Many aquatic animals swim by undulatory body movements and understanding the diversity of these movements could unlock the potential for designing better underwater robots. Here, we analyzed the steady swimming kinematics of a diverse group of fish species to investigate whether their undulatory movements can be represented using a series of interconnected multi-segment models, and if so, to identify the key factors driving the segment configuration of the models. Our results show that the steady swimming kinematics of fishes can be described successfully using parsimonious models, 83% of which had fewer than five segments. In these models, the anterior segments were significantly longer than the posterior segments, and there was a direct link between segment configuration and swimming kinematics, body shape, and Reynolds number. The models representing eel-like fishes with elongated bodies and fishes swimming at high Reynolds numbers had more segments and less segment length variability along the body than the models representing other fishes. These fishes recruited their anterior bodies to a greater extent, initiating the undulatory wave more anteriorly. Two shape parameters, related to axial and overall body thickness, predicted segment configuration with moderate to high success rate. We found that head morphology was a good predictor of its segment length. While there was a large variation in head segments, the length of tail segments was similar across all models. Given that fishes exhibited variable caudal fin shapes, the consistency of tail segments could be a result of an evolutionary constraint tuned for high propulsive efficiency. The bio-inspired multi-segment models presented in this study highlight the key bending points along the body and can be used to decide on the placement of actuators in fish-inspired robots, to model hydrodynamic forces in theoretical and computational studies, or for predicting muscle activation patterns during swimming.

Pigment dispersing factors (PDFs, or PDHs in crustaceans) form a structurally related group of neuropeptides found throughout the Ecdysozoa and were first discovered as pigmentary effector hormones in crustaceans. In insects PDFs fulfill crucial neuromodulatory roles, most notably as output regulators of the circadian system, underscoring their central position in physiological and behavioral organization of arthropods. Intriguingly, decapod crustaceans express multiple isoforms of PDH originating from separate genes, yet their differential functions are still to be determined. Here, we functionally define two PDH receptors in the crab Carcinus maenas and show them to be selectively activated by four PDH isoforms: PDHR 43673 was activated by PDH-1 and PDH-2 at low nanomolar doses whilst PDHR 41189 was activated by PDH-3 and an extended 20 residue e-PDH. Detailed examination of the anatomical distribution of all four peptides and their cognate receptors indicate that they likely perform different functions as secreted hormones and/or neuromodulators, with PDH-1 and its receptor 43,673 implicated in an authentic hormonal axis. PDH-2, PDH-3, and e-PDH were limited to non-neurohemal interneuronal sites in the CNS; PDHR 41189 was largely restricted to the nervous system suggesting a neuromodulatory function. Notably PDH-3 and e-PDH were without chromatophore dispersing activity. This is the first report which functionally defines a PDHR in an endocrine system in a crustacean and to indicate this and other putative roles of this physiologically pivotal peptide group in these organisms. Thus, our findings present opportunities to further examine the endocrine and circadian machinery in this important arthropod phylum.

Neuropeptides represent the largest single class of signal compounds and are involved in regulation of development, growth, reproduction, metabolism and behavior of insects. Over the last few years there has been a tremendous increase in our knowledge of neuropeptide signaling due to genome sequencing, peptidomics, gene micro arrays, receptor characterization and targeted gene interference combined with physiological and behavior analysis. In this chapter we review the current knowledge of structure and distribution of insect neuropeptides and their receptors, as well as their diverse functions. We also discuss peptide biosynthesis, processing and expression, as well as classification of insect neuropeptides. Special attention is paid to the role insect neuropeptides play as potential targets for pest management and as a basis for development of insect control agents employing the rational/structural design approaches.

In fish, as in other vertebrates, the gonads are stimulated by two pituitary gonadotropic hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH). LH and FSH secretion are regulated by the gonadal steroids acting either directly on the pituitary or indirectly via the hypothalamus. Both positive and negative gonadal feedback mechanisms are present in fishes. Plasma and pituitary LH and FSH levels are higher in Atlantic salmon parr males that have been sham-operated in spring than in castrated fish, when sampled in the periods of gonadal growth and breeding, indicated the existence of a physiological positive feedback involved in the control of LH and FSH secretion. However, for FSH also a physiological negative feedback controlling FSH secretion was found in the early phase of sexual maturation in early summer.

The involvement of different steroids and the role of aromatization in feedback systems controlling reproduction has also been studied by administration of different androgens and aromatase inhibitors in vivo. Testosterone (T) stimulates LH by an aromatase-dependent positive feedback. Also 11-androgens exert a positive effect on LH, though weaker than T. FSH appears to be controlled both by a negative aromatase dependent feedback and a negative non-aromatase dependent T effect, as well as possibly a positive aromatase-dependent feedback. FSH is also controlled by both a negative and a positive feedback of 11-androgens. In addition, treatment with different aromatase inhibitors increased the proportion of fish maturing.

Positive gonadal feedback eventually separate gonadotropic functions between rematuring high level and non-rematuring low level fish. However, FSH secretion appears not to be the only responsible factor for the onset of maturation in Atlantic salmon male parr, since immunoassayable plasma FSH levels are equally high in non-rematuring and rematuring fish during the onset of gonadal growth in early summer.

The decline of androgens at the beginning of breeding season is not due to a suppressive action of elevated 17,20P at either the pituitary or testicular level in salmon.

Moreover, administration of T at the end of spawning season diminished the postbreeding decline of 17,20P, testes weights, milt production and sperm motility, possibly via a positive feedback on LH secretion.

Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophilag the regulation of DILP production and release in adult fruit flies is poorly understood. Here we investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR and NKD, in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, we show DTK-immunoreactive axon terminations close to the presumed dendrites of the IPCs, and DTKR immunolabeling in these cells. Second, we utilized targeted RNA interference to knock down expression of the DTK receptor, DTKR, in IPCs and monitored the effects on Dilp transcript levels in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. We also measured trehalose and lipid levels as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, we targeted the IPCs with RNAi or ectopic expression of the other DTK receptor, NKD, but found no effect on survival at starvation. Our results suggest that DTK signaling, via DTKR, regulates the brain IPCs.

Proctolin-immunoreactive neurons in all neuromers of the ventral nerve cord of Tenebrio molitor L. have been quantitatively demonstrated and mapped throughout metamorphosis. Each neuromer contains an anterior and a posterior group of neurons with light and dark staining properties as revealed by peroxidase-antiperoxidase labeling. Serial homologous subsets of dark staining neurons with central and peripheral projections have been identified and found to persist during morphogenetic changes from the larva to the adult. Most neurons maintain their topological and structural characteristics throughout metamorphosis. The identified proctolin-immunoreactive neurons exhibit structures similar to those described in other insect species; some may correspond known motoneurons.

The fruitfly, Drosophila, is dependent on its olfactory sense in food search and reproduction. Processing of odorant information takes place in the antennal lobes, the primary olfactory center in the insect brain. Besides classical neurotransmitters, earlier studies have indicated the presence of a few neuropeptides in the olfactory system. In the present study we made an extensive analysis of the expression of neuropeptides in the Drosophila antennal lobes by direct profiling using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunocytochemistry. Neuropeptides from seven different precursor genes were unambiguously identified and their localization in neurons was subsequently revealed by immunocytochemistry. These were short neuropeptide F, tachykinin related peptide, allatostatin A, myoinhibitory peptide, SIFamide, IPNamide, and myosuppressin. The neuropeptides were expressed in subsets of olfactory sensory cells and different populations of local interneurons and extrinsic (centrifugal) neurons. In some neuron types neuropeptides were colocalized with classical neurotransmitters. Our findings suggest a huge complexity in peptidergic signaling in different circuits of the antennal lobe.

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ~21 pM throughout the molt cycle, showing a peak of ~89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.

Background

Neuropeptides are key players in information transfer and act as important regulators of development, growth, metabolism, and reproduction within multi-cellular animal organisms (Metazoa). These short protein-like substances show a high degree of structural variability and are recognized as the most diverse group of messenger molecules. We used transcriptome sequences from the 1KITE (1K Insect Transcriptome Evolution) project to search for neuropeptide coding sequences in 24 species from the non-pterygote hexapod lineages Protura (coneheads), Collembola (springtails), Diplura (two-pronged bristletails), Archaeognatha (jumping bristletails), and Zygentoma (silverfish and firebrats), which are often referred to as “basal” hexapods. Phylogenetically, Protura, Collembola, Diplura, and Archaeognatha are currently placed between Remipedia and Pterygota (winged insects); Zygentoma is the sistergroup of Pterygota. The Remipedia are assumed to be among the closest relatives of all hexapods and belong to the crustaceans.

Results

We identified neuropeptide precursor sequences within whole-body transcriptome data from these five hexapod groups and complemented this dataset with homologous sequences from three crustaceans (including Daphnia pulex), three myriapods, and the fruit fly Drosophila melanogaster. Our results indicate that the reported loss of several neuropeptide genes in a number of winged insects, particularly holometabolous insects, is a trend that has occurred within Pterygota. The neuropeptide precursor sequences of the non-pterygote hexapods show numerous amino acid substitutions, gene duplications, variants following alternative splicing, and numbers of paracopies. Nevertheless, most of these features fall within the range of variation known from pterygote insects. However, the capa/pyrokinin genes of non-pterygote hexapods provide an interesting example of rapid evolution, including duplication of a neuropeptide gene encoding different ligands.

Conclusions

Our findings delineate a basic pattern of neuropeptide sequences that existed before lineage-specific developments occurred during the evolution of pterygote insects.

Ecological physiologists and biomechanists have investigated swimming performance in a diversity of fishes; however, the connection between form, function, and energetics of locomotion has been rarely evaluated in the same system and under climate change scenarios. In this perspective, I argue that working within the framework of “EcoPhysioMechanics,” i.e. integrating energetics and biomechanics tools, to measure locomotor performance and behavior under different abiotic factors, improves our understanding of the mechanisms, limits and costs of movement. To demonstrate how EcoPhysioMechanics can be applied to locomotor studies, I outline how linking biomechanics and physiology allows us to understand how fishes may modulate their movement to achieve high speeds or reduce the costs of locomotion. I also discuss how the framework is necessary to quantify swimming capacity under climate change scenarios. Finally, I discuss current dearth of integrative studies and gaps in empirical datasets that are necessary to understand fish swimming under changing environments. 

Fishes exhibit an astounding diversity of locomotor behaviors from classic swimming with their body and fins to jumping, flying, walking, and burrowing. Fishes that use their body and caudal fin (BCF) during undulatory swimming have been traditionally divided into modes based on the length of the propulsive body wave and the ratio of head:tail oscillation amplitude: anguilliform, subcarangiform, carangiform, and thunniform. This classification was first proposed based on key morphological traits, such as body stiffness and elongation, to group fishes based on their expected swimming mechanics. Here, we present a comparative study of 44 diverse species quantifying the kinematics and morphology of BCF-swimming fishes. Our results reveal that most species we studied share similar oscillation amplitude during steady locomotion that can be modeled using a second-degree order polynomial. The length of the propulsive body wave was shorter for species classified as anguilliform and longer for those classified as thunniform, although substantial variability existed both within and among species. Moreover, there was no decrease in head:tail amplitude from the anguilliform to thunniform mode of locomotion as we expected from the traditional classification. While the expected swimming modes correlated with morphological traits, they did not accurately represent the kinematics of BCF locomotion. These results indicate that even fish species differing as substantially in morphology as tuna and eel exhibit statistically similar two-dimensional midline kinematics and point toward unifying locomotor hydrodynamic mechanisms that can serve as the basis for understanding aquatic locomotion and controlling biomimetic aquatic robots.

Research on crustacean peptides has concentrated mainly on decapods and isopods, and a growing number of >200 peptides have been sequenced from these two groups, the majority from decapods, but recently, the annotation of the Daphnia pulexgenome has contributed many more novel peptides many of which were also sequenced de novo. Identified and bioactive crustacean peptides — the only ones reported here — regulate a large range of physiological functions, including color change, activities of heart, exoskeletal and visceral muscles, metabolic function, development, metamorphosis, and reproduction.

Insect ionic and fluid homeostasis relies upon the Malpighian tubules (MT) and different hindgut compartments. Primary urine formed in MTs is finally modified by ion, solute and water reabsorptive processes primarily in the hindgut under the control of several large peptide hormones. One of these, the ion transport peptide (ITP), is a chloride transport-stimulating and acid secretion-inhibiting hormone similar to crustacean hyperglycaemic hormones (CHHs). In locusts, moths and fruit flies, ITP together with the slightly longer ITPL isoforms, inactive in hindgut bioassays, arise by alternative splicing from very similar itp genes. ITP and ITPL are differentially distributed in (1) pars lateralis/retrocerebral complex neurosecretory cells (NSCs) containing both splice forms, (2) interneurons with either one of the splice forms, (3) hindgut-innervating abdominal ITP neurons (in Drosophila only), and (4) intrinsic, putative sensory NSCs in peripheral neurohaemal perisympathetic/perivisceral organs or transverse nerves (usually containing ITPL). Both splice forms occur as hormones released into the haemolymph in response to feeding or stress stimuli. ITPL mainly released from the peripheral NSCs is discussed as a competitive inhibitor (as established in vitro) of ITP action on yet to be identified hindgut ITP receptors. Furthermore, some evidence has been provided for possible ecdysis-related functions of ITP and/or ITPL in moths. The comparative data on the highly similar gene, precursor and primary structures and similar differential distributions in insect and crustacean NSCs suggest that CHH/ITP and ITPL neuropeptide-producing cells and their gene products share common phylogenetic ancestry.

We identified previously two long (DrmITPL1 and -L2) and one amidated short isoform (DrmITP) of insect ion transport peptides (ITPs) as products derived by alternatively splicing from the Drosophila itp-gene (CG13586). The peptides are members of a large family of arthropod neuropeptides incl. crustacean hyperglycemic hormones (CHH/ITP-family), but similar ITPs are only known in locusts to have antidiuretic bioactivity on the hindgut. We localised the peptides by in situ hybridisation and immunocytochemistry with isoform-specific antibodies in the nervous system of larval (L3) and adult Drosophila melanogaster and screened Gal4-lines specific for peptidergic cells. Four neurosecretory cells in brain-corpora cardiaca/allata putatively release DrmITP as a hormone in all stages. DrmITP also occurs in interneurons in the brain/ventral ganglia and in neurons efferent towards the hindgut. Some interneurons are identical to well-known circadian clock neurons for which the effector molecules were elusive but are responsible for the evening bouts of locomotor activity in flies. DrmITPL1 and -L2 were found only in peripheral lateral bipolar and putative sensory neurons which are likely to play a role in the control of growth, hindgut ion transport and heart beat. With DrmITP identified in brain neurosecretory cells, hindgut-innervating neurons in the abdominal ganglia and one pair in the abdomen close to the larval anal organ or innervating the adult rectal pads, both chloride-transporting organs, we are facing an enormous complexity in multiple functions of differentially expressed ITP/Ls derived from a single gene. Preliminary results using Gal4-driven RNAi in distinct peptidergic neurons look promising to find deficiency phenotypes.

Crustacean cardioactive peptide-immunoreactive neurons occur in the entire central nervous system of Locusta migratoria. The present paper focuses on mapping studies in the ventral nerve cord and on peripheral projection sites. Two types of contralaterally projecting neurons occur in all neuromers from the subesophageal to the seventh abdominal ganglia. One type forms terminals at the surface of the thoracic nerves 6 and 1, the distal perisympathetic organs, the lateral heart nerves, and on ventral and dorsal diaphragm muscles. Two large neurons in the anterior part and several neurons of a different type in the posterior part of the terminal ganglion project into the last tergal nerves. In the abdominal neuromers 1–7, two types of ipsilaterally projecting neurons occur, one of which gives rise to neurosecretory terminals in the distal perisympathetic organs, in peripheral areas of the transverse, stigmata and lateral heart nerves. Four subesophageal neurons have putative terminals in the neurilemma of the nervus corporis allati II, and in the corpora allata and cardiaca. In addition, several immunoreactive putative interneurons and other neurons were mapped in the ventral nerve cord. A new in situ whole-mount technique was essential for elucidation of the peripheral pathways and targets of the identified neurons, which suggest a role of the peptide in the control of heartbeat, abdominal ventilatory and visceral muscle activity.

We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, a first crustacean allatotropin, Ala7-CCAP, one CCHamide, Arg7-corazonin, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin- and one each of three IGF-related peptides, two alternative splice forms of short and long ion transport peptide (ITP), one each of two N-terminally elongated ITPs, myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met4-proctolin, one novel short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, three tachykinins. Two genes encode orcokinins, three genes different allatostatins-C. Paired gene clusters occur for two novel eclosion hormones; bursicons alpha, beta; glycoproteins GPA2, GPB5; and two of the allatostatin-C genes. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.

Ion transport peptides (ITPs) belong to a large arthropod neuropeptide family including crustacean hyperglycaemic hormones and are antidiuretic hormones in locusts. Because long and short ITP isoforms are generated by alternative splicing from a single gene in locusts and moths, we investigated whether similarly spliced gene products occur in the nervous system of Drosophila melanogaster throughout postembryogenesis. The itp gene CG13586 was reanalyzed, and we found three instead of the two previously annotated alternatively spliced mRNAs. These give rise to three different neuropeptides, two long C-terminally carboxylated isoforms (DrmITPL1 and DrmITPL2, both 87 amino acids) and one short amidated DrmITP (73 amino acids), which were partially identified biochemically. Immunocytochemistry and in situ hybridization reveal nine larval and 14 adult identified neurons: four pars lateralis neurosecretory neurons, three hindgut-innervating neurons in abdominal ganglia, and a stage-specific number of interneurons and peripheral bipolar neurons. The neurosecretory neurons persist throughout postembryogenesis, form release sites in corpora cardiaca, and invade corpora allata. One type of ITP-expressing interneuron exists only in the larval and prepupal subesophageal ganglia, whereas three types of interneurons in the adult brain arise in late pupae and invade circumscribed neuropils in superior median and lateral brain areas. One peripheral bipolar and putative sensory neuron type occurs in the larval, pupal, and adult preterminal abdominal segments. Although the neurosecretory neurons may release DrmITP and DrmITPL2 into the haemolymph, possible physiological roles of the hindgut-innervating and peripheral neurons as well as the interneurons are yet to be identified.

Insulin-like peptides (ILPs) regulate growth, reproduction, metabolic homeostasis, life span and stress resistance in worms, flies and mammals. A set of insulin producing cells (IPCs) in the Drosophila brain that express three ILPs (DILP2, 3 and 5) have been the main focus of interest in hormonal DILP signaling. Little is, however, known about factors that regulate DILP production and release by these IPCs. Here we show that the IPCs express the metabotropic GABA(B) receptor (GBR), but not the ionotropic GABA(A) receptor subunit RDL. Diminishing the GBR expression on these cells by targeted RNA interference abbreviates life span, decreases metabolic stress resistance and alters carbohydrate and lipid metabolism at stress, but not growth in Drosophila. A direct effect of diminishing GBR on IPCs is an increase in DILP immunofluorescence in these cells, an effect that is accentuated at starvation. Knockdown of irk3, possibly part of a G protein-activated inwardly rectifying K(+) channel that may link to GBRs, phenocopies GBR knockdown in starvation experiments. Our experiments suggest that the GBR is involved in inhibitory control of DILP production and release in adult flies at metabolic stress and that this receptor mediates a GABA signal from brain interneurons that may convey nutritional signals. This is the first demonstration of a neurotransmitter that inhibits insulin signaling in its regulation of metabolism, stress and life span in an invertebrate brain.

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABA_AR) for fast inhibitory transmission and on G-protein-coupled ones (GABA_BR) for slow and modulatory action. We used immunocytochemistry to map GABA_BR sites in the Drosophila CNS and compared the distribution with that of the GABA_AR subunit RDL. To identify GABAergic synapses, we raised an antiserum to the vesicular GABA transporter (vGAT). For general GABA distribution, we utilized an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 to drive green fluorescent protein. GABA_BR-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABA_BR-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA_BR-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA_BR and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA_BR distribution and identified neurons in adults and larvae. Our findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites. J. Comp. Neurol. 505:18–31, 2007.

Insect brains are known to express a high degree of experience-dependent structural plasticity. One brain structure in particular, the mushroom body (MB), has been attended to in numerous studies as it is implicated in complex cognitive processes such as olfactory learning and memory. It is, however, poorly understood to what extent sensory input per se affects the plasticity of the mushroom bodies. By performing unilateral blocking of olfactory input on immobilized butterflies, we were able to measure the effect of passive sensory input on the volumes of antennal lobes (ALs) and MB calyces. We showed that the primary and secondary olfactory neuropils respond in different ways to olfactory input. ALs show absolute experience-dependency and increase in volume only if receiving direct olfactory input from ipsilateral antennae, while MB calyx volumes were unaffected by the treatment and instead show absolute age-dependency in this regard. We therefore propose that cognitive processes related to behavioural expressions are needed in order for the calyx to show experience-dependent volumetric expansions. Our results indicate that such experience-dependent volumetric expansions of calyces observed in other studies may have been caused by cognitive processes rather than by sensory input, bringing some causative clarity to a complex neural phenomenon.

Specific floral resources may help bees to face environmental challenges such as parasite infection, as recently shown for sunflower pollen. Whereas this pollen diet is known to be unsuitable for the larval development of bumble bees, it has been shown to reduce the load of a trypanosomatid parasite (Crithidia bombi) in the bumble bee gut. Recent studies suggested it could be due to phenolamides, a group of compounds commonly found in flowering plants. We, therefore, decided to assess separately the impacts of sunflower pollen and its phenolamides on a bumble bee and its gut parasite. We fed Crithidia-infected and -uninfected microcolonies of Bombus terrestris either with a diet of willow pollen (control), a diet of sunflower pollen (natural diet) or a diet of willow pollen supplemented with sunflower phenolamides (supplemented diet). We measured several parameters at both microcolony (i.e., food collection, parasite load, brood development and stress responses) and individual (i.e., fat body content and phenotypic variation) levels. As expected, the natural diet had detrimental effects on bumble bees but surprisingly, we did not observe any reduction in parasite load, probably because of bee species-specific outcomes. The supplemented diet also induced detrimental effects but by contrast to our a priori hypothesis, it led to an increase in parasite load in infected microcolonies. We hypothesised that it could be due to physiological distress or gut microbiota alteration induced by phenolamide bioactivities. We further challenged the definition of medicinal effects and questioned the way to assess them in controlled conditions, underlining the necessity to clearly define the experimental framework in this research field.

The leading-edge vortex (LEV) formation on the caudal fin (CF) has been identified as playing a key role in efficient lift-based thrust production of fish-like propulsion. The enhancement of the CF LEV through its interaction with vortices formed upstream due to a median fin with a distinct shape is the focus of this paper. High-speed, high-fidelity videos and particle imaging velocimetry (PIV) were obtained from rainbow trout during steady forward swimming to visualize the undulatory kinematics and two-dimensional flow behavior. Body kinematics are quantified using a traveling-wave formulation that is used to prescribe the motion of a high-fidelity three-dimensional surface model of the fish body for a computational fluid dynamics (CFD) study. The pressure field of the CFD result is compared and validated with the PIV result from the experiment. Using CFD, the vortex forming and shedding behaviors of the anal fin (AF) and their capturing and interaction with the trunk (TK) and the CF are visualized and examined. Coherent AF-bound LEVs are found to form periodically, leading to thrust production of the AF. The vortices subsequently shed from the AF are found to help stabilize and reinforce the LEV formation on the CF by aiding LEV initiation at stroke reversal and enhancing LEV during a tail stroke, which leads to enhancement of lift-based thrust production. The CF is found to shed vortex tubes (VTs) that create backward-facing jets, and the ventral-side VT and the associated backward jets are both strengthened by vortices shed by the AF. An additional benefit of the AF is found to be reduction of body drag by reducing the lateral crossflow that leads to loss of beneficial pressure gradient across the body. Through varying AF-CF spacing and AF height, we find that CF thrust enhancement and TK drag reduction due to the AF are both affected by the position and size of the AF. The position and area of the AF that led to the most hydrodynamic benefit are found to be the original, anatomically accurate position and size. In this paper, we demonstrate the important effect of vortex interaction among propulsive surfaces in fish-like propulsion.

Global warming has been identified as a key driver of bee declines around the world. While it is clear that elevated temperatures during the spring and summer months—the principal activity period of many bee species—is a factor in this decline, exactly how temperature affects bee survival is unknown. In vertebrates, there is clear evidence that elevated ambient temperatures impair cognition but whether and how heat affects the cognitive abilities of invertebrates remains unclear. Cognitive skills in bees are essential for their survival as, to supply the hive with nutrition, workers must be able to learn and remember the location of the most rewarding floral resources. Here, we investigate whether temperature-related cognitive impairments could be a driver of bee declines by exploring the effect of short-term increases in ambient temperature on learning and memory. We found that, in comparison to bees that were tested at 25°C (a temperature that they would typically experience in summer), bees that were exposed to 32°C (a temperature that they will becoming increasingly exposed to during heatwave events) were significantly worse at forming an association between a coloured light and a sucrose reward and that their capacity to remember this association after just 1 h was abolished. This study provides novel experimental evidence that even just a few hours of exposure to heatwave-like temperatures can severely impair the cognitive performance of insects. Such temperature-induced cognitive deficits could play an important role in explaining recent and future bee population declines.

In a context of rapid global change, understanding how environmental stressors can impact phenotypic variation, and which phenotypic traits are predominantly affected can be particularly relevant. Indeed, potential phenotypic modifications could affect the functionality of traits from taxa that are in decline but that are keystone species in many ecosystems. In this study, we assessed the impact of environmental drivers and agricultural intensification on two crucial pollinator species: the honeybee (Apis mellifera) and the buff-tailed bumblebee (Bombus terrestris). Among eight countries representing four major European biogeographical regions [i.e., Boreal (Sweden and Estonia), Atlantic (Ireland and United Kingdom), Continental (Germany and Switzerland) and Mediterranean (Spain and Italy)] and two type of crops (i.e., apple orchards and oilseed rape) we assessed how landscape structure, latitude and pesticide management could impact their wing morphology. Two sampling sessions were conducted: the first one when the hives/nests were settled on the field (T0) and a second sampling session after the potential effect of agricultural intensification (T1). Using a dataset of more than 7238 wings, we measured the wing size, shape and asymmetry. We observed that, in several countries, a shift in most of the morphological traits occurred between T0 and T1. When focusing on the drivers of phenotypic variation in T1, the levels of significance for some potential drivers were sometimes high, while most of the variation remained unexplained. The latitude and, more rarely, grassland cover were found to partly explain the wing modifications. In light of these results, we conclude that phenotypic shifts can occur in a very short period, after encountering new field conditions. Further studies should be conducted to better understand which alternative drivers could explain morphological changes in the agro-ecosystem after crop exposition, as well as the potential consequences of these changes on foraging performance or pollination efficiency.

Bee foraging behavior provides a pollination service that has both ecological and economic benefits. However, bee population decline could directly affect the efficiency of this interaction. Among the drivers of this decline, global warming has been implicated as an emerging threat but exactly how increasing temperatures affect bee foraging behavior remains unexplored. Here, we assessed how exposure to elevated temperatures during development affects the foraging behavior and morphology of workers from commercial and wild Bombus terrestris colonies. Workers reared at 33 °C had a higher visiting rate and shorter visiting time than those reared at 27°C. In addition, far fewer workers reared at 33 °C engaged in foraging activities and this is potentially related to the drastic reduction in the number of individuals produced in colonies exposed to 33 °C. The impact of elevated developmental temperature on wild colonies was even stronger as none of the workers from these colonies performed any foraging trips. We also found that rearing temperature affected wing size and shape. Our results provide the first evidence that colony temperature can have striking effects on bumblebee foraging behavior. Of particular importance is the drastic reduction in the number of workers performing foraging trips, and the total number of foraging trips made by workers reared in high temperatures. Further studies should explore if, ultimately, these observed effects of exposure to elevated temperature during development lead to a reduction in pollination efficiency.

The impact of global warming on wild bee decline threatens the pollination services they provide. Exposure to temperatures above optimal during development is known to reduce adult body size but how it affects the development and scaling of body parts remains unclear. In bees, a reduction in body size and/or a reduction in body parts, such as the antennae, tongue and wings, and how they scale with body size (i.e. their allometry) could severely affect their fitness. To date, it remains unclear how temperature affects body size and the scaling of morphological traits in bees. To address this knowledge gap, we exposed both males and workers of Bombus terrestris to elevated temperature during development and assessed the effects on (i) the size of morphological traits and (ii) the allometry between these traits. Colonies were exposed to optimal (25°C) or stressful (33°C) temperatures. We then measured the body size, wing size, antenna and tongue length, as well as the allometry between these traits. We found that workers were smaller and the antennae of both castes were reduced at the higher temperature. However, tongue length and wing size were not affected by developmental temperature. The allometric scaling of the tongue was also affected by developmental temperature. Smaller body size and antennae could impair both individual and colony fitness, by affecting foraging efficiency and, consequently, colony development. Our results encourage further exploration of how the temperature-induced changes in morphology affect functional traits and pollination efficiency. 

Foraging behavior is driven by diverse factors, notably life history traits. Foraging strategies are particularly complex among eusocial species such as bumblebees, because they depend primarily on the needs of the colony, rather than on individual's needs. Colony size, i.e. the number of workers in a colony vary a lot among eusocial insects. While a large colony can be adaptive, several drivers can strongly decrease colony size, like pesticides or high temperatures. In this study, we used the bumblebee Bombus terrestris to assess if workers adapted their foraging behavior to such rapid decreases in colony size. We conducted the foraging experiments with two plant species commonly used by bumblebees: Borago officinalis and Echium plantagineum. Several foraging parameters were measured: foraging time, number of foraging trips, number of workers foraging, handling time and visiting rate. Despite a drastic reduction in colony size, nearly all the foraging behavior parameters were unaffected by the colony size reduction. Colonies that were subject to a large decrease in workers instead displayed high resilience and behavioral plasticity by quickly increasing the proportion of foragers. Ultimately, further research should assess if this consistency in foraging behavior also allows bumblebee colonies to maintain both the efficiency of the resources collection and pollination.

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.

Comparative studies on cellular and molecular clock mechanisms have revealed striking similarities in the organization of the clocks among different animal groups. To gain evolutionary insight into the properties of the clock network within the Drosophila genus, we analyzed sequence identities and similarities of clock protein homologues and immunostained brains of 10 different Drosophila species using antibodies against vrille (VRI), PAR-protein domain1 (PDP1), and cryptochrome (CRY). We found that the clock network of both subgenera Sophophora and Drosophila consists of all lateral and dorsal clock neuron clusters that were previously described in Drosophila melanogaster. Immunostaining against CRY and the neuropeptide pigment-dispersing factor (PDF), however, revealed species-specific differences. All species of the Drosophila subgenus and D. pseudoobscura of the Sophophora subgenus completely lacked CRY in the large ventrolateral clock neurons (lLNvs) and showed reduced PDF immunostaining in the small ventrolateral clock neurons (sLNvs). In contrast, we found the expression of the ion transport peptide (ITP) to be consistent within the fifth sLNv and one dorsolateral clock neuron (LNd) in all investigated species, suggesting a conserved putative function of this neuropeptide in the clock. We conclude that the general anatomy of the clock network is highly conserved throughout the Drosophila genus, although there is variation in PDF and CRY expression. Our comparative study is a first step toward understanding the organization of the circadian clock in Drosophila species adapted to different habitats.

Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromosomal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements.

Eusocial insect colonies act as a superorganism, which can improve their ability to buffer the negative impact of some anthropogenic stressors. However, this buffering effect can be affected by anthropogenic factors that reduce their colony size. A reduction in colony size is known to negatively affect several parameters like brood maintenance or thermoregulation, but the effects on behaviour and cognition have been largely overlooked. It remains unclear how a sudden change in group size, such as that which might be caused by anthropogenic stressors, affects individual behaviour within a colony. In this study, the bumblebee Bombus terrestris was used to study the effect of social group size on behaviour by comparing the associative learning capabilities of individuals from colonies that were unmanipulated, reduced to a normal size (a colony of 100 workers) or reduced to a critically low but functional size (a colony of 20 workers). The results demonstrated that workers from the different treatments performed equally well in associative learning tasks, which also included no significant differences in the learning capacity of workers that had fully developed after the colony size manipulation. Furthermore, we found that the size of workers had no impact on associative learning ability. The learning abilities of bumblebee workers were thus resilient to the colony reduction they encountered. Our study is a first step towards understanding how eusocial insect cognition can be impacted by drastic reductions in colony size. Significance statement While anthropogenic stressors can reduce the colony size of eusocial insects, the impact of this reduction is poorly studied, particularly among bumblebees. We hypothesised that colony size reduction would affect the cognitive capacity of worker bumblebees as a result of fewer social interactions or potential undernourishment. Using differential conditioning, we showed that drastic reductions in colony size have no effect on the associative learning capabilities of the bumblebee Bombus terrestris and that this was the same for individuals that were tested just after the colony reduction and individuals that fully developed under the colony size reduction. We also showed that body size did not affect learning capabilities. This resilience could be an efficient buffer against the ongoing impacts of global change.

Orcokinins are a family of myotropic neuropeptides identified in various decapod crustaceans and recently in a cockroach. Their presence in the crustacean nervous system and hemolymph suggests that they act as hormones and as locally acting neuromodulators. To provide further evidence for the existence of orcokinins in insects, we identified a novel orcokinin-related peptide in the locust Schistocerca gregaria and used an antiserum against Asn13-orcokinin for immunostaining in the brains of selected dicondylian insects, including a silverfish, three polyneopteran species (a cockroach and two locusts), and three endopterygote species (a moth, a bee, and a fly). As analyzed by MALDI-TOF spectrometry and nanoelectrospray Q-TOF, the locust orcokinin is a novel tetradecapeptide with striking sequence similarity to crustacean orcokinins. Orcokinin immunostaining was widespread and occurred in similar patterns in the brain of the silverfish and the polyneopteran species. Prominent immunostaining was detected in the optic lobe, especially in the medulla and in the accessory medulla, in local interneurons of the antennal lobe, and in extrinsic and intrinsic mushroom-body neurons. All parts of the central complex and many other areas of the brains were densely stained. In the silverfish, the cockroach, and the locusts, processes in the corpora cardiaca showed orcokinin immunoreactivity, suggesting that orcokinins also serve a hormonal role. In contrast to the case in polyneopteran species, immunostaining was completely lacking in the brains of the honeybee, fruitfly, and sphinx moth. This indicates that orcokinins either are modified considerably or may be completely absent in the brains of endopterygote insects.

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.

The protein interacting with C kinase 1 (PICK1) protein was first identified as a novel binding partner for protein kinase C. PICK1 contains a membrane-binding BAR domain and a PDZ domain interacting with many synaptic proteins, including the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR2 and the dopamine transporter. PICK1 is strongly implicated in GluR2 trafficking and synaptic plasticity. In mammals, PICK1 has been characterized extensively in cell culture studies. To study PICK1 in an intact system, we characterized PICK1 expression immunohistochemically in the adult and larval Drosophila central nervous system. PICK1 was found in cell bodies in the subesophageal ganglion, the antennal lobe, the protocerebrum, and the neuroendocrine center pars intercerebralis. The cell types that express PICK1 were identified using GAL4 enhancer trap lines. The PICK1-expressing cells form a subpopulation of neurons. PICK1 immunoreactivity was neither detected in glutamatergic nor in dopaminergic neurons. Also, we observed PICK1 expression in only a few GABAergic neurons, located in the antennal lobe. In contrast, we detected robust PICK1 immunolabeling of peptidergic neurons in the neuroendocrine system, which express the transcription factor DIMM and the amidating enzyme peptidylglycine-alpha-hydroxylating monooxygenase (PHM). The PICK1-positive cells include neurosecretory cells that produce the insulin-like peptide dILP2. PICK1 expression in insulin-producing cells also occurs in mammals, as it was also observed in a rat insulinoma cell line derived from pancreatic beta-cells. At the subcellular level, PICK1 was found in the perinuclear zone but surprisingly not in synaptic domains. We conclude that PICK1 may serve an important role in the neuroendocrine system both in insects and vertebrates.

About 150 clock neurons are clustered in different groups in the brain of Drosophila. Among these clock neurons, some pigment-dispersing factor (PDF)-positive and PDF-negative lateral neurons (LNs) are principal oscillators responsible for bouts of activity in the morning and evening, respectively. The full complement of neurotransmitters in these morning and evening oscillators is not known. By using a screen for candidate neuromediators in clock neurons, we discovered ion transport peptide (ITP) and short neuropeptide F (sNPF) as novel neuropeptides in subpopulations of dorsal (LN(d)s) and ventral (s-LN(v)s) LNs. Among the six LN(d)s, ITP was found in one that coexpresses long neuropeptide F (NPF) and cryptochrome. We detected sNPF in two LN(d)s that also express cryptochrome; these cells are distinct from three LN(d)s expressing NPF. Thus, we have identified neuropeptides in five of the six LN(d)s. The three LN(d)s expressing cryptochrome, with either ITP or sNPF, are the only ones with additional projections to the accessory medulla. Among the five s-LN(v)s in the adult brain, ITP was detected in the fifth neuron that is devoid of PDF and sNPF in the four neurons that also express PDF. By using a choline acetyltransferase (Cha) Gal4, we detected Cha expression in the two sNPF producing LN(d)s and in the fifth s-LN(v). In the larval brain, two of the four PDF-producing s-LN(v)s coexpress sNPF. Our findings emphasize that the LN(d)s are heterogeneous both anatomically and with respect to content of neuropeptides, cryptochrome, and other markers and suggest diverse functions of these neurons.

By use of newly developed enzyme immunoassays for ecdysteroids (ECD) and crustacean cardioactive peptide (CCAP) it was found (1) that in the isopod Oniscus asellus a peak occurs of hemolymph (HL)-ECD contents in prcmoult stages followed by a second but smaller peak 1 h after posterior ecdysis, and (2) that concomitantly with a drop in the first ECD-peak, CCAP-contcnts of the ventral nerve cord rise tenfold basal level around posterior ecdysis. This suggests that CCAP plays a role in isopod moulting processes.

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

Insulin signaling in Drosophila regulates major physiological processes such as stress resistance, growth, carbohydrate and lipid metabolism, aging, reproduction and possibly diapause. Adipokinetic hormone (AKH) signaling originating from the corpora cardiaca (CC) cells, is another crucial regulator of hemolymph carbohydrate levels in Drosophila. These two systems are suggested to have opposing effects on lipid and sugar metabolism in Drosophila, reminiscent of the mammalian insulin and glucagon hormones. We studied the possible functional relationship between the insulin producing cells (IPCs) and the CC cells by interfering with insulin receptor (InR) and AKH receptor (AKH-R) expression on these cells. Our experiments revealed increased carbohydrate and lipid levels after knocking down InR in the CC cells. We also showed that diminished InR levels on the IPCs lead to increased starvation resistance, however we did not observe any changes in carbohydrate or lipid levels. So far we can only suggest action of insulins via its receptor on the IPCs and the CC cells, but we do not have conclusive data for AKH action on IPCs or CC cells.

Insulin/IGF-like signaling regulates the development, growth, fecundity, metabolic homeostasis, stress resistance and lifespan in worms, flies and mammals. Eight insulin-like peptides (DILP1-8) are found in Drosophila. Three of these (DILP2, 3 and 5) are produced by a set of median neurosecretory cells (insulin-producing cells, IPCs) in the brain. Activity in the IPCs of adult flies is regulated by glucose and several neurotransmitters and neuropeptides. One of these, short neuropeptide F (sNPF), regulates food intake, growth and Dilp transcript levels in IPCs via the sNPF receptor (sNPFR1) expressed on IPCs. Here we identify a set of brain neurons that utilizes sNPF to activate the IPCs. These sNPF-expressing neurons (dorsal lateral peptidergic neurons, DLPs) also produce the neuropeptide corazonin (CRZ) and have axon terminations impinging on IPCs. Knockdown of either sNPF or CRZ in DLPs extends survival in flies exposed to starvation and alters carbohydrate and lipid metabolism. Expression of sNPF in DLPs in the sNPF mutant background is sufficient to rescue wild-type metabolism and response to starvation. Since CRZ receptor RNAi in IPCs affects starvation resistance and metabolism, similar to peptide knockdown in DLPs, it is likely that also CRZ targets the IPCs. Knockdown of sNPF, but not CRZ in DLPs decreases transcription of Dilp2 and 5 in the brain, suggesting different mechanisms of action on IPCs of the two co-released peptides. Our findings indicate that sNPF and CRZ co-released from a small set of neurons regulate IPCs, stress resistance and metabolism in adult Drosophila.

Background: The adaptive significance of phenotypic changes elicited by environmental conditions experienced early in life has long attracted attention in evolutionary biology. In this study, we used Drosophila melanogaster to test whether the developmental diet produces phenotypes better adapted to cope with similar nutritional conditions later in life. To discriminate among competing hypotheses on the underlying nature of developmental plasticity, we employed a full factorial design with several developmental and adult diets. Specifically, we examined the effects of early- and late-life diets (by varying their yeast and sugar contents) on reproductive fitness and on the amount of energy reserves (fat and glycogen) in two wild-caught populations.

Results: We found that individuals that had developed on either low-yeast or high-sugar diet showed decreased reproductive performance regardless of their adult nutritional environment. The lower reproductive fitness might be caused by smaller body size and reduced ovariole number. Overall, these results are consistent with the silver spoon concept, which posits that development in a suboptimal environment negatively affects fitness-associated traits. On the other hand, the higher amount of energy reserves (fat) in individuals that had developed in a suboptimal environment might represent either an adaptive response or a side-effect of compensatory feeding.

Conclusion: Our findings suggest that the observed differences in the adult physiology induced by early-life diet likely result from inevitable and general effects of nutrition on the development of reproductive and metabolic organs, rather than from adaptive mechanisms.

Whether the character of developmental plasticity is adaptive or non-adaptive has often been a matter of controversy. Although thermal developmental plasticity has been studied in Drosophila for several traits, it is not entirely clear how it affects reproductive fitness. We, therefore, investigated how developmental temperature affects reproductive performance (early fecundity and egg-to-adult viability) of wild-caught Drosophila melanogaster. We have tested competing hypotheses on the character of developmental thermal plasticity using a full factorial design with three developmental and adulthood temperatures within the natural thermal range of this species. To account for potential intraspecific differences, we examined flies from tropical (India) and temperate (Slovakia) climate zones. Our results show that flies from both populations raised at intermediate developmental temperature (25°C) have comparable or higher early fecundity and fertility at all tested adulthood temperatures, while lower (17°C) or higher developmental temperatures (29°C) did not entail any advantage under the tested thermal regimes. Importantly, the superior thermal performance of flies raised at 25°C is apparent even after taking two traits positively associated with reproductive output into account – body size and ovariole number. Thus, in Drosophila melanogaster, development at a given temperature does not necessarily provide any advantage at this thermal environment in terms of reproductive fitness. Our findings strongly support the optimal developmental temperature hypothesis which claims that at different thermal environments the highest fitness is achieved when an organism is raised at its optimal developmental temperature.

We reveal the neuroanatomy of the optic ganglia and central brain in the water flea Daphnia magna by use of classical neuroanatomical techniques such as semi-thin sectioning and neuronal backfilling, as well as immunohistochemical markers for synapsins, various neuropeptides and the neurotransmitter histamine. We provide structural details of distinct neuropiles, tracts and commissures, many of which were previously undescribed. We analyse morphological details of most neuron types, which allow for unravelling the connectivities between various substructural parts of the optic ganglia and the central brain and of ascending and descending connections with the ventral nerve cord. We identify 5 allatostatin-A-like, 13 FMRFamide-like and 5 tachykinin-like neuropeptidergic neuron types and 6 histamine-immunoreactive neuron types. In addition, novel aspects of several known pigment-dispersing hormone-immunoreactive neurons are re-examined. We analyse primary and putative secondary olfactory pathways and neuronal elements of the water flea central complex, which displays both insect- and decapod crustacean-like features, such as the protocerebral bridge, central body and lateral accessory lobes. Phylogenetic aspects based upon structural comparisons are discussed as well as functional implications envisaging more specific future analyses of ecotoxicological and endocrine disrupting environmental chemicals.

This study aims to assess effects of 96 h goldfish exposure to 1, 10 and 100 mg/L of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), on metabolic indices and free radical process markers in white muscle of a commercial fish, the goldfish Carassius auratus L. Most oxidative stress markers and antioxidant enzymes were not affected at 2,4-D fish treatment. 2,4-D fish exposure induced the elevated levels of total (by 46% and 40%) and reduced (by 77% and 73%) glutathione in muscles of goldfish of 10 mg/L 2,4-D and recovery (after 100 mg/L of 2,4-D exposure) groups, respectively. However, in muscles of 100 mg/L 2,4-D exposed goldfish these parameters were depleted (by 47% and 64%). None of investigated parameters of protein and carbohydrate metabolisms changed in white muscles of 2,4-D exposed fish, with exception of lactate dehydrogenase activity, which was slightly (by 11-15%) elevated in muscles of goldfish exposed to 10-100 mg/L of 2,4-D, but also recovered. Thus, the short term exposure of goldfish to the selected concentrations of 2,4-D does not substantially affect their white muscle, suggesting the absence of any effect under the environmentally relevant concentrations. .

Many animals adjust their reproductive behaviour according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signalling, which regulates many aspects of insect physiology and behaviour, also affects reproduction in females. We show that insulin signalling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide 5 (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signalling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, we identified fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor. Together, our results suggest that the interaction of insulin peptides determines the tuning of female sexual behaviour, either by acting on pheromone perception or directly in the central nervous system.