence mass of daughter workers, and no proteins in the spermathecal fluid were differentially expressed. These experiments consistently show that pesticides commonly found in wax have no direct impact on queen performance, reproduction, or quality metrics at the doses tested. We suggest that previously reported associations between high levels of pesticide residues in wax and queen failure are most likely driven by indirect effects of worker exposure (either through wax or other hive products) on queen care or queen perception.

Factors Influencing the Reproductive Ability of Male Bees: Current Knowledge and Further Directions.

Simple SummaryBumblebees and honeybees are well known as the dominant and most important pollinators in natural and agricultural ecosystems. The quality characteristics of their colonies depend greatly on the reproductive ability/quality of the parents (queens and drones). Male bees, despite their exclusive reproductive role and ability to determine colony quality, have been less considered than female bees, especially bumblebees. We reviewed the current studies on environmental factors and inherent characteristics that affect the mating success and fecundity of male honeybees and bumblebees. Temperature, nutrients, pesticides, body size, weight and age affect reproduction in male bees and consequently the progeny colony quality. However, more studies, especially in male bumblebees, are still needed to address the impacts of these factors in detail to confront the requirements of agricultural pollination and declining wild bee pollinators worldwide.Bumblebees and honeybees are very important pollinators and play a vital role in agricultural and natural ecosystems. The quality of their colonies is determined by the queens and the reproductive drones of mother colonies, and mated drones transmit semen, including half of the genetic materials, to queens and enhance their fertility. Therefore, factors affecting drone fecundity will also directly affect progeny at the colony level. Here, we review environmental and bee-related factors that are closely related to drone reproductive ability. The environmental factors that mainly affect the sperm count and the viability of males include temperature, nutrients and pesticides. In addition, the inherent characteristics of male bees, such as body size, weight, age, seminal fluid proteins and proteins of the spermathecal fluid, contribute to mating success, sperm quality during long-term storage in the spermathecae and the reproductive behaviors of queens. Based on the results of previous studies, we also suggest that the effects of somatotype dimorphism in bumblebee males on sperm quality and queen fecundity and the indispensable and exploitable function of gland proteins in the fecundity of males and queens should be given more attention in further studies.

Trade-offs between sperm viability and immune protein expression in honey bee queens (Apis mellifera)

Queens of many social hymenoptera keep sperm alive within their specialized storage organ, the spermatheca, for years, defying the typical trade-off between lifespan and reproduction. However, whether honey bee (Apis mellifera) queens experience a trade-off between reproduction and immunity is unknown, and the biochemical processes underlying sperm viability are poorly understood. Here, we survey quality metrics and viral loads of honey bee queens from nine genetic sources. Queens rated as ‘failed’ by beekeepers had lower sperm viability, fewer sperm, and higher levels of sacbrood virus and black queen cell virus. Quantitative proteomics on N = 123 spermathecal fluid samples shows, after accounting for sperm count, health status, and apiary effects, five spermathecal fluid proteins significantly correlating with sperm viability: odorant binding protein (OBP)14, lysozyme, serpin 88Ea, artichoke, and heat-shock protein (HSP)10. The significant negative correlation of lysozyme—a conserved immune effector—with sperm viability is consistent with a reproduction vs. immunity trade-off in honey bee queens.

Protein-Level Interactions as Mediators of Sexual Conflict in Ants*

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

Histological Structure of Reproductive Organ of Freshwater Female Crab, Barytelphusa Cunicularis (West-Wood)

<em>Mercury concentrations were recorded in water and tissue of Barytelphusa cunicularis from Pimpalwadi site (Jaikwadi Dam) near Aurangabad. The level of heavy metals in the ovary and spermatheca of crabs was investigated using Atomic Absorption Spectrophotometer (AAS). The mean concentration of mercury in the crab was 0.9 ±0.001 µg/g. A histopathological alteration in ovary and spermatheca was also studied. Several histological changes were noted in the ovary tissue i. e. Distortion of yolk granules, vacuolization, slight necrosis in the oocytes in the ovary and vacuolization observe in lumen, granular substances, sperm mass and spermathecal fluid was evenly distributed in the crab was observed after exposed to sublethal concentration (24<sup>th</sup> of LC<sub>50</sub>:1/5<sup>th</sup> 0.208 ppm) of mercuric chloride.</em>

Sperm use economy of honeybee (Apis mellifera) queens.

The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long‐lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10−6 to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker‐destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness.

A scientific note on amoeboid movement of honey bee semen

honeybee /Apismellifera /spermatozoa /amoeboidmovement /spermcooperationHoney bee (Apis mellifera) semen is a tan, viscousliquid containing tightly packed and slowly movingspermatozoa (Stort & Goncalves 1986). Immediatelyafter transfer of spermatozoa to the queen’s sperma-theca, they move faster, but after 1 h, the movementstops (Kerr et al. 1962) and later during storage thespermatozoa remain immotile (Lensky & Schindler1967). The immotility is reversible as long as thespermatozoa are alive (Verma 1978). In nature, honeybee semen is diluted with spermathecal fluid but thespermatozoa survive also in many human madediluents (Moritz 1984; Taylor et al. 2009).Here, I report that diluted honey bee semenbehaves in an unusual way; it produces pseudopodiaand is able to move. The movement differs from theamoeboid movement of a single spermatozoon(Nelson & Ward 1981) and involves the coordinatedbehaviour of a group of spermatozoa.Honey bee drones were collected at hive entrancesand were stored in a cage attended by workers. Toinitiate ejaculation, the abdomen of a drone waspressed with fingers, a standard method (Laidlaw &Page 1997). The semen was collected with a syringedesigned for instrumental insemination of honey beequeens. The semen was evacuated from the syringeonto a microscopic slide. From there, it was collectedpassively into a MEDLAB glass capillary (insidediameter 0.7 mm, outside diameter 1.3 mm andlength 50 mm). The semen inside the capillary wasdiluted 1:1 (v/v) with spermathecal fluid obtained froma young uninseminated honey bee queen. Its sperma-theca wasdissectedfrom the queen immediately beforethe experiment and crushed on a microscope slide. Thespermathecalfluidwascollectedusingtheinseminationsyringe, and a droplet of it was evacuated onto anothercleanmicroscopicslide.Whenthecapillarywithsemenwas brought into contact with the droplet, it was drawninto the capillary and diffused through the semen. Thecapillary was positioned horizontally, and it wasphotographed every hour with a Nikon D70 digitalcameratodeterminethespeedofsemenmovement.Theexperiment was repeated 10 times using the semen ofdifferent drones.The behaviour of diluted semen on a microscopicslide was observed with a Nikon Eclipse E600microscope. Semen movement was recorded with aJVC colour video camera (resolution 720×576) at-tached to the microscope. In some experiments, thedroplet of diluted semen was surrounded by six smalldroplets of spermathecal fluid. There was a distance ofabout 0.2 mm between the droplet of semen and thedroplets of spermathecal fluid. If a pseudopodiumformed near a droplet of diluent, it was video recorded.In all experiments, the capillaries or microscopeslides with semen were kept in a transparent plasticcontainer with a tissue wetted with distilled water. Thewet tissue provided high humidity and prevented thesemen from drying. In some experiments, the semenwasdilutedinKievbufferinsteadofspermathecalfluid.The Kiev buffer consisted of 0.3 g D+ glucose, 0.41 g

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee ( Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.

Insights into female sperm storage from the spermathecal fluid proteome of the honeybee Apis mellifera.

A proteomic and metabolic network analysis of honeybee queen spermathecal fluid provides insights into female long-term sperm storage mechanisms.

Proteins in spermathecal gland secretion and spermathecal fluid and the properties of a 29 kDa protein in queens of Apis mellifera

One and two dimensional SDS-PAGE were used to characterize the protein pattern of the spermathecal gland secretion and spermathecal fluid in Apis mellifera queen pupae and emerged queens of different ages. The concentration of protein varied from 8.5 and 15.3 mg/mL in the spermathecal fluid, and from 5 to 8.5 mg/mL in the secretion. Development of the protein pattern of the gland secretion and spermathecal fluid was identical from pupae until the age of 3 days. In sexually mature queens (10 days or older) the gland secretion and spermathecal fluid each had one additional band at 79 kDa and at 29 kDa respectively. The 29 kDa protein was N-terminal blocked but several peptide fragments were sequenced after digestion with LysC protease. Only 2 of the sequences showed a distinct homology to the N-terminal half of the glycolytic enzyme triosephosphate isomerase (TPI). TPI antibody reacted with the 29 kDa protein, but the enzymatic activity was only 1/100 compared to TPI of hemolymph. The possible function of the protein is discussed. spermatheca / sperm storage / protein pattern / 29 kDa protein / Apis mellifera

Instrumental insemination of Apis mellifera queens with hetero- and conspecific spermatozoa results in different sperm survival

Sixty three queens of Apis mellifera were inseminated each with about 8 million spermatozoa from either 1 A. mellifera drone, 8 A. cerana, 5 A. dorsata or 20 A. florea drones. Spermatozoa were collected from vesiculae seminales, diluted in buffer and re-concentrated at 1,000 g for 10 minutes. Between 1.4% and 2.8% of the spermatozoa reached the spermatheca. Motility of spermatozoa of A. mellifera and A. cerana did not change within 4 weeks, it was nearly 100%. The motility of A. florea spermatozoa decreased to 83.4% after 3 days and to 33.9% after 4 weeks and motility of A. dorsata spermatozoa decreased to 61.2% after 3 days and to 26% after 4 weeks. Fertilization of A. mellifera eggs was 57% by A. mellifera spermatozoa. Calculation based on non-hatching eggs showed that about 40% were fertilized by A. cerana and A. florea and less than 20% by A. dorsata spermatozoa. The composition of spermathecal fluid seems to be different within the species and its significance for long term sperm storage is discussed. sperm survival / spermathecal fluid / hetero specific insemination / Apis mellifera queens / Apis species

Analysis and Content of ATP in Spermatozoa of Honeybees

SummaryAdenosine triphosphate (ATP) concentration in sperm of honeybee (Apis mellifera) was determined by using a firefly-luciferase-luminescence method. Sperm from drone ejaculates contained 136·6 nmol ATP/109 cells. Sperm recovered from queen spermathecae contained 209·7 nmol ATP/109 cells. The ATP content of the spermathecal wall and fluid increased as queens became older. The methods described for ATP measurement of sperm extracts provide an alternative to the haemocytometer method for estimating concentrations of sperm from honeybees and possibly other insect species as well.

Inorganic ions in spermathecal fluid and their transport across the spermathecal membrane of the queen bee, Apis mellifera

Micropuncture and microanalytical methods were employed to investigate the rôle of the spermathecal epithelium of the honey queen-bee in providing the appropriate conditions for the prolonged storage of spermatozoa. It was found that the epithelium maintains large concentration gradients of inorganic ions, generates an electrical potential difference of 21 mV, lumen positive, and produces a pH difference of up to 2.4 pH units between spermathecal fluid (SF) and haemolymph (H). The SF H concentration ratios for K +, Na +, Ca ++, Cl −1, HPO 4 −−, H 2PO 4 − and amino acids were 7.7; 0.5; 0.8; 0.4; 1.03; 0.004; 0.3, respectively. While the pH value of haemolymph was constant at 6.17, the pH of SF increased with age from 7.3 to 8.6 over the first 3 days. The calculated electrochemical potential differences suggest that the epithelium of the spermathecal wall secretes K + (and possibly HCO 3 − or OH −) actively into the lumen, but handles Na + passively. This pattern conforms with the organization of ion transport in other insects.

Biology of the Spermatozoon ofApis Mellifera.I. Influence Of Diluents and pH

SummaryA relationship between the pH of the spermathecal fluid and the age of Apis mellifera virgin queens was found. When the queens were 5 days old the pH was 9·7 and it is at this age that mating flights start. It is suggested that spermatozoa migrate to the spermatheca by chemotoxis, due to the difference in pH of the semen (7·) and spermathecal fluid. Coconut water, pH 7·, was a very good preserving agent and diluent of A. mellifera sperm.

The structure and maturation of the spermatozoa of Sciara coprophila.

AbstractThe axial filament of Sciara coprophila does not conform to the usual 9 + 2 filament pattern but consists, rather, of as many as 76 pairs of filaments which decrease in number from the anterior to the posterior region of the sperm. It is first seen at the base of the head in the shape of an indented oval. The axial filament varies in configuration along the remaining length of the sperm as one whorl or two connected whorls of filament pairs.The other structures of the sperm revealed by the light and electron microscopes are a homogeneous, dense, spear‐shaped nucleus, a row of spherical dense bodies in the middle piece enclosed by the axial filament and of unknown nature and function and a single mitochondrial derivative.The mitochondrial nebenkern derivative consists of a large electron transparent region bordered by cristae and a smaller paracrystalline region located adjacent to the axial filament. The derivative arises as paracrystalline material in a medial nuclear indentation. The electron transparent material is first seen at the anterior end of the middle piece.Unlike other known insect sperm, but reminiscent of sperm capacitation in mammals, sperm maturation is completed in the spermathecae of Sciara 7 to 9 hours after insemination. It consists of the acquisition of sperm motility and elimination of the electron transparent region of the mitochondrial nebenkern derivative. The electron microscope reveals in mature sperm that the axial filament doublets have changed configuration and consist of a single whorl which encloses the paracrystalline rod. The process by which the major portion of the nebenkern derivative is eliminated occurs in four identifiable stages. Since sperm maturation does not appear to be intrinsically controlled, factors in the spermathecal fluid may play a role in its completion.