Origin and rearing season of honeybee queens affect some of their physiological and reproductive characteristics

Queen honeybees of Apis mellifera ligustica and Apis mellifera syriaca were raised to investigate physiological and reproductive characteristics and to determine the most suitable time for queen rearing under semi‐arid conditions in Jordan. The queen rearing season as well as the origin of the queens affected the queens’ weight, acceptance, preoviposition period, volume of the spermatheca, and quantity and quality of sperm in the spermatheca. Italian bees were heavier than Syrian bees at emergence. The introduced queen acceptance rate appeared to be a genetic influence of the queen: A. m. ligustica virgin queens were accepted at a higher rate than were A. m. syriaca queens. There were large seasonal variations in the acceptance rate. Experimental bee colonies accepted their virgin queens during spring with good honey flows at a higher rate compared to the other rearing periods. The greatest mating success was achieved in May and the smallest was during July and August. The preoviposition period was shorter in the Syrian than in the Italian queens, and was longer during summer for both honeybee subspecies. The volume of the spermatheca was smaller in Syrian bees and the spermatheca had lower numbers of spermatozoa compared with Italian bees. Thus, under semi‐arid Mediterranean region conditions, it is highly recommended to raise virgin queens in the spring months only to obtain their highest quality.     

The responses of Apis mellifera jemenitica to different artificial queen rearing techniques

In the current study, we investigated if any variations exist in acceptance rate of grafted larvae and quality of queens reared in different queen cell cup sizes, between wet and dry grafting and between queen right and queen less conditions of A. m. jemenitica colonies. The acceptance rate of grafted larvae in different queen cell cup sizes (7.0 mm, 7.5 mm, 8.0 mm, 8.5 mm) were varying from 69 to 71% and the variations were not significant among the different queen cups sizes but averagely lower than the acceptances recorded for other races. Out of the 172 dry grafted larvae, only 56.4% of them were accepted while in wet grafting out of 174 grafted larvae 77.01% were accepted. Regarding the rate of sealing, 48.84% and 71.84% of them sealed for dry and wet grafts, respectively. The observed variation in the rate of acceptance and sealing were significant (N = 346, df = 1, P < 0.0001) between the two techniques. However, there was no significant difference in fresh weight of emerged queens between the two grafting methods. Out of the 324 grafted larvae given to queen right and queen less starter colonies each; 106 (32.72%) and 252 (73.68%) were accepted in queen right and queen less starter colonies, respectively and the variation was highly significant at P < 0.0001. The total number of sealed pupae were 82 (25.31%) and 216 (63.16%) for queen right and queen less colonies, respectively and the variations was significant at P < 0.0001. From the study it can be concluded that A. m. jemenitica colonies can rear significantly more queens under wet grafting and in queen less colonies conditions than dry grafting and queen right conditions     

Circular movement of honey bee spermatozoa inside spermatheca

It is believed that in honey bees spermatozoa stored inside the spermatheca remain motionless, however, some studies have reported the contrary. To observe behaviour of spermatozoa inside spermathecae, we have instrumentally inseminated queens with spermatozoa stained with fluorescent stains. During the first 8 h after insemination movement of the spermatozoa was stationary, without fast forward movement. Later, we observed circular movement of the spermatozoa inside spermathecae. Numerous circles were visible at one time. The circles were located close to the spermathecal wall. Movement of the spermatozoa was also observed in spermathecae of naturally inseminated queens. The marble-like pattern of the spermathecae was changing. The changes were slow and well visible only when video recordings were played at high speed.     

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.     

First record of intersex in neotenic reproductives of the termite Cryptotermes brevis (Isoptera: Kalotermitidae)

Drywood termites of the family Kalotermitidae present a very flexible developmental pattern, in which pseudergates are totipotent and may become reproductives. In this study, three colonies of Cryptotermes brevis headed by neotenic reproductives were used: (i) colony A, with a primary king and a neotenic queen; (ii) colony B, with a primary queen and a neotenic king; and (iii) colony C, with neotenic king and queen. The primary king from colony A and the neotenic king from colony B presented well‐developed reproductive systems, with conspicuous testicular lobes and cysts containing spermatozoa also observed in the seminal vesicles. The neotenic queen of colony A and the primary queen of colony B had ovaries with several oocytes in early developmental stages with some of them already vitellogenic and terminal, which suggests egg‐laying activity. Both of these queens presented the spermathecae lumen filled with spermatozoa. The queen of colony C had vitellogenic oocytes; however, the oocytes were not terminal and the lumen of spermathecae was devoid of spermatozoa and secretions. The seminal vesicles of colony C neotenic king have no spermatozoa. This king displayed a previtellogenic oocyte among its testicular lobes, which suggested that this individual was an intersex.     

Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones

The current study aimed to investigate the important reproductive biology and morphology of A.m. jemenitica queens and drones through measuring the weight of virgin and mated queens, size and weight of spermathecae, weight of ovaries, number of ovarioles, quantity and viability of semen in queen and drones. Accordingly, the average weights of 0.139 ± 0.01 g and 0.143 ± 0.013 g recorded for virgin and mated queens respectively. The sizes of spermathecae were 1.248 ± 0.103 mm and 1.25 ± 0.022 mm for virgin and mated queens respectively. The mean weight of ovaries was 0.013 ± 0.003 g and the numbers of ovarioles varied from 124 to 163 with the mean of 142.9 ± 9.47 and with no significant difference between virgin and mated queens. The average number of stored sperm per spermathecae of mated queen was estimated to be 4.202 ± 0.613 million with the viability of 80.39%. The average number of sperm per drone recorded was 8,763,950 ± 1,633,203.15 with viability of 79.54 ± 6.70%. In general, the current study revealed that the values recorded for reproductive biology and morphological characters of A. m. jemenitica queens and drones were relatively lower than values recorded for other Apis mellifera races. This mainly could be associated with the body size of the race which is known to be the smallest race among A. mellifera races. Moreover, the harsh environmental conditions of the regions, high temperature, low humidity and limited resources may have contributed for the smaller biological and morphological values. The information will serve as a base in future selection and breeding of program of the race.     

Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors

Queen health is closely linked to colony performance in honey bees as a single queen is normally responsible for all egg laying and brood production within the colony. In the U. S. in recent years, queens have been failing at a high rate; with 50% or greater of queens replaced in colonies within 6 months when historically a queen might live one to two years. This high rate of queen failure coincides with the high mortality rates of colonies in the US, some years with >50% of colonies dying. In the current study, surveys of sperm viability in US queens were made to determine if sperm viability plays a role in queen or colony failure. Wide variation was observed in sperm viability from four sets of queens removed from colonies that beekeepers rated as in good health (n = 12; average viability = 92%), were replacing as part of normal management (n = 28; 57%), or where rated as failing (n = 18 and 19; 54% and 55%). Two additional paired set of queens showed a statistically significant difference in viability between colonies rated by the beekeeper as failing or in good health from the same apiaries. Queens removed from colonies rated in good health averaged high viability (ca. 85%) while those rated as failing or in poor health had significantly lower viability (ca. 50%). Thus low sperm viability was indicative of, or linked to, colony performance. To explore the source of low sperm viability, six commercial queen breeders were surveyed and wide variation in viability (range 60–90%) was documented between breeders. This variability could originate from the drones the queens mate with or temperature extremes that queens are exposed to during shipment. The role of shipping temperature as a possible explanation for low sperm viability was explored. We documented that during shipment queens are exposed to temperature spikes (<8 and > 40°C) and these spikes can kill 50% or more of the sperm stored in queen spermathecae in live queens. Clearly low sperm viability is linked to colony performance and laboratory and field data provide evidence that temperature extremes are a potential causative factor.     

CO2麻醉对中华蜜蜂处女蜂王卵巢发育的影响

【目的】明确中华蜜蜂Apis cerana cerana蜂王羽化后生殖系统的动态变化,探索CO₂麻醉对中华蜜蜂处女蜂王卵巢发育的影响。【方法】通过人工育王技术和蜂王的储存获取不同日龄的中华蜜蜂处女蜂王,观察蜂王羽化后生殖系统的形态,测定生殖系统各项形态学指标(体重、卵巢重、受精囊直径和卵巢管数目)。中华蜜蜂处女蜂王连续2 d(分别于5和6日龄时)接受不同浓度(50%, 75%与90%)CO₂麻醉8 min, 以及不同日龄处女蜂王接受75% CO₂麻醉1~3次(每次8 min),待蜂王发育至10日龄时,利用石蜡切片技术与HE染色法观察蜂王卵巢形态、测定蜂王卵巢重和蜂王卵巢最大卵母细胞长度,通过实时荧光定量PCR(RT-qPCR)测定10日龄蜂王头部卵黄原蛋白基因(Vg)的相对表达量。【结果】中华蜜蜂蜂王的卵巢管数目与受精囊直径不会随着日龄发生明显的波动,但蜂王卵巢重在6-12日龄明显增加,之后将趋于稳定。与对照组(未接受CO₂麻醉处理)相比,50%, 75%与90% CO₂麻醉后中华蜜蜂蜂王卵巢重与最大卵母细胞长度均增加,并且头部Vg基因的相对表达量上调。其中CO₂浓度为75%,麻醉次数2次显著促进了中华蜜蜂蜂王的卵巢发育。【结论】中华蜜蜂蜂王羽化后卵巢会发生明显的发育变化,而CO2麻醉能加速卵巢的发育。     

Team swimming in ant spermatozoa

In species where females mate promiscuously, competition between ejaculates from different males to fertilize the ova is an important selective force shaping many aspects of male reproductive traits, such as sperm number, sperm length and sperm–sperm interactions. In eusocial Hymenoptera (bees, wasps and ants), males die shortly after mating and their reproductive success is ultimately limited by the amount of sperm stored in the queen''s spermatheca. Multiple mating by queens is expected to impose intense selective pressure on males to optimize the transfer of sperm to the storage organ. Here, we report a remarkable case of cooperation between spermatozoa in the desert ant Cataglyphis savignyi. Males ejaculate bundles of 50–100 spermatozoa. Sperm bundles swim on average 51% faster than solitary sperm cells. Team swimming is expected to increase the amount of sperm stored in the queen spermatheca and, ultimately, enhance male posthumous fitness.     

Coagulation parameters do not change during luteal phase and pregnancy in cats

Changes in coagulation parameters depending on reproductive status and pregnancy have been previously reported in both human and other veterinary species. The objective of this study was to determine if different reproductive status affects coagulation parameters in queens. Blood samples from 66 queens submitted to spay surgery were obtained. A hemostatic panel including platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and D-dimer and also progesterone concentrations were measured before surgery. According to progesterone results and embryo vesicles diameter, four groups were established: (1) nonpregnant queens with low (≤1 ng/mL) progesterone concentration (LP) (n = 33); (2) nonpregnant queens with high (≥2 ng/mL) progesterone concentration (n = 8) (HP); (3) first half of pregnancy (n = 12); and (4) second half of pregnancy (n = 13). None of the evaluated parameters showed statistically significant differences among the different groups. There was no significant linear correlation between progesterone values and coagulation parameters. In conclusion, neither the presence of the embryo nor the higher values of progesterone concentration induced statistically significant changes in the coagulation profile studied.     

Variation in the quality of Vespula vulgaris (L.) queens (Hymenoptera: Vespidae) and its significance in wasp population dynamics

Abstract

Density‐related variation in queen quality has been proposed as a possible mechanism regulating population fluctuations in Vespula species. We investigated annual variation in the quality (size, weight, and fat content) of adult V. vulgaris queens representing four stages of their life cycle (spring, founding, developing, and emerged) taken from six sites in beech forest, South Island, New Zealand. For each queen the dry weight, head width, and thorax length was measured. For a subsample of queens, the fat content was determined by ether extraction. The size of queen cells was measured from a subsample of nests. Size, weight, and fat content of queens varied between wasp colonies and sites. The smallest juvenile queens were under‐represented in the reproductive population. There was no direct link between body size and food supply. Size and weight of developing queens increased as the number of cells in the nest increased. The size of the queen cells varied significantly among layers in a nest and among nests. The under‐representation of small queens in the reproductive population suggests that queen quality may affect survival and/or competitive ability by increasing winter fat storage, nest building activity, and/or success in usurpation disputes.     

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 characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.     

Assessing the mating 'health' of commercial honey bee queens

Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices.     

Effects of fluvalinate and coumaphos on queen honey bees (Hymenoptera: Apidae) in two commercial queen rearing operations

We conducted research on the potential impacts of fluvalinate and coumaphos on honey bee, Apis mellifera L., queen viability and health. Queens were reared in colonies that had been treated with differing amounts of both fluvalinate and coumaphos. Pre- and posttreatment samples of both wax and bees were collected from all of the colonies and analyzed for total concentrations of fluvalinate and coumaphos. All queens were measured for queen weight, ovarial weight, and number of sperm in the spermathecae. The queens treated with high doses of fluvalinate weighed significantly less than low-dose or control queens, but otherwise appeared to develop normally. The highest fluvalinate concentrations were observed in the wax and queen cells of the high-dose group. The developing queens in colonies treated with as little as one coumaphos-impregnated strip for more than 24 h suffered a high mortality rate. Several of the queens showed sublethal effects from the coumaphos, including physical abnormalities and atypical behavior. The queens exposed to coumaphos weighed significantly less and had lower ovary weights than the control group queens. The highest coumaphos concentrations were observed in the queen cells and wax of the high-dose groups.     

Hydroxyflutamide alters the characteristics of live boar spermatozoa

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 μg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the “C-Ruch” computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P < 0.05) in sperm mitochondrial membrane potential and a concomitant increase (P < 0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 μg OH-Flu compared with the respective controls and other doses used (P < 0.05). The adverse effects of OH-Flu become strengthened over time (P < 0.05). Notably, 50 and 100 μg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P < 0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P < 0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P < 0.05) only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility.     

Queen-worker differences in spermatheca reservoir of phylogenetically basal ants

Ant queens mate when young and store sperm in their spermatheca to fertilize eggs for several years until their death. In contrast, workers in most species never mate. We have compared the histological organization of spermathecae in 25 poneromorph species exhibiting various degrees of queen-worker dimorphism. The spermathecae of both castes in all species are similar in having a reservoir connected by a sperm duct to the ovary, and a paired gland opening into this duct. The reservoir of queens typically has a columnar epithelium in the hilar region (near the opening of the sperm duct), whereas the epithelium in the distal region is cuboidal. Abundant mitochondria together with apical microvilli and basal invaginations indicate an osmoregulatory function. In contrast, the reservoir epithelium of workers is flattened throughout and lacks these transport characteristics. This single difference shows the importance of a columnar epithelium in the reservoir for sperm storage. However, our data have not revealed inter-specific variations in the development of the hilar region linked with higher fecundity. We have found no consistent differences in associated structures, such as the spermatheca gland or sperm ducts, or in the musculature between queens and workers.This work was funded by IWT, FWO, KULeuven OT and JSPS.     

Sperm competition and last-male precedence in the honeybee

Five microsatellite loci were used to determine paternities in six Apis mellifera colonies headed by naturally mated queens. The last inseminating males were identified by collecting and genotyping the mating sign left in the genital tract of each queen. Significant differences in paternity frequencies were observed between males, but the proportion of worker and queen offspring sired by the last inseminating drone did not differ significantly from those of other drones. Each male kept his rank of precedence for the different cohorts, although the variance in subfamily proportions decreased over time, most notably in the colony displaying the lowest level of polyandry. These results suggest that, if sperm competition exists in the honeybee, it does not significantly increase the fitness of the last inseminating drone. The spermatozoa of the different inseminating drones are not totally mixed before they reach the spermatheca, in particular when only few males mate with the queen. The weak difference in the subfamily proportions observed between queen and worker samples confirms that nepotistic interactions are rare. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

The caste system of the dolichoderine antTechnomyrmex albipes (Hymenoptera: Formicidae): morphological description of queens,workers and reproductively active intercastes

Females ofTechnomyrmex albipes consist of winged queens, intercastes and workers. In established colonies, reproduction is performed by many intercastes (wingless females which have intermediate phenotypes between queen and worker characters). Dissection and morphological examination revealed that intercastes had a spermatheca, but workers did not. Intercastes can be divided into three classes: major intercastes with three ocelli, medium intercastes with one ocellus, and minor intercases without ocelli. Workers have no ocelli. The thoracic structure of intercastes gradually becomes more complex from minors to majors. The body size of intercastes gradually increases from minors to majors, and so does the number of overioles. The body size distributions of minor intercastes and workers overlap considerably, but the distributions of ovariole numbers overlap less. Winged queens had distinctly larger body sizes, more ovarioles and larger spermathecae than intercastes. Most intercastes were inseminated with developed ovaries and appeared to reproduce. The caste system and reproductive division of labour inT. albipes is compared to those of ant species in which permanently wingless females reproduce.     

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 (Chi² = 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.