Density of mushroom body synaptic complexes limits intraspecies brain miniaturization in highly polymorphic leaf-cutting ant workers

Hymenoptera possess voluminous mushroom bodies (MBs), brain centres associated with sensory integration, learning and memory. The mushroom body input region (calyx) is organized in distinct synaptic complexes (microglomeruli, MG) that can be quantified to analyse body size-related phenotypic plasticity of synaptic microcircuits in these small brains. Leaf-cutting ant workers (Atta vollenweideri) exhibit an enormous size polymorphism, which makes them outstanding to investigate neuronal adaptations underlying division of labour and brain miniaturization. We particularly asked how size-related division of labour in polymorphic workers is reflected in volume and total numbers of MG in olfactory calyx subregions. Whole brains of mini, media and large workers were immunolabelled with anti-synapsin antibodies, and mushroom body volumes as well as densities and absolute numbers of MG were determined by confocal imaging and three-dimensional analyses. The total brain volume and absolute volumes of olfactory mushroom body subdivisions were positively correlated with head widths, but mini workers had significantly larger MB to total brain ratios. Interestingly, the density of olfactory MG was remarkably independent from worker size. Consequently, absolute numbers of olfactory MG still were approximately three times higher in large compared with mini workers. The results show that the maximum packing density of synaptic microcircuits may represent a species-specific limit to brain miniaturization.     

Mmp1 Processing of the PDF Neuropeptide Regulates Circadian Structural Plasticity of Pacemaker Neurons

In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.     

Mapping the cellular network of the circadian clock in two cockroach species

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.     

RNA-Interference Knockdown of Drosophila Pigment Dispersing Factor in Neuronal Subsets: The Anatomical Basis of a Neuropeptide's Circadian Functions

Background

In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila.

Methodology/Principal Findings

Here we investigate the anatomical basis for PDF''s various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of the central brain and not in the abdominal ganglion for normal activity rhythms. Differential knockdown of PDF in the large or small vLNs indicates that PDF from the small vLNs is likely responsible for the maintenance of free-running activity rhythms and that PDF is not required in the large vLNs for normal behavior. PDF''s role in setting the period of free-running activity rhythms and the proper timing of evening activity under light:dark cycles emanates from both subtypes of vLN, since PDF in either class of vLN was sufficient for these aspects of behavior.

Conclusions/Significance

These results reveal the neuroanatomical basis PDF''s various circadian functions and refine our understanding of the clock neuron circuitry of Drosophila.     

Collective control of nest climate parameters in bumblebee colonies

We examined two aspects of the social control of nest climate in bumblebee colonies: which parameters of nest climate bumblebees actively down-regulate by fanning and the dynamics of the colony response as colony size increased. Colonies of Bombus terrestris were exposed to an increase in carbon dioxide, temperature or relative humidity. We performed 70 temperature trials (six colonies), 58 CO₂ trials (four colonies) and four humidity trials (two colonies). An increase in CO₂ concentration and temperature elicited a fanning response whereas an increase in relative humidity did not. This is the first report of fanning in bumblebee colonies to control respiratory gases. The number of fanning bees increased with stimulus intensity. The colony response to a CO₂ concentration of 3.2% was comparable to the colony response to a temperature of 30°C. A marked fanning response occurred at 1.6% CO₂, a concentration never exceeded in a large field nest during a pilot measurement of 10 days. We investigated the colony response over a wide range of colony sizes (between 10 and 119 workers). The proportion of the total workforce invested by colonies in nest ventilation did not change significantly; thus, the number of fanning workers increased with colony size. Furthermore, as colony size increased, the dynamics of the colony response changed: colonies responded faster to perturbations of their environment when they were large (60 or more individuals) than when they were small. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Development of the circadian clock in the German cockroach, Blattella germanica

The cell distribution and immunoreactivity (ir) against period (PER), pigment dispersing factor (PDF) and corazonin (CRZ), were compared between adults and nymphs in the central nervous system of the German cockroach. Although PER-ir cells in the optic lobes (OL) were expressed in the nymphs from the first instar, the links between major clock cells became more elaborated after second/third instar. A circadian rhythm of locomotion was initiated at the fourth/fifth instar. The results suggest that the clock was running from hatching, but the control network needed more time to develop. In addition, the putative downstream regulators, PDF-ir and CRZ-ir, are co-localized in various regions of the brain, indicating potential output routes of the circadian clock. CRZ-ir cells with typical morphology of neurosecretory cells in the dorsolateral protocerebrum send out three neural fibers to reach the ipsilateral corpora cardiaca (CC), the antennal lobe and two hemispheres of the protocerebrum. Based on co-localization with some PER-ir/PDF-ir cells, the CRZ-ir cells have the potential to serve as a bridge between circadian neural signals and endocrine regulation. Based on PDF's role in the regulation of locomotion, our results support the finding that the locomotor circadian rhythm is possibly controlled by a hormonal route.     

Corazonin- and PDF-immunoreactivities in the cephalic ganglia of termites

Antisera against the pigment-dispersing factor (PDF) and corazonin (Crz) reacted with distinct sets of neurons in the cephalic ganglia of termites. The locations of immunoreactive cells were similar but their numbers differed among the eight species examined: PDF-ir occurred in 0-6 cells in each optic lobe and 1-2 pairs of cells in the subosophageal ganglion (SOG), and Crz-ir in 0-2 pairs of cells in the pars intecerebralis, 3-14 cells in each lateral protocerebrum, and 0-6 pairs of cells in the SOG. Staining patterns were identical in the pseudergates, soldiers, and substitutive reproductives of Prorhinotermes simplex. Workers and soldiers were compared in the remaining 7 species. The only caste divergence was detected in Coptotermes formosanus, in which the soldiers differed from the workers by lack of 4 Crz-ir perikarya in the pars intercerebralis and occasionally also by the absence of 2 Crz-ir perikarya in the SOG. Diurnal changes in PDF-ir and Crz-ir were examined in P. simplex kept under long day (18:6 h light:darkness) or short day (10:14 h) photoperiods. No circadian fluctuations in the distribution or the intensity of immunostaining were found in the pseudergates and soldiers that were sacrificed in 4 h intervals or in the male and female substitutive reproductives examined in 6 h intervals.     

Can alloethism in workers of the bumblebee, Bombus terrestris, be explained in terms of foraging efficiency?

Bumblebee workers vary greatly in size, unlike workers of most other social bees. This variability has not been adequately explained. In many social insects, size variation is adaptive, with different-sized workers performing different tasks (alloethism). Here we established whether workers of the bumblebee, Bombus terrestris (L.) (Hymenoptera; Apidae), exhibit alloethism. We quantified the size of workers engaging in foraging compared to those that remain in the nest, and confirmed that it is the larger bees that tend to forage (X±SE thorax widths 4.34±0.01 mm for nest bees and 4.93±0.02 mm for foragers). We then investigated whether large bees are better suited to foraging because they are able to transport heavier loads of food back to the nest. Both pollen and nectar loads of returning foragers were measured, demonstrating that larger bees do return with a heavier mass of forage. Foraging trip times were inversely related to bee size when collecting nectar, but were unrelated to bee size for bees collecting pollen. Overall, large bees brought back more nectar per unit time than small bees, but the rate of pollen collection appeared to be unrelated to size. The smallest foragers had a nectar foraging rate close to zero, presumably explaining why foragers tend to be large. Why might larger bees be better at foraging? Various explanations are considered: larger bees are able to forage in cooler conditions, may be able to forage over larger distances, and are perhaps also less vulnerable to predation. Conversely, small workers are presumably cheaper to produce and may be more nimble at within-nest tasks. Further research is needed to assess these possibilities. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Potential host shift of the small hive beetle (Aethina tumida) to bumblebee colonies (Bombus impatiens)

Here we explored the potential for host shift from honeybee, Apis mellifera, colonies to bumblebee, Bombus impatiens, colonies by the small hive beetle, a nest parasite/scavenger native to sub-Saharan Africa. We investigated small hive beetle host choice, bumblebee colony defence as well as individual defensive behaviour of honeybee and bumblebee workers. Our findings show that in its new range in North America, bumblebees are potential alternate hosts for the small hive beetle. We found that small hive beetles do invade bumblebee colonies and readily oviposit there. Honeybee colonies are not preferred over bumblebee colonies. But even though bumblebees lack a co-evolutionary history with the small hive beetle, they are able to defend their colonies against this nest intruder by removal of beetle eggs and larvae and stinging of the latter. Hence, the observed behavioural mechanisms must be part of a generalistic defence system suitable for defence against multiple attackers. Nevertheless, there are quantitative (worker force) and qualitative differences (hygienic behaviour) between A. mellifera and B. impatiens. Received 16 July 2007; revised 16 January 2008; accepted 17 January 2008.     

The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells—the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into “circadian signalosomes,” whose compositions differ between E and M pacemaker cell types.     

Pigment-dispersing factor (PDF) has different effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.     

Distribution of corazonin and pigment-dispersing factor in the cephalic ganglia of termites

Distribution of neurones detectable with antisera to the corazonin (Crz) and the pigment-dispersing factor (PDF) was mapped in the workers or pseudergates of 10 species representing six out of seven termite families. All species contained two triads of Crz-immunoreactive (Crz-ir) neurones in the protocerebrum. Their fibres were linked to the opposite hemisphere, formed a network in the fronto-lateral protocerebrum, and projected to the corpora cardiaca (CC); in most species the fibres also supplied the deuto- and tritocerebrum and the frontal ganglion. Some species possessed additional Crz-ir perikarya in the protocerebrum and the suboesophageal ganglion (SOG). The PDF-ir somata were primarily located in the optic lobe (OL) and SOG. OL harboured a group (3 groups in Coptotermes) of 2-6 PDF-ir cells with processes extending to the medulla, connecting to the contralateral OL, forming 1-2 networks in the protocerebrum, and in most species running also to CC. Such a PDF-ir system associated with the OL was missing in Reticulitermes. Except for Mastotermes, the termites contained 1-2 PDF-ir cell pairs in the SOG and two species had additional perikarya in the protocerebrum. The results are consistent with the view of a monophyletic termite origin and demonstrate how the Crz-ir and PDF-ir systems diversified in the course of termite phylogeny.     

The police are not the army: context-dependent aggressiveness in a clonal ant

Animals often exhibit particular ‘personalities’, i.e. their behaviour is correlated across different situations. Recent studies suggest that this limitation of behavioural plasticity may be adaptive, since continuous adjustment of one''s behaviour may be time-consuming and costly. In social insects, particularly aggressive workers might efficiently take over fighting in the contexts of both nest defence and ‘policing’, i.e. the regulation of kin conflict in the society. Here, we examine whether workers who engage in aggressive policing in the ant Platythyrea punctata play a prominent role also in nest defence against intruders. The participation of individuals in policing and nest defence was highly skewed and a minority of workers exhibited most of the aggression. Workers who attacked reproductives after experimental colony fusion were less active during nest defence and vice versa. This suggests that workers show situation-dependent behavioural plasticity rather than consistently aggressive personalities.     

Using citizen science to monitor Bombus populations in the UK: nesting ecology and relative abundance in the urban environment

Citizen science can provide a valuable tool for collecting large quantities of ecological data over a larger geographic area than would otherwise be possible. Here, data were collected on 1,022 bumblebee nests by means of a public survey in which participants were asked to record attributes of bumblebee nests discovered in their gardens. All commonly reported species appeared to be generalist in their nest site selection and though species-specific differences in nest site choice were evident, there was a high degree of overlap in nesting habitat between most species. There was little evidence supporting the hypothesis that bumblebees tend to nest in the same site in consecutive years. A comparison of the contributions made by different species to the total nests reported in this and previous similar surveys suggests that the common bumblebee species Bombus pascuorum may have declined over the past 20?years relative to other species, comprising ~21% of colonies discovered in a survey conducted in 1989–1991, but just 8–9% of colonies in 2007–2009. This was accompanied by a reduction in the proportion of nests on the ground surface (the preferred position of this species). This is the first quantitative evidence of potential declines in the one of the UK’s ‘big six’ common bumblebee species.     

Drosophila free-running rhythms require intercellular communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

A circadian neuropeptide,pigment-dispersing factor-PDF,in the last-summer cicada Meimuna opalifera: cDNA cloning and immunocytochemistry

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neuromodulator functioning downstream of the insect brain's circadian clock, modulating daily rhythms of locomotor activity. Recently, we found that PDF precursors of the cricket Gryllus bimaculatus comprise a nuclear localization signal (NLS). Moreover, the nuclear localization of PDF immunoreactivity and the translocation of GFP-fused PDF precursor into the nucleus have both been demonstrated. These suggest a fundamental role for PDF peptide in the circadian clock system within the nucleus, in addition to its role in downstream neural events. In the present study, we carried out the cDNA cloning of PDF from adult brains of the last-summer cicada Meimuna opalifera, and found that an isolated clone (545 bp) encodes an ordinary PDF precursor protein. PDF peptide itself shows a high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to the crustacean beta-PDH peptides. The computer-assisted sequence analysis of PDF precursor revealed a possible translocation into the nucleus, despite the lack of a definite NLS-like sequence. Using immunocytochemistry, the optic lobes of M. opalifera revealed PDF-immunoreactive neurons in both the medulla and lamina neuropiles. All these PDF cells exhibited prominent immunolabeling of both their perikarya and axons, but not their nuclei. Our results provide the first structural and immunocytochemical identification of PDF neurons in Hemiptera.     

Morphological differences between extranidal and intranidal workers in the ant Temnothorax rugatulus,but no effect of body size on foraging distance

Most ant genera are thought to have monomorphic workers, indicating perhaps a high degree of flexibility in task allocation, and the well-studied genus Temnothorax is an example of this. However, considerable size variation may exist between individuals. In addition, though workers can show flexible behavior, it has been shown that individuals may consistently differ in their task profiles. Here we test whether body size variation among workers affects foraging behavior. Two main hypotheses were tested: first, whether larger ants forage at greater distance from the nest, and second, whether larger individuals show a higher propensity to work outside of the nest. Our results showed that ant body size does not significantly affect foraging distance. However, larger ants were more likely to be found outside the nest. Though Temnothorax ants are a common model system, this is the first study demonstrating task allocation based on body size, which is fixed in adults. Our study suggests that particularly small species may have to be examined carefully for body size variation before concluding that body size is uniform and therefore irrelevant for task allocation.     

Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.     

Identification and characterization of an Egr ortholog as a neural immediate early gene in the European honeybee (Apis mellifera L.)

To date, there are only few reports of immediate early genes (IEGs) available in insects. Aiming at identifying a conserved IEG in insects, we characterized an Egr homolog of the honeybee (AmEgr: Apis mellifera Egr). AmEgr was transiently induced in whole worker brains after seizure induction. In situ hybridization for AmEgr indicated that neural activity of a certain mushroom body (a higher brain center) neuron subtype, which is the same as that we previously identified using another non-coding IEG, termed kakusei, is more enhanced in forager brains. These findings suggest that Egr can be utilized as an IEG in insects.