- Email: [email protected]
New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction
IP 3080
No. of Pages 7, Model 5G
3 May 2013 Journal of Insect Physiology xxx (2013) xxx–xxx 1
Contents lists available at SciVerse ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 5 6
New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction
3 4 7 8 9 10 12 11 13 1 2 5 9 16 17 18 19 20 21 22 23 24 25 26 27 28
Q3
Jakob Wegener a,⇑, Zachary Y. Huang b, Matthias W. Lorenz c, Judith I. Lorenz c, Kaspar Bienefeld a a
Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany Department of Entomology, Michigan State University, East Lansing, MI 48824, USA c Department of Animal Ecology 1, University of Bayreuth, D-95440 Bayreuth, Germany b
a r t i c l e
i n f o
Article history: Received 18 December 2012 Received in revised form 16 April 2013 Accepted 18 April 2013 Available online xxxx Keywords: Juvenile hormone Makisterone Spermathecal gland Vitellogenin Dimorphism Caste
a b s t r a c t In workers of the Western honeybee, Apis mellifera, juvenile hormone (JH) and ecdysteroids regulate many aspects of age polyphenism. Here we investigated whether these derived functions in workers have developed by an uncoupling of endocrine mechanisms in adult queens and workers, or whether parallels can be found between the roles of the two hormones in both castes. We looked at yolk protein metabolism as a process central to the physiology of both queens and workers, and at sperm storage as a feature of the queen alone. Queens of differing fertility status (virgin, virgin but CO2-treated, inseminated, freshly laying and 1–2 years-old) were compared regarding vitellogenin (Vg), JH and ecdysteroid-titers in their hemolymph, as well as ovarian yolk protein and spermathecal gland composition. Our results showed that hormone titres were unrelated to the composition of spermathecal glands. JH-concentrations in the hemolymph were low in the groups of queens characterized by yolk uptake into the ovaries, and high in pre-vitellogenic queens or animals that were forced to interrupt egg-laying by caging. Ecdysteroidconcentrations were higher in untreated virgins than after insemination or during egg-laying. They were not affected by the caging of queens. These patterns of hormone changes were parallel to those known from worker bees. Together, these findings suggest a conserved role for JH as repressor of vitellogenin uptake into tissues, and for ecdysteroids in preparing tissues for this process. An involvement of the two hormones in the regulation of sperm storage seems unlikely. Our results add to the view that JH and ecdysteroids act similarly on the yolk protein metabolism of both castes of A. mellifera. This may imply that it was the biochemical versatility of Vg rather than that of hormonal regulatory circuits that allowed for the functional separation of the two castes. Ó 2013 Published by Elsevier Ltd.
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52
1. Introduction
53
In most insect species studied so far, including several social Hymenoptera, JH and ecdysteroids are involved in ovary activation, vitellogenin (Vg) synthesis and/or oogenesis in adult females (reviewed in Hagedorn and Kunkel, 1979; Raikhel et al., 2005; Hartfelder and Emlen, 2005; Gilbert, 2012). In the honey bee (Apis mellifera) however, JH and ecdysteroids are important elements for behavioral regulation in the quasi-sterile worker caste (Amdam et al., 2003, 2005; Nelson et al., 2007; Paul et al., 2005). Hemolymph concentrations of JH are low in hive bees and high in foragers (Huang et al., 1994). JH interacts with Vg to regulate longevity and behavioral maturation (Amdam et al., 2003, 2005; Nelson et al., 2007). Although a minimum concentration of the hormone
54 55 56 57 58 59 60 61 62 63 64
⇑ Corresponding author. Tel.: +49 3303 293830; fax: +49 3303 293840. E-mail addresses: [email protected] (J. Wegener), [email protected] (Z.Y. Huang), [email protected] (M.W. Lorenz), [email protected] (J.I. Lorenz), [email protected] (K. Bienefeld).
appears to be required for initiating Vg synthesis (Rutz and Lüscher, 1974; Hartfelder and Emlen, 2005), injections of larger doses of JH or analogs lead to the dropping of Vg titers in the hemolymph (Rutz et al., 1976; Pinto et al., 2000) and to the early onset of foraging (Jaycox et al., 1974). Allatectomized workers show reduced flight performance and metabolic rates, indicating additional and maybe more fundamental roles of this hormone (Sullivan et al., 2003). The role of ecdysteroids in worker behavior has only recently been recognized. The ovaries are a likely source of ecdysteroids or at least participate in their synthesis in workers (Amdam et al., 2010; Yamazaki et al., 2011), and individuals with larger or supernumerary ovaries have been shown to forage earlier than individuals with smaller ones (Amdam et al., 2010; Wang et al., 2010). The expression of genes involved in ecdysteroid signaling in the mushroom bodies of worker brains also suggests a role of these hormones in behavioral development (Paul et al., 2005; Yamazaki et al., 2011). The discovery of these new roles of JH and ecdysteroids in honey bee workers leads to the question of how they are reconciled
0022-1910/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
IP 3080
No. of Pages 7, Model 5G
3 May 2013 2 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
with the persistent need for regulation of reproductive processes in queens. Results on JH titers in queens are inconsistent. While Fluri et al. (1981) found no relationship with mating status, Fahrbach et al. (1995) reported higher titers in newly emerged and banked queens than in sexually mature virgins and actively laying queens. Both in vivo and in vitro analyses suggest a loss of the typical role of JH as a stimulant for Vg synthesis (reviewed in Engels and Imperatriz-Fonseca, 1990; Hartfelder and Emlen, 2005), similar to the situation in workers. Corona et al. (2007) suggested that JH may interact with Vg and elements of the insulin signaling pathway to regulate queen longevity. In the case of ecdysteroids, one study (Robinson et al., 1991) showed a positive correlation between fertility and hormone titers, while another (Hartfelder et al., 2002) failed to detect this relationship and came to the conclusion that in higher eusocial bees, ecdysteroids may have lost their role in reproduction. Our first aim was therefore to resolve the discrepancies of earlier studies regarding the phenotypic link between hemolymph titers of JH and ecdysteroids on one hand and Vg and queen reproductive status on the other. The second aim was to use this knowledge regulatory mechanisms in the queen caste to look for physiological patterns that are similar to those described for the worker caste. Our third aim was to investigate possible relationships between the two hormones and sperm storage by A. mellifera queens. The capacity for long-term storage of male gametes tends to be correlated with the level of sociality in several groups of ants as well as bees (Ito and Ohkawara, 1994; Martins and Serrao, 2002). While non-social species typically store sperm for periods of hours to weeks, eusocial species store male gametes for months or even years (Gobin et al., 2006). It can therefore be argued that long-term sperm storage in the Hymenoptera likely presents an adaptation to sociality in the queen caste. Attraction of sperm to the spermatheca, their conservation and reactivation for fertilization are thought to be mediated by different classes of secretions from the spermathecal glands (Ruttner and Koeniger, 1971; Lensky and Schindler, 1967; Koeniger, 1970; Verma and Shuel, 1973; Dallai, 1975; Collins et al., 2006; Wegener et al., 2013). As sperm storage is functionally closely linked to mating and oogenesis, we hypothesized that the classical insect gonadotropins JH and ecdysteroids may be involved in the regulation of spermathecal glands as well. Hence, we looked for associations between JH and ecdysteroid titers and changes in the chemical composition of the spermathecal glands. As far as we know, endocrine control of the spermathecal glands has never been studies in any insect before.
128
2. Materials and methods
129
2.1. Queens
130
Queens of the subspecies A. mellifera carnica were reared in strong, queenless colonies according to standard techniques (Ruttner, 1976). All were daughters of the same, multiply mated mother. Shortly after emergence, they were transferred into mating nuclei of approximately 1500–2000 workers. The entrances of the hives were equipped with queen excluder grids to prevent natural mating. When the queens were 10–11 days old, all but 5 were narcotized with CO2 for seven minutes and reintroduced into their nuclei. Eighteen to 24 h later, all but 7 of the previously CO2-treated queens were artificially inseminated with semen of mixed origin (7 ll/queen) following standard procedures (Ruttner, 1983). Insemination involved a second CO2-narcosis of 3–7 min. Another set of 10 of the inseminated queens (12–13 days old, 24 h after insemination) were caught from their nuclei and samples for the determination of Vg and hormone titers were taken as described
131 132 133 134 135 136 137 138 139 140 141 142 143 144
below. The uninseminated, unnarcotized as well as the uninseminated but CO2-treated queens were sampled in the same way. The remaining 9 queens were allowed to initiate egg-laying inside their nuclei, which they did after 4–10 days. On the 24th to 25th day after emergence, they were caught and samples were taken from them as well. In addition to the queens reared especially for this experiment, two outgroups were included in the analyses. Both consisted of 1–2 year-old, fertile A. mellifera carnica-queens taken from colonies of various sizes. These queens were of diverse genetic origins, although all belonged to the same subspecies, A. mellifera carnica. In one group, hemolymph and ovary samples were taken directly (<10 min) after removal from the colonies. In a second group, queens were prevented from egg-laying for 4–10 h by caging in wire mesh cages (3 4 0.8 cm) before sampling. They were accompanied by 4–6 workers. The cages were supplied with a drop of honey and kept at room temperature in the dark. Altogether, the following groups of queens were compared:
145
(A) 12–13 days post-emergence, no CO2-treatment, no insemination (n = 5). (B) 12–13 days post-emergence, 24-36 h after CO2-treatment (n = 7). (C) 12–13 days post-emergence, 24 h after insemination (n = 10). (D) 24–25 days post-emergence, shortly after onset of egg-laying (n = 9). (E) 1–2 years old, actively egg-laying (n = 9). (F) 1–2 years old, caged for 4–10 h before dissection (n = 9).
162
146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161
163 164 165 166 167 168 169 170 171 172
2.2. Sample preparation
173
Queens were cold-anaesthetized on ice. For the measurement of hormone and Vg-titers, two samples of hemolymph (1–3 and 5– 6 ll) were collected into glass capillaries from small incisions in the intersegmental membrane between the last two abdominal segments. The larger sample was diluted in 500 ll ice-cold methanol and later used for the measurement of JH. The smaller sample, used for Vg and ecdysteroid quantification, was expelled into 500 ll of ice-cold Tris-buffer (0.05 mol/l TRIS, 0.16 mol/l NaCl, 0.1 mmol/l phenylmethylsulfonyl fluoride, pH 8.5). This sample was further divided into two subsamples: one percent was used for the estimation of Vg and 99% was used for the estimation of ecdysteroids. The queens were decapitated and their ovaries placed into 500 ll of Tris-buffer for the measurement of ovarian Vg/vitellin. The spermathecae together with the adhering spermathecal glands were placed on a microscope slide. The glands were removed with a pair of forceps and frozen in 300 ll of TRIS-buffer.
174
2.3. Quantification of JH, ecdysteroids and Vg
191
The samples intended for JH-measurement were shipped on dry ice from Germany to Michigan (USA). JH III was extracted and JH titers were estimated according to well-established procedures used in honey bees (Huang et al., 1994; Jassim et al., 2000; Slone et al., 2012). Liquid scintillation counting was performed using a Q2 Tricarb2100TR (Packard), which gave the radioactivity in disintegrations per min for each sample. A standard curve with various amounts (0, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 pg) of standard JH-III (Sigma) was obtained each day that samples were measured, following the protocol of Huang and Robinson (1996). Each sample was determined in duplicates. The amount of JH in the samples was corrected by dividing by two, because the racemic mixture of JH standard overestimates the 10R JH in biological samples by a factor of two.
192
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190
193 194 195 196 197 198 199 200 201 202 203 204 205
IP 3080
No. of Pages 7, Model 5G
3 May 2013 3
216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237
238 239
2.4. Determination of the chemical composition of the spermathecal glands
246
Spermathecal glands were homogenized by use of a hand-held motorized pestle. The amounts of free carbohydrate, glycogen, lipid, and protein were measured according to Lorenz (2003) with the modifications described in Lorenz (2007). In addition, to increase the sensitivity of the method, which was necessary due to the small size of the spermathecal glands, the solvent volumes of the procedure were down-scaled by a factor of 5.
247
2.5. Statistical analysis
248
251
IBM-SPSS 19 was used for data analysis. Differences between the treatment groups were analyzed using the Kruskal-Wallis nonparametric ANOVA followed by stepwise step-down multiple testing (SPSS, 2010) to identify homogenous groups of treatments.
252
3. Results
253
Fig. 1 summarizes the measurements of hormones and Vg. Vg was present already in the hemolymph of virgins not treated with CO2. Hemolymph protein titers did not differ significantly among different treatments (Fig. 1A, n = 48; df = 5; Z = 1.83; P = 0.87), whereas ovarian Vg/vitellin varied strongly (n = 48; df = 5; Z = 26.9; P < 0.001). Average quantities were low (<4 mg Vg-equivalent/ovary) in untreated, CO2-treated and freshly inseminated animals. They were much higher (17–28 mg/ovary) in fertile animals (treatments D–F). Among the groups of fertile queens, age or caging did not significantly affect the quantities of the two proteins in the ovaries (P > 0.05). Levels of JH also differed significantly among the treatments (Fig. 1C, n = 49; df = 5; Z = 33.9; P < 0.001).
240 241 242 243 244 245
249 250
254 255 256 257 258 259 260 261 262 263 264
a 100
50
0 40
B
b
30
b b
20
10
a
a
a
0 80
C
60
a
a
a
40
b bc
20
c 0 150
D
a ab
100
bc
abc c
c
50
0
1-2 years, caged
215
a
1-2 years, fertile
214
a
a
after onset of egg-laying
213
a
24h after insemination
212
a 150
24h after CO2
210 211
A
virgin
209
200
Vitellin per ovary [mg vitellogenin-equivalents]
208
The samples intended for the quantification of Vg and ecdysteroids were transported within Germany from Berlin to Bayreuth on dry ice. The hemolymph-buffer-mixture was transferred from the original reaction tubes that had been used for hemolymph sampling into 6.5 ml polypropylene tubes (Sarstedt, Nümbrecht, Germany). The original tubes were rinsed twice with 500 ll of methanol each by vigorous vortexing and a 5 min treatment in an ultrasonic bath. Rinsing solutions were combined with the hemolymph-buffer-mixture and 3 ml isooctane was added for defatting. After vortexing and treatment in the ultrasonic bath, samples were centrifuged (5500g, 4 °C, 10 min). After discarding the top phase (isooctane containing the lipids), the remaining methanol-buffer mixture was transferred into a new 6.5 ml tube, mixed with 2 ml of water and concentrated to a volume of 500 ll in a Speed Vac. The following fractionation of ecdysteroids by solid phase separation (to obtain free ecdysteroids) as well as their quantification by radioimmuno assay were performed as described in Lorenz et al. (1997) with the following modifications: DBL-1 antiserum, which displays a high cross-reactivity for makisterone A, was used. The standard curve was obtained by using makisterone A (AG Scientific, USA) as the non-labeled standard (0–10,000 pg). Vg/vitellin in queen hemolymph and ovaries was determined by ELISA as in Wegener et al. (2009a). Ovarian tissue was homogenized in the TRIS-buffer for 35 s with a fast-spinning motorized pestle. Each sample was measured in two dilutions, 2000- and 10,000-fold. Protein measured in the ovaries is probably a mixture of Vg and vitellin. In the honey bee, the two are known to be electrophoretically indistinguishable (Engels et al., 1990). It is known from previous studies that the antibody used shows affinity to both Vg and vitellin (Wegener et al., 2009a, 2010). As only Vg was used for the standard curves, results are given in Vg-equivalents.
Hemolymph juvenile hormone [pg/µl]
207
Hemolymph ecdysteroids [pgkisterone A-equ./µl]
206
Hemolymph vitellogenin [µg/µl]
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
Fertility status of queens Fig. 1. Ecdysteroids and Juvenile hormone in hemolymph and vitellin/vitellogenin in hemolymph and ovaries of queens of different reproductive status. Bars show means ± SE. A total of 49 queens were used for the analyses. Different letters on top of bars indicate significant differences between treatments (P < 0.05).
They were moderate in untreated virgins and remained so after CO2-treatment. They were lower in freshly inseminated queens, and even lower in queens that had initiated oviposition
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
265 266 267
IP 3080
No. of Pages 7, Model 5G
3 May 2013
276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
296
4. Discussion
297
4.1. A caste-transgressing role for JH in A. mellifera?
298
Juvenile hormone is a stimulant of reproductive dominance, Vg synthesis and/or oogenesis in several groups of social Hymenoptera, including Vespidae (reviewed by Strambi, 1990), Halictidae (Bell, 1973; Smith et al., 2013), and Apidae (reviewed in Hartfelder, 2000). Even in the closely related bumble bee Bombus terrestris, JH titers are positively linked to ovary development and social status of queenless workers (Röseler, 1977; Röseler and Röseler, 1988; Bloch et al., 2000a). In adult A. mellifera however, JH has long been thought to have lost its classical role as stimulant of gonadic activity in favor of a new role as ‘‘behavioral pacemaker’’ (Robinson
300 301 302 303 304 305 306 307
a
2
0 8
B
b b
6 b
b
ab
4
a
2
0
13
C
b
ab
ab
ab
10 a a
8 5 3 0 20
D c
15
d
10 a
ab ab b
5
0
virgin
299
a
a
4
1-2 years, caged
275
a a
1-2 years, fertile
274
6
after onset of egg-laying
273
a
24h after insemination
272
A
24h after CO2
271
8
Glycogen [µg/pair of glands]
270
(treatments C and D, P < 0.05 in both cases). JH-titers significantly increased if queens were caged before sampling (P < 0.05). Hemolymph titers of ecdysteroids varied among treatment groups (n = 47; df = 5; Z = 16.83; P = 0.005; Fig. 1D). They were higher in untreated virgins than in old, fertile queens regardless of caging (P < 0.05), while animals from the other treatment groups showed intermediate values. The ovaries of all caged queens contained yellowish bodies towards the posterior end of the ovarioles (Fig. 2), indicative of follicles in the process of degeneration (Gauthier et al., 2011) or resorption (Hitchcock, 1956; Flanders, 1959). They did not contain any mature eggs as were found in the ovaries of most laying queens. Fig. 3 shows the results of the analyses of spermathecal glands. Glycogen titers did not vary according to queen reproductive status (Fig. 3A, n = 49; df = 5; Z = 9.1; P = 0.078). Free carbohydrate contents did vary (Fig. 3B, n = 49; df = 5; Z = 15.2; P = 0.01), showing a peak in freshly inseminated animals and dropping sharply after the onset of egg-laying. Lipids also varied in quantity (Fig. 3C, n = 49; df = 5; Z = 22.0; P = 0.001), with lower amounts found in CO2-treated and freshly inseminated animals than in all other treatment groups (P < 0.05). Protein titers in the glands displayed the clearest variations between the treatments (Fig. 3D, n = 49; df = 5; Z = 47.1; P < 0.001). They dropped slightly after insemination, but then increased again after the onset of egg-laying (P < 0.05). They were still higher in 1–2 year-old, fertile animals, and slightly dropped if animals were caged before sampling (P < 0.05).
Free carbohydrates [µg/pair of glands]
269
Lipid [µg/pair of glands]
268
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
Protein [µg BSA-equivalents/pair of glands]
4
Fertility status of queens Fig. 3. Chemical composition of spermathecal glands in queens of different reproductive status. Bars show means ± SE. A total of 49 queens were used for the analyses. Different letters on top of bars indicate significant differences between treatments groups (P < 0.05). No significant differences between treatment groups were observed regarding glycogen content.
Fig. 2. Degeneration/resorption of follicles in caged queens. Stereomicroscopic image of the dissected abdomen of a fertile queen bee after 6 h of caging. Arrows indicate yellowish bodies representing follicles in the process of resorption/ degeneration.
et al., 1991; Robinson and Vargo, 1997; Hartfelder, 2000; Engels et al., 1990; Pinto et al., 2000). This view has been changed by the finding that JH and ecdysteroids do participate in the initiation
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
308 309 310
IP 3080
No. of Pages 7, Model 5G
3 May 2013 J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364
365 366 367 368 369 370 371 372 373
of Vg-synthesis, but that this event has shifted in time from the Q4 adult to the late pupal stage (Barchuk et al., 2002). The pattern of JH-concentrations we observed in the hemolymph of adult queens is generally in good accordance with the data of Robinson et al. (1991) and Fahrbach et al. (1995). In contrast, Fluri et al. (1981) detected no differences in JH-titers between virgin, non-reproductive and mated, laying queens. This may be due to the fact that in their experiment, all queens were caged for >1 day before sampling. Results from our caged animals suggest that this procedure is likely to have influenced the outcome. We show that in adult queens, changes in hemolymph titers of JH coincide with changes in the reproductive status. JH-titers were found to be low in females that are engaged in active oogenesis, regardless of their chronological age and mating status, and high in queens where this is not the case. Caging induced a sudden stop of oogenesis, as evidenced by the occurrence of degenerating/ partly resorbed follicles or oocytes and the absence of mature eggs. This stop coincided with a sharp rise in JH-concentrations. Double treatment with CO2, which is known to lead to the initiation of Vg synthesis and oogenesis in honeybee queens (Mackensen, 1947; Engels and Ramamurty, 1976), also led to a drop in JH titers. To summarize, JH concentrations were low in the hemolymph of animals with starting or ongoing vitellogenesis, and high in previtellogenic animals or animals that showed signs of yolk resorption. A similar negative relationship between JH and Vg uptake into tissues can also be found in workers, where hemolymph concentrations of JH are low in nurses and winter bees, both of which take up Vg into their hypopharyngeal glands, as well as in individuals with vitellogenic ovaries. Foragers, in which neither ovaries nor hypopharyngeal glands are known to take up Vg, have higher hemolymph titers of JH (Fluri et al., 1982; Huang et al., 1994). Together, these findings strongly suggest a role of JH as suppressor of Vg-synthesis and/or uptake in both castes of A. mellifera. Treatment of young workers with the JH-analog pyriproxyfen leads to the suppression of Vg synthesis (Pinto et al., 2000), which seems to suggest that the effect of JH lies at the level of synthesis. Corona et al. (2007) showed that expression of the Vg-gene in queen heads is reduced by administration of a high dose of another JH-analog, methoprene, or of an unspecified dose of JH III. On the other hand, the same authors show that abdominal concentrations of VgmRNA are highest in queens shortly after emergence, when JH-titers are known to peak as well (Fahrbach et al., 1995). An earlier study found that injections of JH III prevent synthesis only at high doses, while low doses stimulate Vg production (Rutz et al., 1976). Engels et al. (1990) reported that application of JH III to queens or incubation of queen fat bodies with this hormone did not affect Vg synthesis. The fact that in the untreated virgin queens of our study, high titers of JH coincided with the presence of relatively high levels of Vg in the hemolymph also seems to contradict a universal role for JH as suppressor of yolk protein synthesis. Therefore, we suggest that the primary role of JH in Vg metabolism is the suppression of uptake by target tissues rather than the suppression of synthesis.
4.2. Functions of ecdysteroids in honey bee queens – similarities to the worker caste In B. terrestris, the role of ecdysteroids in reproduction is less clear than for JH, although higher concentrations in the hemolymph are linked with dominance status in queenless workers (Bloch et al., 2000b; Geva et al., 2005). In adults of this species, the ovaries are an important source of ecdysteroids (Geva et al., 2005). In workers of A. mellifera however, only the early steps of ecdysteroid synthesis seem to be located in the ovary, whereas
5
the later stages mainly take place in the brain and fat body (Yamazaki et al., 2011). Earlier studies regarding ecdysteroids in queen honey bees have given inconsistent results. Robinson et al. (1991) found higher titers in laying queens than in functionally sterile or laying workers, whereas Hartfelder et al. (2002) found no differences between newly-emerged and actively laying queens, or between queens and workers of any reproductive status. Here we found higher titers in sexually mature virgin than in laying queens. While these findings are insufficient to resolve the discrepancies between the two earlier reports, they show that elevated ecdysteroid titers in queens are a transient state, explaining why different studies, using animals of differing reproductive status, may come to seemingly contradictive results. Ecdysteroids in workers bees are known to promote general protein synthesis by the fat body of queens (Engels et al., 1990), and titers are higher in reproductive animals of both castes than they are in non-reproductive workers (Robinson et al., 1991). On the other hand, there is no continuous relationship between individual levels of ecdysteroids and development of either ovaries or hypopharyngeal glands in workers (Hartfelder et al., 2002; Wegener et al., 2009b). Instead there is a peak of hemolymph ecdysteroid concentration on day three of adult life (Hartfelder et al., 2002), i.e. shortly before the onset of secretory activity of the hypopharyngeal glands, while titers are low during the phase of intensive secretion of the glands (approximately days 4–14). In queenless worker bees, high titers have been found in individuals whose ovaries were on the brink of vitellogenesis (Wegener et al., 2009b), while individuals with undeveloped or vitellogenic ovaries had lower titers (Hartfelder et al., 2002; Wegener et al., 2009b). In the present study, we found high ecdysteroid levels in queens with pre-vitellogenic ovaries, as evidenced by a low Vg/ vitellin-content, and lower levels in animals with vitellogenic ovaries. Taken together, these results seem to suggest a role for ecdysteroids in preparing tissues for Vg uptake. Robinson et al. (1991) showed that the ecdysteroid-levels measured in laying queens are still much higher than those present in queenright nurses and foragers. This could mean that in addition to its proposed role in preparing organs for Vg uptake, sustained levels of ecdysteroids are required for oogenesis. This hypothesis is supported by results of Paul et al. (2005), who detected transcripts of an ecdysteroiddependent transcription regulator gene, AmE74, in pre-vitellogenic and vitellogenic egg chambers of actively laying queens. In vitellogenic egg chambers, the transcripts are found in the follicle cells, which in A. mellifera are known to take up Vg and channel it to the surface of the oocyte (Fleig, 1995). However, no transcripts of AmE74 were detected in the heads of active nurse bees, so this gene is either not or only transiently involved in Vg-uptake by the hypopharyngeal glands.
374
4.3. Role of JH and ecdysteroids in the regulation of the spermathecal glands
423
Sociality can evolve independently of long-term sperm storage, and not all sperm-storing species are social. Nevertheless, the capacity to store male gametes in great numbers for prolonged periods of time is a characterizing feature of the queens of social Hymenoptera (Gobin et al., 2006), indicating that it may be adaptive within the context of hymenopteran sociality. Sperm storage is not a static state, but a process, involving the steps of attracting sperm to the site of storage, keeping them alive, and activating them for fertilization. In A. mellifera, the spermathecal glands are thought to be involved in all of these steps, and different classes of substances from the glands have been suggested to play a role at different stages of the process (Lensky and Schindler, 1967; Koeniger, 1970; Verma and Shuel, 1973; Dallai, 1975; Collins
425
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422
424
426 427 428 429 430 431 432 433 434 435 436 437
IP 3080
No. of Pages 7, Model 5G
3 May 2013 6
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
466
et al., 2006; Baer et al., 2009; Wegener et al., 2013). Given that sperm storage is a process and that it has to be coordinated with other processes like mating and oogenesis, it likely needs regulation, and the classical insect gonadotropins JH and ecdysteroids seemed possible candidate signal molecules. However, the fluctuations observed in the chemical composition of the glands did not follow the same pattern as those of the two groups of hormones, so a direct control of the glandular activity by JH or ecdysteroids seems unlikely. Instead, some of our results could shed a light on possible functions of different classes of substances during sperm storage. The fact that free carbohydrates tended to accumulate in the glands after CO2-treatment and insemination and then sharply dropped after the onset of egg-laying may indicate that these substances could be involved in the transfer of sperm into the spermatheca. Energy generation in freshly ejaculated honey bee sperm is known to be sugar-driven (Verma, 1978), while sperm stored inside the theca are thought to catabolize lipids (Verma and Shuel, 1973), which would be in accordance with the observed rise in lipid contents in the glands of newly-laying queens. Dallai (1975) observed lipid droplets in the glands of mated, but not of virgin honeybee queens. Interestingly, strong differences in gland composition were observed between queens that had recently begun oviposition and animals that had been fertile for >1 year. This change was most marked in the case of protein. Enzymes from the spermathecal glands are known to support energy metabolism of stored sperm and provide protection against oxidative stress (Collins et al., 2006; Baer et al., 2009). Our results suggest that the importance of this enzymatic support system grows as spermatozoa increase in age.
467
4.4. Implications for the physiology of A. mellifera caste dimorphism
468
489
West-Eberhard (1996) suggested that in eusocial species, the role of regulatory mechanisms derived from those of solitary ancestors is modulated by the nutritional status of individuals, resulting in a splitting between (well-nourished) queens and (less well nourished) nurses (‘‘split function’’-hypothesis). We here present an example of how functions of regulatory circuits in queens and workers are split regarding their effects on the life history of individuals, while remaining the same at a fundamental physiological level. Whether (or to which degree) these circuits are based on those of solitary ancestors cannot be deduced from our findings. Like in many solitary species (reviewed in Hartfelder, 2000), JH and ecdysteroids in A. mellifera queens seem to be involved in reproductive processes. On the other hand, the negative association between Vg uptake and JH is clearly atypical of the situation in the Hymenoptera as a whole (reviewed in Robinson and Vargo, 1997). At present, the caste-transgressing roles for JH and ecdysteroids regarding Vg uptake we propose here are mostly based on correlations only and have to be verified by an inductive approach. If confirmed, they would add to the view that the supposed loss of function of the two groups of hormones in honey bee reproduction has to be reconsidered.
490
Acknowledgements
491
We thank Anja Rogge for excellent apicultural assistance. This work was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation. Some of the equipment used was financed through the European Fund for Regional Development (EFRE).
438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488
492 493 494 495 496 497
References
498
Amdam, G.V., Norberg, K., Hagen, A., Omholt, S.W., 2003. Social exploitation of vitellogenin. Proceedings of the National Academy of Sciences of the United States of America 100, 1799–1802. Amdam, G.V., Aase, A.L.T.O., Seehuus, S.C., Fondrk, M.K., Norberg, K., Hartfelder, K., 2005. Social reversal of immunosenescence in honey bee workers. Experimental Gerontology 40, 939–947. Amdam, G., Page, R.E., Fondrk, M., Brent, C., 2010. Hormone response to bidirectional selection on social behavior. Evolution and Development 12, 428–436. Baer, B., Heazlewood, J.L., Taylor, N.L., Eubel, H., Millar, A.H., 2009. The seminal fluid proteome of the honeybee Apis mellifera. Proteomics 9, 2085–2097. Bell, W.J., 1973. Factors controlling initiation of vitellogenesis in a primitively social bee, Lasioglossum zephyrum (Hymenoptera: Halictidae). Insectes Sociaux 20, 253–260. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Cnaani, J., Hefetz, A., 2000a. Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. Journal of Insect Physiology 46, 47– 57. Bloch, G., Hefetz, A., Hartfelder, K., 2000b. Ecdysteroid titer, ovary status, and dominance in adult worker and queen bumble bees (Bombus terrestris). Journal of Insect Physiology 46, 1033–1040. Collins, A.M., Caperna, T.J., Williams, V., Garrett, W.M., Evans, J.D., 2006. Proteomic analyses of male contributions to honey bee sperm storage and mating. Insect Molecular Biology 15, 541–549. Corona, M., Velarde, R.A., Remolina, S.R., Moran-Lauter, A., Wang, Y., Hughes, K.A., Robinson, G.E., 2007. Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proceedings of the Academy of Sciences of the United States of America 104, 7128–7133. Dallai, R., 1975. Fine structure of the spermatheca of Apis mellifera. Journal of Insect Physiology 21, 89–109. Engels, W., Ramamurty, P.S., 1976. Initiation of oögenesis in allatectomised virgin honeybee queens by carbon dioxide treatment. Journal of Insect Physiology 22, 1427–1432. Engels, W., Imperatriz-Fonseca, V.L., 1990. Caste development, reproductive strategies, and control of fertility in honey bees and stingless bees. In: Engels, W. (Ed.), Social Insects: An Evolutionary Approach to Castes and Reproduction. Springer, Berlin, pp. 167–230. Engels, W., Kaatz, H., Zillikens, A., Simoes, Z.L.P., Trube, A., Braun, R., Dittrich, F., 1990. Honey bee reproduction: vitellogenin and caste-specific regulation of fertility. In: Hoshi, M., Yamashita, O. (Eds.), Advances in Invertebrate Reproduction, Elsevier, vol. 5. London, New York, pp. 495–502. Fahrbach, S., Giray, T., Robinson, G.E., 1995. Volume changes in the mushroom bodies of adult honey bee queens. Neurobiology of Learning and Memory 63, 181–191. Flanders, S.E., 1959. Embryonic starvation. An explanation of the defective honeybee egg. Journal of Economic Entomology 52, 166–167. Fleig, R., 1995. Role of the follicle cells for yolk uptake in ovarian follicles of the honey bee Apis mellifera L. (Hymenoptera: Apidae). International Journal of Insect Morphology and Embryology 24, 427–433. Fluri, P., Sabatini, A.G., Vecchi, M., Wille, H., 1981. Blood juvenile hormone, protein and vitellogenin titres in laying and non-laying queen honeybees. Journal of Apicultural Research 20, 221–225. Fluri, P., Lüscher, M., Wille, H., Gerig, L., 1982. Changes of weight of the pharyngeal gland and haemolymph titres of juvenile hormone, protein and vitellogenin in worker honey bees. Journal of Insect Physiology 28, 61–68. Gauthier, L., Ravallec, M., Tournaire, M., Cousserans, F., Bergoin, M., Dainat, B., Miranda, J.D., 2011. Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One 6, e16217, doi: 16210.11371/ journal.pone.0016217. Geva, S., Hartfelder, K., Bloch, G., 2005. Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris. Journal of Insect Physiology 51, 811–823. Gilbert, L.E., 2012. Insect Endocrinology. Academic Press, London, Waltham, San Diego. Gobin, B., Ito, F., Peeters, C., Billen, J., 2006. Queen-worker differences in spermatheca reservoir of phylogenetically basal ants. Cell and Tissue Research 326, 169–178. Hagedorn, H.H., Kunkel, J.G., 1979. Vitellogenin and vitellin in insects. Annual Review of Entomology 24, 475–505. Hartfelder, K., 2000. Insect juvenile hormone: from ‘status quo’ to high society. Brazilian Journal of Medical and Biological Research 33, 157–177. Hartfelder, K., Bitondi, M.M.G., Santana, W.C., Simoes, Z.L.P., 2002. Ecdysteroid titer and reproduction in queens and workers of the honey bee and of a stingless bee: loss of ecdysteroid function at increasing levels of sociality? Insect Biochemistry and Molecular Biology 32, 211–216. Hartfelder, K., Emlen, D.J., 2005. Endocrine control of insect polymorphism. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Insect Molecular Science, vol. 3. Elsevier, Oxford, pp. 651–703. Hitchcock, J.D., 1956. Honey bee queens whose eggs all fail to hatch. Journal of Economic Entomology 49, 11–14. Huang, Z.-Y., Robinson, G., Borst, D., 1994. Physiological correlates of division of labor among similarly aged honey bees. Journal of Comparative Physiology A 174, 731–739.
499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
IP 3080
No. of Pages 7, Model 5G
3 May 2013 J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634
Huang, Z.-Y., Robinson, G.E., 1996. Regulation of honey bee division of labor by colony age demography. Behavioral Ecology and Sociobiology 39, 147–158. Ito, F., Ohkawara, K., 1994. Spermatheca size differentiation between queens and workers in primitive ants – relationship with reproductive structure of colonies. Naturwissenschaften 81, 138–140. Jassim, O., Huang, Z.Y., Robinson, G.E., 2000. Juvenile hormone profiles of worker honey bees during normal and accelerated behavioral development. Journal of Insect Physiology 46, 243–249. Jaycox, E.R., Skowronek, W., Guynn, G., 1974. Behavioral changes in worker honeybees (Apis mellifica L.) induced by injections of a juvenile hormone mimic. Annals of the Entomological Society of America 67, 529–535. Koeniger, G., 1970. Bedeutung der Tracheenhülle und der Anhangdrüse der Spermatheka für die Befruchtungsfähigkeit der Spermatozoen der Bienenkönigin (Apis mellifica L.). Apidologie 1, 55–71. Lensky, Y., Schindler, H., 1967. Motility and reversible inactivation of honeybee spermatozoa in vivo and in vitro. Annales de l’Abeille 10, 5–16. Lorenz, J.I., Lorenz, M.W., Hoffmann, K.H., 1997. Factors regulating juvenile hormone and ecdysteroid biosynthesis in Gryllus bimaculatus (Ensifera: Gryllidae). European Journal of Entomology 94, 369–379. Lorenz, M.W., 2003. Adipokinetic hormone inhibits the formation of energy stores and egg production in the cricket Gryllus bimaculatus. Comparative Biochemistry and Physiology B Comparative Biochemistry 136, 197–206. Lorenz, M.W., 2007. Oogenesis-flight syndrome in crickets: age-dependent egg production, flight performance, and biochemical composition of the flight muscles in adult female Gryllus bimaculatus. Journal of Insect Physiology 53, 819–832. Mackensen, O., 1947. Effect of CO2 on initial oviposition of artificially inseminated and virgin queen bees. Journal of Economic Entomology 40, 344–349. Martins, G.F., Serrao, J.E., 2002. A comparative study of the spermatheca in bees. Sociobiology 40, 711–720. Nelson, C.M., Ihle, K.E., Fondrk, M.K., Page, R.E., Amdam, G.V., 2007. The gene vitellogenin has multiple coordinating effects on social organization. Plos Biology 5, 673–677. Paul, R.K., Takeuchi, H., Matsuo, Y., Kubo, T., 2005. Gene expression of ecdysteroidregulated gene E74 of the honeybee in ovary and brain. Insect Molecular Biology 14, 9–15. Pinto, L.Z., Bitondi, M.M., Simoes, Z.L.P., 2000. Inhibition of vitellogenin synthesis in Apis mellifera workers by juvenile hormone analogue pyriproxyfen. Journal of Insect Physiology 46, 153–160. Raikhel, A.S., Brown, M.R., Belles, X., 2005. Hormonal control of reproductive processes. In: Gilbert, L.I. (Ed.), Comprehensive Molecular Insect Science, vol. 3. Elsevier Pergamon, Oxford, pp. 433–491. Robinson, G.E., Strambi, C., Strambi, A., Feldlaufer, M.F., 1991. Comparison of juvenile hormone and ecdysteroid haemolymph titers in adult worker and queen honey bees (Apis mellifera). Journal of Insect Physiology 37, 929–937. Robinson, G.E., Vargo, E.L., 1997. Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker. Archives of Insect Biochemistry and Physiology 35, 559–583. Röseler, P.F., 1977. Juvenile hormone control of oögenesis in Bumblebee workers, Bombus terrestris. Journal of Insect Physiology 23, 985–992. Röseler, P.F., Röseler, I., 1988. Influence of juvenile hormone on fat body metabolism in ovariectomized queens of the bumblebee, Bombus terrestris. Insect Biochemistry 18, 557–563.
7
Ruttner, F., Koeniger, G., 1971. Filling of spermatheca of honeybee queen – active migration or passive transport of spermatozoa. Zeitschrift für Vergleichende Physiologie 72, 411–422. Ruttner, F., 1976. The Instrumental Insemination of the Queen Bee, 2nd ed. Apimondia, Bukarest. Ruttner, F., 1983. Queen Rearing – Biological Basis and Technical Instructions, 2nd ed. Apimondia, Bucharest. Rutz, W., Lüscher, L., 1974. The occurrence of vitellogenin in workers and queens of Apis mellifica and the possibility of its transmission to the queen. Journal of Insect Physiology 20, 897–909. Rutz, W., Gerig, L., Wille, H., Lüscher, M., 1976. The function of juvenile hormone in adult worker honey bees, Apis mellifera. Journal of Insect Physiology 22, 1485– 1491. Slone, J.D., Stout, T.L., Huang, Z.Y., Schneider, S.S., 2012. The influence of drone physical condition on the likelihood of receiving vibration signals from worker honey bees, Apis mellifera. Insectes Sociaux 59, 101–107. Smith, A.R., Kapheim, K.M., Pérez-Ortega, B., Brent, C.S., Wcislo, W.T., 2013. Juvenile hormone levels reflect social opportunities in the facultatively eusocial sweat bee Megalopa genalis (Hymenoptera: Halictidae). Hormones and Behavior 63, 1–4. SPSS, 2010. IBM SPSS Statistics 19 core system user’s guide, IBM-SPSS. Strambi, A., 1990. Physiology and reproduction in social wasps. In: Engels, W. (Ed.), Social Insects – an Evolutionary Approach to Caste and Reproduction. Springer, Berlin, pp. 59–75. Sullivan, J., Fahrbach, S., Harrison, J., Capaldi, E., Fewell, J., Robinson, G., 2003. Juvenile hormone and division of labor in honeybee colonies: effects of allatectomy on flight behavior and metabolism. Journal of Experimental Biology 206, 2287–2296. Verma, L.R., Shuel, R.W., 1973. Respiratory metabolism of the semen of the honeybee, Apis mellifera. Journal of Insect Physiology 19, 97–103. Verma, L.R., 1978. Biology of honeybee spermatozoa 2. Effect of washing and sugars on respiration as measured by the cartesian diver technique. Journal of Apicultural Research 17, 176–181. Wang, Y.J., Kaftanoglu, O., Siegel, A., Page, R.E., Amdam, G., 2010. Surgically increased ovarian mass in the honey bee confirms link between reproductive physiology and worker behavior. Journal of Insect Physiology 56, 1816–1824. Wegener, J., Lorenz, M.W., Bienefeld, K., 2009a. Physiological consequences of prolonged nursing in the honey bee. Insectes Sociaux 56, 85–93. Wegener, J., Huang, Z.Y., Lorenz, M.W., Bienefeld, K., 2009b. Regulation of hypopharyngeal gland activity and oogenesis in honey bee (Apis mellifera) workers. Journal of Insect Physiology 55, 716–725. Wegener, J., Lorenz, M.W., Bienefeld, K., 2010. Differences between queen- and worker-laid eggs of the honey bee (Apis mellifera). Apidologie 41, 116–126. Wegener, J., Zschörnig, K., Onischke, K., Fuchs, B., Schiller, J., Müller, K., 2013. Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen – a TLC/MALDI-TOF MS study. Experimental Gerontology 48, 213–222. West-Eberhard, M.J., 1996. In: Wasp societies as microcosms for the study of development and evolution. In: Turillazzi, S., West-Eberhard, M.J. (Eds.), . Natural History and Evolution of Paper Wasps. Oxford Press, New York, pp. 290– 317. Yamazaki, Y., Kiuchi, M., Takeuchi, H., Kubo, T., 2011. Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L. Insect Biochemistry and Molecular Biology 41, 283–293.
Q5
635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
No. of Pages 7, Model 5G
3 May 2013 Journal of Insect Physiology xxx (2013) xxx–xxx 1
Contents lists available at SciVerse ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 5 6
New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction
3 4 7 8 9 10 12 11 13 1 2 5 9 16 17 18 19 20 21 22 23 24 25 26 27 28
Q3
Jakob Wegener a,⇑, Zachary Y. Huang b, Matthias W. Lorenz c, Judith I. Lorenz c, Kaspar Bienefeld a a
Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany Department of Entomology, Michigan State University, East Lansing, MI 48824, USA c Department of Animal Ecology 1, University of Bayreuth, D-95440 Bayreuth, Germany b
a r t i c l e
i n f o
Article history: Received 18 December 2012 Received in revised form 16 April 2013 Accepted 18 April 2013 Available online xxxx Keywords: Juvenile hormone Makisterone Spermathecal gland Vitellogenin Dimorphism Caste
a b s t r a c t In workers of the Western honeybee, Apis mellifera, juvenile hormone (JH) and ecdysteroids regulate many aspects of age polyphenism. Here we investigated whether these derived functions in workers have developed by an uncoupling of endocrine mechanisms in adult queens and workers, or whether parallels can be found between the roles of the two hormones in both castes. We looked at yolk protein metabolism as a process central to the physiology of both queens and workers, and at sperm storage as a feature of the queen alone. Queens of differing fertility status (virgin, virgin but CO2-treated, inseminated, freshly laying and 1–2 years-old) were compared regarding vitellogenin (Vg), JH and ecdysteroid-titers in their hemolymph, as well as ovarian yolk protein and spermathecal gland composition. Our results showed that hormone titres were unrelated to the composition of spermathecal glands. JH-concentrations in the hemolymph were low in the groups of queens characterized by yolk uptake into the ovaries, and high in pre-vitellogenic queens or animals that were forced to interrupt egg-laying by caging. Ecdysteroidconcentrations were higher in untreated virgins than after insemination or during egg-laying. They were not affected by the caging of queens. These patterns of hormone changes were parallel to those known from worker bees. Together, these findings suggest a conserved role for JH as repressor of vitellogenin uptake into tissues, and for ecdysteroids in preparing tissues for this process. An involvement of the two hormones in the regulation of sperm storage seems unlikely. Our results add to the view that JH and ecdysteroids act similarly on the yolk protein metabolism of both castes of A. mellifera. This may imply that it was the biochemical versatility of Vg rather than that of hormonal regulatory circuits that allowed for the functional separation of the two castes. Ó 2013 Published by Elsevier Ltd.
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52
1. Introduction
53
In most insect species studied so far, including several social Hymenoptera, JH and ecdysteroids are involved in ovary activation, vitellogenin (Vg) synthesis and/or oogenesis in adult females (reviewed in Hagedorn and Kunkel, 1979; Raikhel et al., 2005; Hartfelder and Emlen, 2005; Gilbert, 2012). In the honey bee (Apis mellifera) however, JH and ecdysteroids are important elements for behavioral regulation in the quasi-sterile worker caste (Amdam et al., 2003, 2005; Nelson et al., 2007; Paul et al., 2005). Hemolymph concentrations of JH are low in hive bees and high in foragers (Huang et al., 1994). JH interacts with Vg to regulate longevity and behavioral maturation (Amdam et al., 2003, 2005; Nelson et al., 2007). Although a minimum concentration of the hormone
54 55 56 57 58 59 60 61 62 63 64
⇑ Corresponding author. Tel.: +49 3303 293830; fax: +49 3303 293840. E-mail addresses: [email protected] (J. Wegener), [email protected] (Z.Y. Huang), [email protected] (M.W. Lorenz), [email protected] (J.I. Lorenz), [email protected] (K. Bienefeld).
appears to be required for initiating Vg synthesis (Rutz and Lüscher, 1974; Hartfelder and Emlen, 2005), injections of larger doses of JH or analogs lead to the dropping of Vg titers in the hemolymph (Rutz et al., 1976; Pinto et al., 2000) and to the early onset of foraging (Jaycox et al., 1974). Allatectomized workers show reduced flight performance and metabolic rates, indicating additional and maybe more fundamental roles of this hormone (Sullivan et al., 2003). The role of ecdysteroids in worker behavior has only recently been recognized. The ovaries are a likely source of ecdysteroids or at least participate in their synthesis in workers (Amdam et al., 2010; Yamazaki et al., 2011), and individuals with larger or supernumerary ovaries have been shown to forage earlier than individuals with smaller ones (Amdam et al., 2010; Wang et al., 2010). The expression of genes involved in ecdysteroid signaling in the mushroom bodies of worker brains also suggests a role of these hormones in behavioral development (Paul et al., 2005; Yamazaki et al., 2011). The discovery of these new roles of JH and ecdysteroids in honey bee workers leads to the question of how they are reconciled
0022-1910/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
IP 3080
No. of Pages 7, Model 5G
3 May 2013 2 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
with the persistent need for regulation of reproductive processes in queens. Results on JH titers in queens are inconsistent. While Fluri et al. (1981) found no relationship with mating status, Fahrbach et al. (1995) reported higher titers in newly emerged and banked queens than in sexually mature virgins and actively laying queens. Both in vivo and in vitro analyses suggest a loss of the typical role of JH as a stimulant for Vg synthesis (reviewed in Engels and Imperatriz-Fonseca, 1990; Hartfelder and Emlen, 2005), similar to the situation in workers. Corona et al. (2007) suggested that JH may interact with Vg and elements of the insulin signaling pathway to regulate queen longevity. In the case of ecdysteroids, one study (Robinson et al., 1991) showed a positive correlation between fertility and hormone titers, while another (Hartfelder et al., 2002) failed to detect this relationship and came to the conclusion that in higher eusocial bees, ecdysteroids may have lost their role in reproduction. Our first aim was therefore to resolve the discrepancies of earlier studies regarding the phenotypic link between hemolymph titers of JH and ecdysteroids on one hand and Vg and queen reproductive status on the other. The second aim was to use this knowledge regulatory mechanisms in the queen caste to look for physiological patterns that are similar to those described for the worker caste. Our third aim was to investigate possible relationships between the two hormones and sperm storage by A. mellifera queens. The capacity for long-term storage of male gametes tends to be correlated with the level of sociality in several groups of ants as well as bees (Ito and Ohkawara, 1994; Martins and Serrao, 2002). While non-social species typically store sperm for periods of hours to weeks, eusocial species store male gametes for months or even years (Gobin et al., 2006). It can therefore be argued that long-term sperm storage in the Hymenoptera likely presents an adaptation to sociality in the queen caste. Attraction of sperm to the spermatheca, their conservation and reactivation for fertilization are thought to be mediated by different classes of secretions from the spermathecal glands (Ruttner and Koeniger, 1971; Lensky and Schindler, 1967; Koeniger, 1970; Verma and Shuel, 1973; Dallai, 1975; Collins et al., 2006; Wegener et al., 2013). As sperm storage is functionally closely linked to mating and oogenesis, we hypothesized that the classical insect gonadotropins JH and ecdysteroids may be involved in the regulation of spermathecal glands as well. Hence, we looked for associations between JH and ecdysteroid titers and changes in the chemical composition of the spermathecal glands. As far as we know, endocrine control of the spermathecal glands has never been studies in any insect before.
128
2. Materials and methods
129
2.1. Queens
130
Queens of the subspecies A. mellifera carnica were reared in strong, queenless colonies according to standard techniques (Ruttner, 1976). All were daughters of the same, multiply mated mother. Shortly after emergence, they were transferred into mating nuclei of approximately 1500–2000 workers. The entrances of the hives were equipped with queen excluder grids to prevent natural mating. When the queens were 10–11 days old, all but 5 were narcotized with CO2 for seven minutes and reintroduced into their nuclei. Eighteen to 24 h later, all but 7 of the previously CO2-treated queens were artificially inseminated with semen of mixed origin (7 ll/queen) following standard procedures (Ruttner, 1983). Insemination involved a second CO2-narcosis of 3–7 min. Another set of 10 of the inseminated queens (12–13 days old, 24 h after insemination) were caught from their nuclei and samples for the determination of Vg and hormone titers were taken as described
131 132 133 134 135 136 137 138 139 140 141 142 143 144
below. The uninseminated, unnarcotized as well as the uninseminated but CO2-treated queens were sampled in the same way. The remaining 9 queens were allowed to initiate egg-laying inside their nuclei, which they did after 4–10 days. On the 24th to 25th day after emergence, they were caught and samples were taken from them as well. In addition to the queens reared especially for this experiment, two outgroups were included in the analyses. Both consisted of 1–2 year-old, fertile A. mellifera carnica-queens taken from colonies of various sizes. These queens were of diverse genetic origins, although all belonged to the same subspecies, A. mellifera carnica. In one group, hemolymph and ovary samples were taken directly (<10 min) after removal from the colonies. In a second group, queens were prevented from egg-laying for 4–10 h by caging in wire mesh cages (3 4 0.8 cm) before sampling. They were accompanied by 4–6 workers. The cages were supplied with a drop of honey and kept at room temperature in the dark. Altogether, the following groups of queens were compared:
145
(A) 12–13 days post-emergence, no CO2-treatment, no insemination (n = 5). (B) 12–13 days post-emergence, 24-36 h after CO2-treatment (n = 7). (C) 12–13 days post-emergence, 24 h after insemination (n = 10). (D) 24–25 days post-emergence, shortly after onset of egg-laying (n = 9). (E) 1–2 years old, actively egg-laying (n = 9). (F) 1–2 years old, caged for 4–10 h before dissection (n = 9).
162
146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161
163 164 165 166 167 168 169 170 171 172
2.2. Sample preparation
173
Queens were cold-anaesthetized on ice. For the measurement of hormone and Vg-titers, two samples of hemolymph (1–3 and 5– 6 ll) were collected into glass capillaries from small incisions in the intersegmental membrane between the last two abdominal segments. The larger sample was diluted in 500 ll ice-cold methanol and later used for the measurement of JH. The smaller sample, used for Vg and ecdysteroid quantification, was expelled into 500 ll of ice-cold Tris-buffer (0.05 mol/l TRIS, 0.16 mol/l NaCl, 0.1 mmol/l phenylmethylsulfonyl fluoride, pH 8.5). This sample was further divided into two subsamples: one percent was used for the estimation of Vg and 99% was used for the estimation of ecdysteroids. The queens were decapitated and their ovaries placed into 500 ll of Tris-buffer for the measurement of ovarian Vg/vitellin. The spermathecae together with the adhering spermathecal glands were placed on a microscope slide. The glands were removed with a pair of forceps and frozen in 300 ll of TRIS-buffer.
174
2.3. Quantification of JH, ecdysteroids and Vg
191
The samples intended for JH-measurement were shipped on dry ice from Germany to Michigan (USA). JH III was extracted and JH titers were estimated according to well-established procedures used in honey bees (Huang et al., 1994; Jassim et al., 2000; Slone et al., 2012). Liquid scintillation counting was performed using a Q2 Tricarb2100TR (Packard), which gave the radioactivity in disintegrations per min for each sample. A standard curve with various amounts (0, 3, 10, 30, 100, 300, 1000, 3000, and 10,000 pg) of standard JH-III (Sigma) was obtained each day that samples were measured, following the protocol of Huang and Robinson (1996). Each sample was determined in duplicates. The amount of JH in the samples was corrected by dividing by two, because the racemic mixture of JH standard overestimates the 10R JH in biological samples by a factor of two.
192
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190
193 194 195 196 197 198 199 200 201 202 203 204 205
IP 3080
No. of Pages 7, Model 5G
3 May 2013 3
216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237
238 239
2.4. Determination of the chemical composition of the spermathecal glands
246
Spermathecal glands were homogenized by use of a hand-held motorized pestle. The amounts of free carbohydrate, glycogen, lipid, and protein were measured according to Lorenz (2003) with the modifications described in Lorenz (2007). In addition, to increase the sensitivity of the method, which was necessary due to the small size of the spermathecal glands, the solvent volumes of the procedure were down-scaled by a factor of 5.
247
2.5. Statistical analysis
248
251
IBM-SPSS 19 was used for data analysis. Differences between the treatment groups were analyzed using the Kruskal-Wallis nonparametric ANOVA followed by stepwise step-down multiple testing (SPSS, 2010) to identify homogenous groups of treatments.
252
3. Results
253
Fig. 1 summarizes the measurements of hormones and Vg. Vg was present already in the hemolymph of virgins not treated with CO2. Hemolymph protein titers did not differ significantly among different treatments (Fig. 1A, n = 48; df = 5; Z = 1.83; P = 0.87), whereas ovarian Vg/vitellin varied strongly (n = 48; df = 5; Z = 26.9; P < 0.001). Average quantities were low (<4 mg Vg-equivalent/ovary) in untreated, CO2-treated and freshly inseminated animals. They were much higher (17–28 mg/ovary) in fertile animals (treatments D–F). Among the groups of fertile queens, age or caging did not significantly affect the quantities of the two proteins in the ovaries (P > 0.05). Levels of JH also differed significantly among the treatments (Fig. 1C, n = 49; df = 5; Z = 33.9; P < 0.001).
240 241 242 243 244 245
249 250
254 255 256 257 258 259 260 261 262 263 264
a 100
50
0 40
B
b
30
b b
20
10
a
a
a
0 80
C
60
a
a
a
40
b bc
20
c 0 150
D
a ab
100
bc
abc c
c
50
0
1-2 years, caged
215
a
1-2 years, fertile
214
a
a
after onset of egg-laying
213
a
24h after insemination
212
a 150
24h after CO2
210 211
A
virgin
209
200
Vitellin per ovary [mg vitellogenin-equivalents]
208
The samples intended for the quantification of Vg and ecdysteroids were transported within Germany from Berlin to Bayreuth on dry ice. The hemolymph-buffer-mixture was transferred from the original reaction tubes that had been used for hemolymph sampling into 6.5 ml polypropylene tubes (Sarstedt, Nümbrecht, Germany). The original tubes were rinsed twice with 500 ll of methanol each by vigorous vortexing and a 5 min treatment in an ultrasonic bath. Rinsing solutions were combined with the hemolymph-buffer-mixture and 3 ml isooctane was added for defatting. After vortexing and treatment in the ultrasonic bath, samples were centrifuged (5500g, 4 °C, 10 min). After discarding the top phase (isooctane containing the lipids), the remaining methanol-buffer mixture was transferred into a new 6.5 ml tube, mixed with 2 ml of water and concentrated to a volume of 500 ll in a Speed Vac. The following fractionation of ecdysteroids by solid phase separation (to obtain free ecdysteroids) as well as their quantification by radioimmuno assay were performed as described in Lorenz et al. (1997) with the following modifications: DBL-1 antiserum, which displays a high cross-reactivity for makisterone A, was used. The standard curve was obtained by using makisterone A (AG Scientific, USA) as the non-labeled standard (0–10,000 pg). Vg/vitellin in queen hemolymph and ovaries was determined by ELISA as in Wegener et al. (2009a). Ovarian tissue was homogenized in the TRIS-buffer for 35 s with a fast-spinning motorized pestle. Each sample was measured in two dilutions, 2000- and 10,000-fold. Protein measured in the ovaries is probably a mixture of Vg and vitellin. In the honey bee, the two are known to be electrophoretically indistinguishable (Engels et al., 1990). It is known from previous studies that the antibody used shows affinity to both Vg and vitellin (Wegener et al., 2009a, 2010). As only Vg was used for the standard curves, results are given in Vg-equivalents.
Hemolymph juvenile hormone [pg/µl]
207
Hemolymph ecdysteroids [pgkisterone A-equ./µl]
206
Hemolymph vitellogenin [µg/µl]
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
Fertility status of queens Fig. 1. Ecdysteroids and Juvenile hormone in hemolymph and vitellin/vitellogenin in hemolymph and ovaries of queens of different reproductive status. Bars show means ± SE. A total of 49 queens were used for the analyses. Different letters on top of bars indicate significant differences between treatments (P < 0.05).
They were moderate in untreated virgins and remained so after CO2-treatment. They were lower in freshly inseminated queens, and even lower in queens that had initiated oviposition
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
265 266 267
IP 3080
No. of Pages 7, Model 5G
3 May 2013
276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
296
4. Discussion
297
4.1. A caste-transgressing role for JH in A. mellifera?
298
Juvenile hormone is a stimulant of reproductive dominance, Vg synthesis and/or oogenesis in several groups of social Hymenoptera, including Vespidae (reviewed by Strambi, 1990), Halictidae (Bell, 1973; Smith et al., 2013), and Apidae (reviewed in Hartfelder, 2000). Even in the closely related bumble bee Bombus terrestris, JH titers are positively linked to ovary development and social status of queenless workers (Röseler, 1977; Röseler and Röseler, 1988; Bloch et al., 2000a). In adult A. mellifera however, JH has long been thought to have lost its classical role as stimulant of gonadic activity in favor of a new role as ‘‘behavioral pacemaker’’ (Robinson
300 301 302 303 304 305 306 307
a
2
0 8
B
b b
6 b
b
ab
4
a
2
0
13
C
b
ab
ab
ab
10 a a
8 5 3 0 20
D c
15
d
10 a
ab ab b
5
0
virgin
299
a
a
4
1-2 years, caged
275
a a
1-2 years, fertile
274
6
after onset of egg-laying
273
a
24h after insemination
272
A
24h after CO2
271
8
Glycogen [µg/pair of glands]
270
(treatments C and D, P < 0.05 in both cases). JH-titers significantly increased if queens were caged before sampling (P < 0.05). Hemolymph titers of ecdysteroids varied among treatment groups (n = 47; df = 5; Z = 16.83; P = 0.005; Fig. 1D). They were higher in untreated virgins than in old, fertile queens regardless of caging (P < 0.05), while animals from the other treatment groups showed intermediate values. The ovaries of all caged queens contained yellowish bodies towards the posterior end of the ovarioles (Fig. 2), indicative of follicles in the process of degeneration (Gauthier et al., 2011) or resorption (Hitchcock, 1956; Flanders, 1959). They did not contain any mature eggs as were found in the ovaries of most laying queens. Fig. 3 shows the results of the analyses of spermathecal glands. Glycogen titers did not vary according to queen reproductive status (Fig. 3A, n = 49; df = 5; Z = 9.1; P = 0.078). Free carbohydrate contents did vary (Fig. 3B, n = 49; df = 5; Z = 15.2; P = 0.01), showing a peak in freshly inseminated animals and dropping sharply after the onset of egg-laying. Lipids also varied in quantity (Fig. 3C, n = 49; df = 5; Z = 22.0; P = 0.001), with lower amounts found in CO2-treated and freshly inseminated animals than in all other treatment groups (P < 0.05). Protein titers in the glands displayed the clearest variations between the treatments (Fig. 3D, n = 49; df = 5; Z = 47.1; P < 0.001). They dropped slightly after insemination, but then increased again after the onset of egg-laying (P < 0.05). They were still higher in 1–2 year-old, fertile animals, and slightly dropped if animals were caged before sampling (P < 0.05).
Free carbohydrates [µg/pair of glands]
269
Lipid [µg/pair of glands]
268
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
Protein [µg BSA-equivalents/pair of glands]
4
Fertility status of queens Fig. 3. Chemical composition of spermathecal glands in queens of different reproductive status. Bars show means ± SE. A total of 49 queens were used for the analyses. Different letters on top of bars indicate significant differences between treatments groups (P < 0.05). No significant differences between treatment groups were observed regarding glycogen content.
Fig. 2. Degeneration/resorption of follicles in caged queens. Stereomicroscopic image of the dissected abdomen of a fertile queen bee after 6 h of caging. Arrows indicate yellowish bodies representing follicles in the process of resorption/ degeneration.
et al., 1991; Robinson and Vargo, 1997; Hartfelder, 2000; Engels et al., 1990; Pinto et al., 2000). This view has been changed by the finding that JH and ecdysteroids do participate in the initiation
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
308 309 310
IP 3080
No. of Pages 7, Model 5G
3 May 2013 J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364
365 366 367 368 369 370 371 372 373
of Vg-synthesis, but that this event has shifted in time from the Q4 adult to the late pupal stage (Barchuk et al., 2002). The pattern of JH-concentrations we observed in the hemolymph of adult queens is generally in good accordance with the data of Robinson et al. (1991) and Fahrbach et al. (1995). In contrast, Fluri et al. (1981) detected no differences in JH-titers between virgin, non-reproductive and mated, laying queens. This may be due to the fact that in their experiment, all queens were caged for >1 day before sampling. Results from our caged animals suggest that this procedure is likely to have influenced the outcome. We show that in adult queens, changes in hemolymph titers of JH coincide with changes in the reproductive status. JH-titers were found to be low in females that are engaged in active oogenesis, regardless of their chronological age and mating status, and high in queens where this is not the case. Caging induced a sudden stop of oogenesis, as evidenced by the occurrence of degenerating/ partly resorbed follicles or oocytes and the absence of mature eggs. This stop coincided with a sharp rise in JH-concentrations. Double treatment with CO2, which is known to lead to the initiation of Vg synthesis and oogenesis in honeybee queens (Mackensen, 1947; Engels and Ramamurty, 1976), also led to a drop in JH titers. To summarize, JH concentrations were low in the hemolymph of animals with starting or ongoing vitellogenesis, and high in previtellogenic animals or animals that showed signs of yolk resorption. A similar negative relationship between JH and Vg uptake into tissues can also be found in workers, where hemolymph concentrations of JH are low in nurses and winter bees, both of which take up Vg into their hypopharyngeal glands, as well as in individuals with vitellogenic ovaries. Foragers, in which neither ovaries nor hypopharyngeal glands are known to take up Vg, have higher hemolymph titers of JH (Fluri et al., 1982; Huang et al., 1994). Together, these findings strongly suggest a role of JH as suppressor of Vg-synthesis and/or uptake in both castes of A. mellifera. Treatment of young workers with the JH-analog pyriproxyfen leads to the suppression of Vg synthesis (Pinto et al., 2000), which seems to suggest that the effect of JH lies at the level of synthesis. Corona et al. (2007) showed that expression of the Vg-gene in queen heads is reduced by administration of a high dose of another JH-analog, methoprene, or of an unspecified dose of JH III. On the other hand, the same authors show that abdominal concentrations of VgmRNA are highest in queens shortly after emergence, when JH-titers are known to peak as well (Fahrbach et al., 1995). An earlier study found that injections of JH III prevent synthesis only at high doses, while low doses stimulate Vg production (Rutz et al., 1976). Engels et al. (1990) reported that application of JH III to queens or incubation of queen fat bodies with this hormone did not affect Vg synthesis. The fact that in the untreated virgin queens of our study, high titers of JH coincided with the presence of relatively high levels of Vg in the hemolymph also seems to contradict a universal role for JH as suppressor of yolk protein synthesis. Therefore, we suggest that the primary role of JH in Vg metabolism is the suppression of uptake by target tissues rather than the suppression of synthesis.
4.2. Functions of ecdysteroids in honey bee queens – similarities to the worker caste In B. terrestris, the role of ecdysteroids in reproduction is less clear than for JH, although higher concentrations in the hemolymph are linked with dominance status in queenless workers (Bloch et al., 2000b; Geva et al., 2005). In adults of this species, the ovaries are an important source of ecdysteroids (Geva et al., 2005). In workers of A. mellifera however, only the early steps of ecdysteroid synthesis seem to be located in the ovary, whereas
5
the later stages mainly take place in the brain and fat body (Yamazaki et al., 2011). Earlier studies regarding ecdysteroids in queen honey bees have given inconsistent results. Robinson et al. (1991) found higher titers in laying queens than in functionally sterile or laying workers, whereas Hartfelder et al. (2002) found no differences between newly-emerged and actively laying queens, or between queens and workers of any reproductive status. Here we found higher titers in sexually mature virgin than in laying queens. While these findings are insufficient to resolve the discrepancies between the two earlier reports, they show that elevated ecdysteroid titers in queens are a transient state, explaining why different studies, using animals of differing reproductive status, may come to seemingly contradictive results. Ecdysteroids in workers bees are known to promote general protein synthesis by the fat body of queens (Engels et al., 1990), and titers are higher in reproductive animals of both castes than they are in non-reproductive workers (Robinson et al., 1991). On the other hand, there is no continuous relationship between individual levels of ecdysteroids and development of either ovaries or hypopharyngeal glands in workers (Hartfelder et al., 2002; Wegener et al., 2009b). Instead there is a peak of hemolymph ecdysteroid concentration on day three of adult life (Hartfelder et al., 2002), i.e. shortly before the onset of secretory activity of the hypopharyngeal glands, while titers are low during the phase of intensive secretion of the glands (approximately days 4–14). In queenless worker bees, high titers have been found in individuals whose ovaries were on the brink of vitellogenesis (Wegener et al., 2009b), while individuals with undeveloped or vitellogenic ovaries had lower titers (Hartfelder et al., 2002; Wegener et al., 2009b). In the present study, we found high ecdysteroid levels in queens with pre-vitellogenic ovaries, as evidenced by a low Vg/ vitellin-content, and lower levels in animals with vitellogenic ovaries. Taken together, these results seem to suggest a role for ecdysteroids in preparing tissues for Vg uptake. Robinson et al. (1991) showed that the ecdysteroid-levels measured in laying queens are still much higher than those present in queenright nurses and foragers. This could mean that in addition to its proposed role in preparing organs for Vg uptake, sustained levels of ecdysteroids are required for oogenesis. This hypothesis is supported by results of Paul et al. (2005), who detected transcripts of an ecdysteroiddependent transcription regulator gene, AmE74, in pre-vitellogenic and vitellogenic egg chambers of actively laying queens. In vitellogenic egg chambers, the transcripts are found in the follicle cells, which in A. mellifera are known to take up Vg and channel it to the surface of the oocyte (Fleig, 1995). However, no transcripts of AmE74 were detected in the heads of active nurse bees, so this gene is either not or only transiently involved in Vg-uptake by the hypopharyngeal glands.
374
4.3. Role of JH and ecdysteroids in the regulation of the spermathecal glands
423
Sociality can evolve independently of long-term sperm storage, and not all sperm-storing species are social. Nevertheless, the capacity to store male gametes in great numbers for prolonged periods of time is a characterizing feature of the queens of social Hymenoptera (Gobin et al., 2006), indicating that it may be adaptive within the context of hymenopteran sociality. Sperm storage is not a static state, but a process, involving the steps of attracting sperm to the site of storage, keeping them alive, and activating them for fertilization. In A. mellifera, the spermathecal glands are thought to be involved in all of these steps, and different classes of substances from the glands have been suggested to play a role at different stages of the process (Lensky and Schindler, 1967; Koeniger, 1970; Verma and Shuel, 1973; Dallai, 1975; Collins
425
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422
424
426 427 428 429 430 431 432 433 434 435 436 437
IP 3080
No. of Pages 7, Model 5G
3 May 2013 6
J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx
466
et al., 2006; Baer et al., 2009; Wegener et al., 2013). Given that sperm storage is a process and that it has to be coordinated with other processes like mating and oogenesis, it likely needs regulation, and the classical insect gonadotropins JH and ecdysteroids seemed possible candidate signal molecules. However, the fluctuations observed in the chemical composition of the glands did not follow the same pattern as those of the two groups of hormones, so a direct control of the glandular activity by JH or ecdysteroids seems unlikely. Instead, some of our results could shed a light on possible functions of different classes of substances during sperm storage. The fact that free carbohydrates tended to accumulate in the glands after CO2-treatment and insemination and then sharply dropped after the onset of egg-laying may indicate that these substances could be involved in the transfer of sperm into the spermatheca. Energy generation in freshly ejaculated honey bee sperm is known to be sugar-driven (Verma, 1978), while sperm stored inside the theca are thought to catabolize lipids (Verma and Shuel, 1973), which would be in accordance with the observed rise in lipid contents in the glands of newly-laying queens. Dallai (1975) observed lipid droplets in the glands of mated, but not of virgin honeybee queens. Interestingly, strong differences in gland composition were observed between queens that had recently begun oviposition and animals that had been fertile for >1 year. This change was most marked in the case of protein. Enzymes from the spermathecal glands are known to support energy metabolism of stored sperm and provide protection against oxidative stress (Collins et al., 2006; Baer et al., 2009). Our results suggest that the importance of this enzymatic support system grows as spermatozoa increase in age.
467
4.4. Implications for the physiology of A. mellifera caste dimorphism
468
489
West-Eberhard (1996) suggested that in eusocial species, the role of regulatory mechanisms derived from those of solitary ancestors is modulated by the nutritional status of individuals, resulting in a splitting between (well-nourished) queens and (less well nourished) nurses (‘‘split function’’-hypothesis). We here present an example of how functions of regulatory circuits in queens and workers are split regarding their effects on the life history of individuals, while remaining the same at a fundamental physiological level. Whether (or to which degree) these circuits are based on those of solitary ancestors cannot be deduced from our findings. Like in many solitary species (reviewed in Hartfelder, 2000), JH and ecdysteroids in A. mellifera queens seem to be involved in reproductive processes. On the other hand, the negative association between Vg uptake and JH is clearly atypical of the situation in the Hymenoptera as a whole (reviewed in Robinson and Vargo, 1997). At present, the caste-transgressing roles for JH and ecdysteroids regarding Vg uptake we propose here are mostly based on correlations only and have to be verified by an inductive approach. If confirmed, they would add to the view that the supposed loss of function of the two groups of hormones in honey bee reproduction has to be reconsidered.
490
Acknowledgements
491
We thank Anja Rogge for excellent apicultural assistance. This work was supported by funds of the German Ministry for Food, Agriculture and Consumer Security (BMELV) through the intermediary of the Federal Office for Agriculture and Food (BLE), within the framework of the program for innovation. Some of the equipment used was financed through the European Fund for Regional Development (EFRE).
438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488
492 493 494 495 496 497
References
498
Amdam, G.V., Norberg, K., Hagen, A., Omholt, S.W., 2003. Social exploitation of vitellogenin. Proceedings of the National Academy of Sciences of the United States of America 100, 1799–1802. Amdam, G.V., Aase, A.L.T.O., Seehuus, S.C., Fondrk, M.K., Norberg, K., Hartfelder, K., 2005. Social reversal of immunosenescence in honey bee workers. Experimental Gerontology 40, 939–947. Amdam, G., Page, R.E., Fondrk, M., Brent, C., 2010. Hormone response to bidirectional selection on social behavior. Evolution and Development 12, 428–436. Baer, B., Heazlewood, J.L., Taylor, N.L., Eubel, H., Millar, A.H., 2009. The seminal fluid proteome of the honeybee Apis mellifera. Proteomics 9, 2085–2097. Bell, W.J., 1973. Factors controlling initiation of vitellogenesis in a primitively social bee, Lasioglossum zephyrum (Hymenoptera: Halictidae). Insectes Sociaux 20, 253–260. Bloch, G., Borst, D.W., Huang, Z.Y., Robinson, G.E., Cnaani, J., Hefetz, A., 2000a. Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris. Journal of Insect Physiology 46, 47– 57. Bloch, G., Hefetz, A., Hartfelder, K., 2000b. Ecdysteroid titer, ovary status, and dominance in adult worker and queen bumble bees (Bombus terrestris). Journal of Insect Physiology 46, 1033–1040. Collins, A.M., Caperna, T.J., Williams, V., Garrett, W.M., Evans, J.D., 2006. Proteomic analyses of male contributions to honey bee sperm storage and mating. Insect Molecular Biology 15, 541–549. Corona, M., Velarde, R.A., Remolina, S.R., Moran-Lauter, A., Wang, Y., Hughes, K.A., Robinson, G.E., 2007. Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proceedings of the Academy of Sciences of the United States of America 104, 7128–7133. Dallai, R., 1975. Fine structure of the spermatheca of Apis mellifera. Journal of Insect Physiology 21, 89–109. Engels, W., Ramamurty, P.S., 1976. Initiation of oögenesis in allatectomised virgin honeybee queens by carbon dioxide treatment. Journal of Insect Physiology 22, 1427–1432. Engels, W., Imperatriz-Fonseca, V.L., 1990. Caste development, reproductive strategies, and control of fertility in honey bees and stingless bees. In: Engels, W. (Ed.), Social Insects: An Evolutionary Approach to Castes and Reproduction. Springer, Berlin, pp. 167–230. Engels, W., Kaatz, H., Zillikens, A., Simoes, Z.L.P., Trube, A., Braun, R., Dittrich, F., 1990. Honey bee reproduction: vitellogenin and caste-specific regulation of fertility. In: Hoshi, M., Yamashita, O. (Eds.), Advances in Invertebrate Reproduction, Elsevier, vol. 5. London, New York, pp. 495–502. Fahrbach, S., Giray, T., Robinson, G.E., 1995. Volume changes in the mushroom bodies of adult honey bee queens. Neurobiology of Learning and Memory 63, 181–191. Flanders, S.E., 1959. Embryonic starvation. An explanation of the defective honeybee egg. Journal of Economic Entomology 52, 166–167. Fleig, R., 1995. Role of the follicle cells for yolk uptake in ovarian follicles of the honey bee Apis mellifera L. (Hymenoptera: Apidae). International Journal of Insect Morphology and Embryology 24, 427–433. Fluri, P., Sabatini, A.G., Vecchi, M., Wille, H., 1981. Blood juvenile hormone, protein and vitellogenin titres in laying and non-laying queen honeybees. Journal of Apicultural Research 20, 221–225. Fluri, P., Lüscher, M., Wille, H., Gerig, L., 1982. Changes of weight of the pharyngeal gland and haemolymph titres of juvenile hormone, protein and vitellogenin in worker honey bees. Journal of Insect Physiology 28, 61–68. Gauthier, L., Ravallec, M., Tournaire, M., Cousserans, F., Bergoin, M., Dainat, B., Miranda, J.D., 2011. Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One 6, e16217, doi: 16210.11371/ journal.pone.0016217. Geva, S., Hartfelder, K., Bloch, G., 2005. Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris. Journal of Insect Physiology 51, 811–823. Gilbert, L.E., 2012. Insect Endocrinology. Academic Press, London, Waltham, San Diego. Gobin, B., Ito, F., Peeters, C., Billen, J., 2006. Queen-worker differences in spermatheca reservoir of phylogenetically basal ants. Cell and Tissue Research 326, 169–178. Hagedorn, H.H., Kunkel, J.G., 1979. Vitellogenin and vitellin in insects. Annual Review of Entomology 24, 475–505. Hartfelder, K., 2000. Insect juvenile hormone: from ‘status quo’ to high society. Brazilian Journal of Medical and Biological Research 33, 157–177. Hartfelder, K., Bitondi, M.M.G., Santana, W.C., Simoes, Z.L.P., 2002. Ecdysteroid titer and reproduction in queens and workers of the honey bee and of a stingless bee: loss of ecdysteroid function at increasing levels of sociality? Insect Biochemistry and Molecular Biology 32, 211–216. Hartfelder, K., Emlen, D.J., 2005. Endocrine control of insect polymorphism. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Insect Molecular Science, vol. 3. Elsevier, Oxford, pp. 651–703. Hitchcock, J.D., 1956. Honey bee queens whose eggs all fail to hatch. Journal of Economic Entomology 49, 11–14. Huang, Z.-Y., Robinson, G., Borst, D., 1994. Physiological correlates of division of labor among similarly aged honey bees. Journal of Comparative Physiology A 174, 731–739.
499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006
IP 3080
No. of Pages 7, Model 5G
3 May 2013 J. Wegener et al. / Journal of Insect Physiology xxx (2013) xxx–xxx 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634
Huang, Z.-Y., Robinson, G.E., 1996. Regulation of honey bee division of labor by colony age demography. Behavioral Ecology and Sociobiology 39, 147–158. Ito, F., Ohkawara, K., 1994. Spermatheca size differentiation between queens and workers in primitive ants – relationship with reproductive structure of colonies. Naturwissenschaften 81, 138–140. Jassim, O., Huang, Z.Y., Robinson, G.E., 2000. Juvenile hormone profiles of worker honey bees during normal and accelerated behavioral development. Journal of Insect Physiology 46, 243–249. Jaycox, E.R., Skowronek, W., Guynn, G., 1974. Behavioral changes in worker honeybees (Apis mellifica L.) induced by injections of a juvenile hormone mimic. Annals of the Entomological Society of America 67, 529–535. Koeniger, G., 1970. Bedeutung der Tracheenhülle und der Anhangdrüse der Spermatheka für die Befruchtungsfähigkeit der Spermatozoen der Bienenkönigin (Apis mellifica L.). Apidologie 1, 55–71. Lensky, Y., Schindler, H., 1967. Motility and reversible inactivation of honeybee spermatozoa in vivo and in vitro. Annales de l’Abeille 10, 5–16. Lorenz, J.I., Lorenz, M.W., Hoffmann, K.H., 1997. Factors regulating juvenile hormone and ecdysteroid biosynthesis in Gryllus bimaculatus (Ensifera: Gryllidae). European Journal of Entomology 94, 369–379. Lorenz, M.W., 2003. Adipokinetic hormone inhibits the formation of energy stores and egg production in the cricket Gryllus bimaculatus. Comparative Biochemistry and Physiology B Comparative Biochemistry 136, 197–206. Lorenz, M.W., 2007. Oogenesis-flight syndrome in crickets: age-dependent egg production, flight performance, and biochemical composition of the flight muscles in adult female Gryllus bimaculatus. Journal of Insect Physiology 53, 819–832. Mackensen, O., 1947. Effect of CO2 on initial oviposition of artificially inseminated and virgin queen bees. Journal of Economic Entomology 40, 344–349. Martins, G.F., Serrao, J.E., 2002. A comparative study of the spermatheca in bees. Sociobiology 40, 711–720. Nelson, C.M., Ihle, K.E., Fondrk, M.K., Page, R.E., Amdam, G.V., 2007. The gene vitellogenin has multiple coordinating effects on social organization. Plos Biology 5, 673–677. Paul, R.K., Takeuchi, H., Matsuo, Y., Kubo, T., 2005. Gene expression of ecdysteroidregulated gene E74 of the honeybee in ovary and brain. Insect Molecular Biology 14, 9–15. Pinto, L.Z., Bitondi, M.M., Simoes, Z.L.P., 2000. Inhibition of vitellogenin synthesis in Apis mellifera workers by juvenile hormone analogue pyriproxyfen. Journal of Insect Physiology 46, 153–160. Raikhel, A.S., Brown, M.R., Belles, X., 2005. Hormonal control of reproductive processes. In: Gilbert, L.I. (Ed.), Comprehensive Molecular Insect Science, vol. 3. Elsevier Pergamon, Oxford, pp. 433–491. Robinson, G.E., Strambi, C., Strambi, A., Feldlaufer, M.F., 1991. Comparison of juvenile hormone and ecdysteroid haemolymph titers in adult worker and queen honey bees (Apis mellifera). Journal of Insect Physiology 37, 929–937. Robinson, G.E., Vargo, E.L., 1997. Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker. Archives of Insect Biochemistry and Physiology 35, 559–583. Röseler, P.F., 1977. Juvenile hormone control of oögenesis in Bumblebee workers, Bombus terrestris. Journal of Insect Physiology 23, 985–992. Röseler, P.F., Röseler, I., 1988. Influence of juvenile hormone on fat body metabolism in ovariectomized queens of the bumblebee, Bombus terrestris. Insect Biochemistry 18, 557–563.
7
Ruttner, F., Koeniger, G., 1971. Filling of spermatheca of honeybee queen – active migration or passive transport of spermatozoa. Zeitschrift für Vergleichende Physiologie 72, 411–422. Ruttner, F., 1976. The Instrumental Insemination of the Queen Bee, 2nd ed. Apimondia, Bukarest. Ruttner, F., 1983. Queen Rearing – Biological Basis and Technical Instructions, 2nd ed. Apimondia, Bucharest. Rutz, W., Lüscher, L., 1974. The occurrence of vitellogenin in workers and queens of Apis mellifica and the possibility of its transmission to the queen. Journal of Insect Physiology 20, 897–909. Rutz, W., Gerig, L., Wille, H., Lüscher, M., 1976. The function of juvenile hormone in adult worker honey bees, Apis mellifera. Journal of Insect Physiology 22, 1485– 1491. Slone, J.D., Stout, T.L., Huang, Z.Y., Schneider, S.S., 2012. The influence of drone physical condition on the likelihood of receiving vibration signals from worker honey bees, Apis mellifera. Insectes Sociaux 59, 101–107. Smith, A.R., Kapheim, K.M., Pérez-Ortega, B., Brent, C.S., Wcislo, W.T., 2013. Juvenile hormone levels reflect social opportunities in the facultatively eusocial sweat bee Megalopa genalis (Hymenoptera: Halictidae). Hormones and Behavior 63, 1–4. SPSS, 2010. IBM SPSS Statistics 19 core system user’s guide, IBM-SPSS. Strambi, A., 1990. Physiology and reproduction in social wasps. In: Engels, W. (Ed.), Social Insects – an Evolutionary Approach to Caste and Reproduction. Springer, Berlin, pp. 59–75. Sullivan, J., Fahrbach, S., Harrison, J., Capaldi, E., Fewell, J., Robinson, G., 2003. Juvenile hormone and division of labor in honeybee colonies: effects of allatectomy on flight behavior and metabolism. Journal of Experimental Biology 206, 2287–2296. Verma, L.R., Shuel, R.W., 1973. Respiratory metabolism of the semen of the honeybee, Apis mellifera. Journal of Insect Physiology 19, 97–103. Verma, L.R., 1978. Biology of honeybee spermatozoa 2. Effect of washing and sugars on respiration as measured by the cartesian diver technique. Journal of Apicultural Research 17, 176–181. Wang, Y.J., Kaftanoglu, O., Siegel, A., Page, R.E., Amdam, G., 2010. Surgically increased ovarian mass in the honey bee confirms link between reproductive physiology and worker behavior. Journal of Insect Physiology 56, 1816–1824. Wegener, J., Lorenz, M.W., Bienefeld, K., 2009a. Physiological consequences of prolonged nursing in the honey bee. Insectes Sociaux 56, 85–93. Wegener, J., Huang, Z.Y., Lorenz, M.W., Bienefeld, K., 2009b. Regulation of hypopharyngeal gland activity and oogenesis in honey bee (Apis mellifera) workers. Journal of Insect Physiology 55, 716–725. Wegener, J., Lorenz, M.W., Bienefeld, K., 2010. Differences between queen- and worker-laid eggs of the honey bee (Apis mellifera). Apidologie 41, 116–126. Wegener, J., Zschörnig, K., Onischke, K., Fuchs, B., Schiller, J., Müller, K., 2013. Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen – a TLC/MALDI-TOF MS study. Experimental Gerontology 48, 213–222. West-Eberhard, M.J., 1996. In: Wasp societies as microcosms for the study of development and evolution. In: Turillazzi, S., West-Eberhard, M.J. (Eds.), . Natural History and Evolution of Paper Wasps. Oxford Press, New York, pp. 290– 317. Yamazaki, Y., Kiuchi, M., Takeuchi, H., Kubo, T., 2011. Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L. Insect Biochemistry and Molecular Biology 41, 283–293.
Q5
635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688
Please cite this article in press as: Wegener, J., et al. New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction. Journal of Insect Physiology (2013), http://dx.doi.org/10.1016/j.jinsphys.2013.04.006