Differential Methylation of Brain DNA in Queens and Workers: http://www.plosbiology.org/article/info ... io.1000506

Erm, being the queen can make your brain DNA differentially methylated

See what I did there?

Excellent! Work on my plan to create an army of drones is proceeding well bwahahahaha!

That's fantastically interesting. Thank you for sharing!

Someone care to explain to me why the authors left out of their references list one of the major papers on diphenic caste selection in honey bees? Seems like a glaring omission to me.

For those who have just choked upon the above phrase ( ), this is the process by which a honey bee larva is destined to become either a worker bee or a queen. Here is the fun part - two honey bee larvae can begin life with identical genes, but can have different fates depending upon the feeding régime. How is this possible?

Allow me to introduce the following scientific paper:

The Making Of A Queen: TOR Pathway Is A Key Player In Diphenic Caste Development by Avani Patel, M. Kim Fondrk, Osman Kaftanoglu, Christine Emore, Greg Hunt, Katy Frederick, Gro V. Amdam, PLoS One, 6: e509 (June 2007), doi:10.1371/journal.pone.0000509

I quote from the paper thus:

Patel, Fondrik et al, 2007 wrote:Background. Honey bees (Apis mellifera) provide a principal example of diphenic development. Excess feeding of female larvae results in queens (large reproductives). Moderate diet yields workers (small helpers). The signaling pathway that links provisioning to female developmental fate is not understood, yet we reasoned that it could include TOR (target of rapamycin), a nutrient- and energy-sensing kinase that controls organismal growth. Methodology/Principal Findings. Here, the role of Apis mellifera TOR (amTOR) in caste determination is examined by rapamycin/FK506 pharmacology and RNA interference (RNAi) gene knockdown. We show that in queen-destined larvae, the TOR inhibitor rapamycin induces the development of worker characters that are blocked by the antagonist FK506. Further, queen fate is associated with elevated activity of the Apis mellifera TOR encoding gene, amTOR, and amTOR gene knockdown blocks queen fate and results in individuals with worker morphology. Conclusions/Significance. A much-studied insect dimorphism, thereby, can be governed by the TOR pathway. Our results present the first evidence for a role of TOR in diphenic development, and suggest that adoption of this ancestral nutrient-sensing cascade is one evolutionary pathway for morphological caste differentiation in social insects.

What the authors did was to test the hypothesis that the kinase enzyme coded for by the TOR gene (in this case, the amTOR variant present in Apis mellifera) influenced development based upon environmental conditions.

Honey bee larvae are fed with a special substance known as 'royal jelly' for the first 48 hours of life. Those larvae destined to become workers (the majority) are then fed after this initial period with a food comprising pollen and other ingredients (nicknamed "bee bread"), and the amount of royal jelly that they receive is reduced significantly. A larva that is destined to become a queen, however, is fed royal jelly throughout its larval live in quantity. A special "queen cell" is constructed to receive the egg, the egg is laid therein, and the cell is stocked with a surplus of royal jelly (continually renewed so that the larva always has more royal jelly present than it can eat at one sitting).

Now, it turns out that the TOR pathway involves a nutrient sensitive kinase enzyme, and this has been found to be of influence in other insects in particular. That staple of genetics research, Drosophila melanogaster, was subject to TOR pathway investigation as cited by the above paper:

Patel, Fondrik et al, 2007 wrote:INTRODUCTION

TOR is the central component of a conserved eukaryotic signaling pathway that regulates cell and organismal growth in response to nutrient status [1,2]. Growth rate correlates with ribosome number and metabolism, and TOR-dependent growth control in yeast and Drosophila involves transcriptional regulation of ribosomal and metabolic genes [3,4]. Suppression of the Drosophila TOR pathway results in prolonged pre-adult development and reduces larval and adult body sizes [2].

So, the authors considered it likely that the TOR pathway played at least some role in the differential development of bee larvae as described above.

Patel, Fondrik et al, 2007 wrote:In sum, these signatures of experimental variation in TOR signaling strikingly resemble the naturally occurring diphenism of the highly eusocial honey bee, where two alternative female phenotypes – the reproductive queen caste and the facultatively sterile worker caste – differentiate through social manipulation of larval nutrient status [5]. Queen-destined individuals, which receive a rich diet of royal jelly as larvae, are from the 3rd instar characterized by accelerated larval growth, upregulation of larval ribosomal and metabolic gene expression, rapid pre-adult development and large body sizes [5–7]. Worker-destined larvae, which receive a moderate diet of less nutritious jelly [5], are characterized by the opposite of all of these. Moreover, honey bee queens and workers diverge in morphological characters, other than organismal size, that involve the differential growth of body parts ([5], see below).

This pattern led us to hypothesize that the evolution of caste diphenism in honey bees involved adoption of TOR signaling as an ancestral mechanism for regulation of phenotypic plasticity in response to variation in nutrient status. Consequently, honey bee caste determination was predicted to be amTOR-dependent, and queen vs. worker development to be conditional on high vs. low amTOR signaling in larvae, respectively.

So, how was this investigated?

Well, it turns out that the TOR enzyme, the nutrient sensitive kinase, is blocked by an antibiotic called rapamycin. Indeed, this property led to the TOR pathway being thus named - Target Of Rapamycin. So, one way of investigating whether or not the TOR pathway is involved in diphenic caste development is to feed queen-destined larvae with rapamycin, and see if the inhibition of the TOR pathway by rapamycin leads to worker characteristics in the adult insect. Which is exactly what happened when subject to experimental test.

Patel, Fondrik et al, 2007 wrote:Here, we first use pharmacology to implicate amTOR in the differentiation of female honey bees into queens and workers. Thereafter, we establish that amTOR is expressed at higher levels in queen-destined females. Finally, we make use of amTOR RNAi combined with double RNAi controls; i) exposure to GFP double stranded RNA and ii) knockdown of the honey bee vitellogenin gene; to demonstrate that queen-fate is blocked and workers develop when amTOR activity is reduced during development. This new insight represents the first evidence for a central role of TOR in a naturally occurring diphenism.

However, the tests to implicate the TOR pathway in diphenic caste development were somewhat more sophisticated than mere rapamycin inhibition, as is described above. First, rapamycin inhibition is used to demonstrate that queen-destined larvae become workers when the TOR pathway is inhibited. Second, another antibiotic, FK506, competitively inhibits rapamycin, and leads to the restoration of the TOR pathway even in the presence of rapamycin. Therefore use of the two antibiotics can be used to provide a robust first-stage test of the connection between the TOR pathway and diphenic caste development.

Patel, Fondrik et al, 2007 wrote:RESULTS

The TOR inhibitor rapamycin induces worker characters in queen-destined individuals

We first exposed colony-reared queen- and worker-destined 4–5th instar larvae to treatments with rapamycin, FK506 and vehicle control (2% ethanol). Pharmacological effects in younger stages could not be examined, as treated 1–3rd instar larvae were rejected by colonies. Rapamycin and FK506 are structural analogues that compete for binding to the highly conserved FK binding protein-12 (FKBP-12). The rapamycin-FKBP-12 complex, but not the FK506-FKBP-12 complex, inhibits TOR activity and, thus, rapamycin-mediated interference with TOR signaling can be antagonized (competitively inhibited) by FK506. This approach has been used with high specificity to study TOR in insects [8]. In queen-destined individuals, rapamycin prolonged pre-adult development (ANOVA: F2,22 = 66.0, P < 0.0001, Fig. 1a), reduced wet-weight (size) at adult emergence (ANOVA: F2,20 = 5.75.0, P < 0.01, Fig. 1b), and caused appearance of corbicula (pollen basket), a worker-specific morphological trait, while ovarian morphology remained queen-like (ANOVA: F2,20 = 0.17.0, P = 0.84, Fig. 1c–d; queens have much larger ovaries than workers). Nutritional manipulation of 4–5th instar larvae, e.g., by transferring queen larvae into worker diet and vice versa, leads to similar intercastes with some traits characteristic of workers and some characteristic of queens (see [9] and refs. therein). Honey bee caste differentiation is a sequential process, with ovary size determined during the 3rd larval instar, and corbicula during the 4–5th larval instars [9]. In queen-destined larvae, the TOR inhibitor rapamycin caused developmental changes toward worker characters; predictably excluding a trait determined prior to the experiment (ovary size) and including a trait determined concomitant with rapamycin treatment (corbicula).

Worker-destined individuals were not affected by rapamycin/ FK506 pharmacology (ANOVA developmental time:
F2,117 =0.51, P = 0.51; wet-weight: F2,41 = 0.64, P = 0.53, ovariole number: F2,43 = 1.68, P = 0.20, Fig. 1e–g), consistent with the prediction that worker determination occurs when amTOR signaling is low in larvae.

So, having established that queen fate is associated with the TOR pathway, and that the kinase enzyme is somehow responsible for determining downstream signalling of other developmental aspects of a queen adult dependent upon royal jelly nutrient levels (details of this will doubtless be the subject of future papers), the next step was to establish that queen fate was due to elevated activity of the gene in question:

Patel, Fondrik et al, 2007 wrote:Queen fate is associated with elevated activity of the Apis mellifera TOR encoding gene, amTOR

Several mechanisms can influence TOR signaling [1,2]; one is the transcript level of TOR mRNA [10]. As a next step, we explored if the critical decision-point of caste determination (3rd larval instar) was characterized by variation in amTOR expression that correlated with developmental fate. Using larvae reared as queens and workers by colonies, we found that 3rd instar queen-destined females had approximately two-fold higher amTOR mRNA levels than 3rd instar worker-destined larvae (ANOVA: F1,12 = 9.46, P < 0.01, Fig. 2a). Moreover, after larval feeding and growth were completed (spinning 5th instar), this difference in amTOR expression was no longer present (ANOVA: F1,11 = 1.55, P = 0.24, Fig. 2b). Relative amTOR levels in larvae, therefore, are consistent with general patterns of growth [5] that characterize queen- and worker-destined individuals. Also, high vs. low amTOR activity in the 3rd instar correlate with queen vs. worker developmental fate, respectively – in agreement with the role we propose for amTOR signaling in honey bee caste determination.

So, the scientists established that elevated expression of the gene was notable in queens, and that the signalling corresponded to the results from the pharmacology manipulation experiments.

As a second test, the scientists then used gene knockdown techniques to examine the effects of eliminating the expression of the gene altogether. To ensure that this was robust, another gene knockdown was used to ensure that the intended gene knockdown technique did not influence the TOR pathway as an artifact of using RNAi knockdown, but was specific to the gene in question.

Patel, Fondrik et al, 2007 wrote:amTOR gene knockdown blocks queen fate and results in workers

To test the effect of amTOR on caste development, we combined in vitro rearing of 1–5th instar larvae (i.e., removed from the colony setting) with suppression of amTOR activity by RNAi. Larvae were given a diet made primarily of royal jelly that yields up to 50% queens. Due to methodological challenges [11], in vitro rearing typically does not yield a higher proportion of queens. The amount of amTOR double-stranded RNA (dsRNA) delivered to larvae was adjusted to yield a transient two-fold reduction of amTOR levels (Fig. 2c–d, 3rd instar ANOVA: F1,21 =32.06, P < 0.00001, Fig. 2c, vs. spinning 5th instar F1,14 = 0.11, P < 0.75, Fig. 2d), thereby mimicking the relative expression pattern observed in colonies (Fig. 2a–b).

Initially, the same amount of dsRNA derived from green fluorescent protein (GFP) sequence was used as control. We found that suppression of amTOR activity reduced the growth of the developing larvae (ANOVA: F1,28 = 99.29, P < 0.00001, Fig. 3a), prolonged pre-adult development (ANOVA: F1,19 = 48.00, P < 0.00001, Fig. 3b–c), reduced wet-weight (size) at adult emergence (ANOVA: F1,19 = 68.28, P < 0.00001, Fig. 3d), and ultimately caused all amTOR knockdowns (n = 10) to emerge with fully developed worker morphology (Fig. 3e). In the control group, the proportion of females with queen morphology was 55% (n = 11), as expected from diet alone. Remaining controls were intercastes (e.g., Fig. 3f–g), a result common with in vitro rearing [11]. Thus, in a nutritional environment that encouraged queen differentiation, a physiologically relevant suppression of amTOR activity was sufficient to silence queen development. Instead, individuals emerged with a worker morphotype.

The specificity of the effect of amTOR was tested in a control experiment where the gene vitellogenin was suppressed by RNAi. vitellogenin is transcribed in male and female honey bee larvae, and the protein is present in low amounts during larval development [12]. In adults, vitellogenin is a major yolk precursor with pleiotropic effects [13,14]. Putative roles in larvae remain unclear, but previous experiments show that vitellogenin knockdown does not obstruct normal development [15,16], suggesting that vitellogenin could serve as a valid RNAi control. Also, we verified that although amTOR RNAi reduces vitellogenin mRNA levels (ANOVA: F1,26 = 6.07, P < 0.02, Fig. 4a) vitellogenin RNAi does not affect amTOR (for vitellogenin ANOVA: F1,15 = 5.25 P < 0.04, Fig. 4b, vs. for amTOR F1,41 = 0.36, P = 0.55, Fig. 4c). This information is consistent with a positive effect of TOR on vitellogenin transcription described in mosquito [8], and further shows that vitellogenin RNAi is not confounded by effects on amTOR.

In comparison to vitellogenin RNAi, amTOR RNAi reduced larval growth (ANOVA: F1,34 = 299.20, P < 0.0001, Fig. 4d); delayed development (ANOVA: F1,8 = 87.48, P < 0.0005, Fig. 4e), and reduced final adult size (ANOVA: F1,8 = 28.67, P < 0.001). As observed for GFP dsRNA control (Fig. 3), vitellogenin knockdowns emerged with queen morphology; amTOR knockdowns emerged as workers (Fig. 4f–g). Thus, the blocking of queen fate that occurs after amTOR knockdown is not an artifact of RNAi.

So, what we have here is evidence that there is a genetic basis for phenotypic plasticity in Apis mellifera, leading to diphenic caste development depending upon the nutrient environment that the larva is reared in, and consequently evidence for the existence of at least one differential developmental process, based upon a specific gene, that can lead to two manifestly different outcomes in an organism on the basis of the effect of environmental influence upon that gene-driven developmental pathway. It is likely that similar interactions are going to be apposite in other developmental pathways (and in other organisms), and that it may transpire that epigenetic phenomena rest upon developmental pathways with built-in environmental cue sensitivity. Indeed, it was the investigation of the effect of the TOR pathway on Drosophila and other organisms in the first place that led to the investigation of the TOR pathway as a suitable control for diphenic caste development.

Development does not have to be gene driven. For example, in sex determination pathways it can be an SRY gene or temperature input that determines gender and sexual dimorphism. eg see Goodfellow :-

Goodfellow, P. N. and R. Lovell-Badge (1993). "SRY and Sex Determination in Mammals." Annual Review of Genetics 27(1): 71-92.
or
Swain, A., V. Narvaez, et al. (1998). "Dax1 antagonizes Sry action in mammalian sex determination." Nature 391(6669): 761-767.

Ospina-Álvarez, N. and F. Piferrer (2008). "Temperature-Dependent Sex Determination in Fish Revisited: Prevalence, a Single Sex Ratio Response Pattern, and Possible Effects of Climate Change." PLoS ONE 3(7): e2837.

In gonochoristic vertebrates, sex determination mechanisms can be classified as genotypic (GSD) or temperature-dependent (TSD). Some cases of TSD in fish have been questioned, but the prevalent view is that TSD is very common in this group of animals, with three different response patterns to temperature. We analyzed field and laboratory data for the 59 fish species where TSD has been explicitly or implicitly claimed so far. For each species, we compiled data on the presence or absence of sex chromosomes and determined if the sex ratio response was obtained within temperatures that the species experiences in the wild. If so, we studied whether this response was statistically significant. We found evidence that many cases of observed sex ratio shifts in response to temperature reveal thermal alterations of an otherwise predominately GSD mechanism rather than the presence of TSD. We also show that in those fish species that actually have TSD, sex ratio response to increasing temperatures invariably results in highly male-biased sex ratios, and that even small changes of just 1–2°C can significantly alter the sex ratio from 1∶1 (males∶females) up to 3∶1 in both freshwater and marine species. We demonstrate that TSD in fish is far less widespread than currently believed, suggesting that TSD is clearly the exception in fish sex determination. Further, species with TSD exhibit only one general sex ratio response pattern to temperature. However, the viability of some fish populations with TSD can be compromised through alterations in their sex ratios as a response to temperature fluctuations of the magnitude predicted by climate change.

And Emlen:-

Emlen, D. J., Q. Szafran, et al. (2006). "Insulin signaling and limb-patterning: candidate pathways for the origin and evolutionary diversification of beetle /`horns/'." Heredity 97(3): 179-191.

Shows that dimorphism can occur even within one sex. The available nutrition determines the size of beetle horns in males, and thus the insulin pathway influences hox timing and expression[dll, al, egfr] etc.

Oh I'm aware of epigenetic patterns of development from my fishkeeping activities. For example, there are several species of riverine African Cichlid that exhibit differential sex ratios upon reproduction, depending upon the pH of the surrounding water. Typical species include Anomalochromis thomasi, Pelvicachromis pulcher, and various Nanochromis species. Not quite sure what causes this, but I can try and find out if anyone has done the relevant research ...

Calilasseia wrote:Oh I'm aware of epigenetic patterns of development from my fishkeeping activities. For example, there are several species of riverine African Cichlid that exhibit differential sex ratios upon reproduction, depending upon the pH of the surrounding water. Typical species include Anomalochromis thomasi, Pelvicachromis pulcher, and various Nanochromis species. Not quite sure what causes this, but I can try and find out if anyone has done the relevant research ...

Ain't nature brilliant! Beats fuckin' mythology every bloody time!

Calilasseia wrote:Oh I'm aware of epigenetic patterns of development from my fishkeeping activities. For example, there are several species of riverine African Cichlid that exhibit differential sex ratios upon reproduction, depending upon the pH of the surrounding water. Typical species include Anomalochromis thomasi, Pelvicachromis pulcher, and various Nanochromis species. Not quite sure what causes this, but I can try and find out if anyone has done the relevant research ...

Yep. In many lizard species the gender ratio of a clutch is determined by the ambient temperature. As a breeder you can actually control the ratio to a very high degree if you just set the temperature in the incubator right.

Mazille wrote:

Calilasseia wrote:Oh I'm aware of epigenetic patterns of development from my fishkeeping activities. For example, there are several species of riverine African Cichlid that exhibit differential sex ratios upon reproduction, depending upon the pH of the surrounding water. Typical species include Anomalochromis thomasi, Pelvicachromis pulcher, and various Nanochromis species. Not quite sure what causes this, but I can try and find out if anyone has done the relevant research ...

Yep. In many lizard species the gender ratio of a clutch is determined by the ambient temperature. As a breeder you can actually control the ratio to a very high degree if you just set the temperature in the incubator right.

So it is YOU that is the nasty lizard GOD! I am going to tell them . Poor buggers!