Kenneth Halberg | University of Glasgow (original) (raw)
Papers by Kenneth Halberg
Developmental Cell
Highlights d Autophagy regulates steroid production d Autophagosomes sequester and traffic choles... more Highlights d Autophagy regulates steroid production d Autophagosomes sequester and traffic cholesterol substrate for steroid production d The Warts effector microRNA bantam regulates autophagy via the EcR and Tor pathways d Autophagy-dependent steroid regulation adjusts growth to nutrient availability
Journal of Morphology, 2009
The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means... more The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means of fluorescent-coupled phalloidin in combination with confocal laser scanning microscopy and computeraided three-dimensional reconstruction, in addition to light microscopy (Nomarski), scanning electron microscopy, and transmission electron microscopy (TEM). The somatic musculature of H. crispae is composed of structurally independent muscle fibers, which can be divided into a dorsal, ventral, dorsoventral, and a lateral musculature. Moreover, a distinct leg musculature is found. The number and arrangement of muscles differ in each leg. Noticeably, the fourth leg contains much fewer muscles when compared with the other legs. Buccopharyngeal musculature (myoepithelial muscles), intestinal musculature, and cloacal musculature comprise the animal's visceral musculature. TEM of stylet and leg musculature revealed ultrastructural similarities between these two muscle groups. Furthermore, microtubules are found in the epidermal cells of both leg and stylet muscle attachments. This would indicate that the stylet and stylet glands are homologues to the claw and claw glands, respectively. When comparing with previously published data on both heterotardigrade and eutardigrade species, it becomes obvious that eutardigrades possess very similar numbers and arrangement of muscles, yet differ in a number of significant details of their myoanatomy. This study establishes a morphological framework for the use of muscular architecture in elucidating tardigrade phylogeny.
Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships... more Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships of Tardigrada. Here, we present new insight into the morphology of the brain in a marine arthrotardigrade, Acti-narctus doryphorus, based on transmission electron microscopy, supported by scanning electron microscopy, conventional light microscopy as well as confocal laser scanning microscopy. Arthrotardigrades contain a large number of plesiomorphic characters and likely represent ancestral tardigrades. They often have segmented body outlines and each trunk segment, with its paired set of legs, may have up to five sensory appendages. Noticeably, the head carries numerous cephalic appendages that are structurally equivalent to the sensory appendages of the trunk segments. Our data reveal that the brain of A. dory-phorus is partitioned into three paired lobes, and that these lobes exhibit a more pronounced separation as compared to that of eutardigrades. The first brain lobe in A. doryphorus is located anteriodorsally, with the second lobe just below it in an anterioventral position. Both of these two paired lobes are located anterior to the buccal tube. The third pair of brain lobes are situated posterioventrally to the first two lobes, and flank the buccal tube. In addition , A. doryphorus possesses a subpharyngeal ganglion, which is connected with the first of the four ventral trunk ganglia. The first and second brain lobes in A. doryphorus innervate the clavae and cirri of the head. The innerva-tions of these structures indicate a homology between, respectively, the clavae and cirri of A. doryphorus and the temporalia and papilla cephalica of eutardigrades. The third brain lobes innervate the buccal lamella and the stylets as described for eutardigrades. Collectively, these findings suggest that the head region of extant tardi-grades is the result of cephalization of multiple segments. Our results on the brain anatomy of Actinarctus dorypho-rus support the monophyly of Panarthropoda.
Peptides, Jan 17, 2016
Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in ins... more Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways. Finally, alt...
Insect Biochemistry and Molecular Biology, 2015
The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response... more The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism.
Insect Biochemistry and Molecular Biology, 2015
Renal function is essential to maintain homeostasis. This is particularly significant for insects... more Renal function is essential to maintain homeostasis. This is particularly significant for insects that undergo complete metamorphosis; larval mosquitoes must survive a freshwater habitat whereas adults are terrestrial, and mature females must maintain ion and fluid homeostasis after blood feeding. To investigate the physiological adaptations required for successful development to adulthood, we studied the Malpighian tubule transcriptome of Anopheles gambiae using Affymetrix arrays. We assessed transcription under several conditions; as third instar larvae, as adult males fed on sugar, as adult females fed on sugar, and adult females after a blood meal. In addition to providing the most detailed transcriptomic data to date on the Anopheles Malpighian tubules, the data provide unique information on the renal adaptations required for the switch from freshwater to terrestrial habitats, on gender differences, and on the contrast between nectar-feeding and haematophagy. We found clear differences associated with ontogenetic change in lifestyle, gender and diet, particularly in the neuropeptide receptors that control fluid secretion, and the water and ion transporters that impact volume and composition. These data were also combined with transcriptomics from the Drosophila melanogaster tubule, allowing meta-analysis of the genes which underpin tubule function across Diptera. To further investigate renal conservation across species we selected four D. melanogaster genes with orthologues highly enriched in the Anopheles tubules, and generated RNAi knockdown flies. Three of these genes proved essential, showing conservation of critical functions across 150 million years of phylogenetic separation. This extensive data-set is available as an online resource, MozTubules.org, and could potentially be mined for novel insecticide targets that can impact this critical organ in this pest species.
Nature communications, 2015
Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, o... more Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuro...
PLoS ONE, 2013
Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted t... more Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted to handle large fluctuations in physical and chemical parameters. Tardigrades are microscopic ecdysozoans well known for their ability to endure hostile conditions, such as complete desiccation -a phenomenon called anhydrobiosis. During dehydration, anhydrobiotic animals undergo a series of anatomical changes. Whether this reorganization is an essential regulated event mediated by active controlled processes, or merely a passive result of the dehydration process, has not been clearly determined. Here, we investigate parameters pivotal to the formation of the so-called "tun", a state that in tardigrades and rotifers marks the entrance into anhydrobiosis. Estimation of body volume in the eutardigrade Richtersius coronifer reveals an 87 % reduction in volume from the hydrated active state to the dehydrated tun state, underlining the structural stress associated with entering anhydrobiosis. Survival experiments with pharmacological inhibitors of mitochondrial energy production and muscle contractions show that i) mitochondrial energy production is a prerequisite for surviving desiccation, ii) uncoupling the mitochondria abolishes tun formation, and iii) inhibiting the musculature impairs the ability to form viable tuns. We moreover provide a comparative analysis of the structural changes involved in tun formation, using a combination of cytochemistry, confocal laser scanning microscopy and 3D reconstructions as well as scanning electron microscopy. Our data reveal that the musculature mediates a structural reorganization vital for anhydrobiotic survival, and furthermore that maintaining structural integrity is essential for resumption of life following rehydration.
Journal of Experimental Biology, 2012
We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotu... more We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1mmoll -1 ; 81% reduction), the Na + /K + -ATPase inhibitor ouabain (10mmoll -1 ; 21% reduction) and the vacuolar H + -ATPase inhibitor bafilomycin (5mmoll -1 ; 21% reduction), and by the organic anions PAH (10mmoll -1 ; 44% reduction) and probenecid (10mmoll -1 ; 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na + /K + -ATPase -subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H + and possibly basal Na + /K + pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades.
Journal of Experimental Biology, 2013
Journal of Experimental Biology, 2009
Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study,... more Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around -20°C, revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350-500 μm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg -1 . Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.
Marine Biology Research, 2013
Tardigrades form an important component of meiofaunal communities across the globe. However, our ... more Tardigrades form an important component of meiofaunal communities across the globe. However, our knowledge on tardigrade ecology is very limited. Here, we report the results of 21 field samplings of the marine tardigrade Halobiotus crispae collected over a period of 74 months at the locality of Vellerup Vig, Denmark, with the aim of providing novel insights into its ecology. Uniquely, H. crispae is characterized by the presence of seasonal cyclic changes in the phenotype of the animal, i.e. cyclomorphosis. Our sampling data include (i) total number of animals extracted, (ii) dominant cyclomorphic stage found, and (iii) important environmental parameters such as temperature, salinity and pH. Our accumulated data constitute a tentative model for the annual fluctuations in animal density, which reveals an annual peak in abundance during the months of February and March. In contrast, tardigrade density appears to decrease in response to increasing temperatures during late spring/early summer. The thermal tolerance of H. crispae was therefore investigated experimentally for the pseudosimplex 2 stage, active stage and pseudosimplex 1 stage, revealing LT min values of 24.68C, 14.28C and 24.78C, LT 50 values of 30.68C, 29.68C and 30.88C, and LT max values of 358C, 35.88C and 358C, respectively. In general, our thermal tolerance data reveal that (i) the entry into the pseudosimplex 1 stage represents an adaptive strategy that enables the survival of the Vellerup Vig population during periods of heat stress, and (ii) temperature is an important factor in limiting the geographic distribution of H. crispae.
Journal of Zoological Systematics and Evolutionary Research, 2011
Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state te... more Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In the anhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in their natural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selected species of limno-terrestrial tardigrades. The primary focus was to assess the effect of cosmic radiation. This was achieved through the RoTaRad (Rotifers, Tardigrades and Radiation) project on the BIOPAN 6 mission, funded by Agenzia Spaziale Italiana under the European Space Agency. To test their tolerance of space environment, tardigrades were sent into low earth orbit, and exposed to cosmic radiation and a microgravity environment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer just after returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmic radiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo were desiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to the applied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in the laboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades in anhydrobiosis are unaffected by these conditions.
Journal of Morphology, 2012
The position of Tardigrada in the animal tree of life is a subject that has received much attenti... more The position of Tardigrada in the animal tree of life is a subject that has received much attention, but still remains controversial. Whereas some think tardigrades should be categorized as cycloneuralians, most authors argue in favor of a phylogenetic position within Panarthropoda as a sister group to Arthropoda or Arthropoda 1 Onychophora. Thus far, neither molecular nor morphological investigations have provided conclusive results as to the tardigrade sister group relationships. In this article, we present a detailed description of the nervous system of the eutardigrade Halobiotus crispae, using immunostainings, confocal laser scanning microscopy, and computer-aided three-dimensional reconstructions supported by transmission electron microscopy. We report details regarding the structure of the brain as well as the ganglia of the ventral nerve cord. In contrast to the newest investigation, we find transverse commissures in the ventral ganglia, and our data suggest that the brain is partitioned into at least three lobes. Additionally, we can confirm the existence of a subpharyngeal ganglion previously called subesophagal ganglion. According to our results, the original suggestion of a brain comprised of at least three parts cannot be rejected, and the data presented supports a sister group relationship of Tardigrada to 1) Arthropoda or 2) Onychophora or 3) Arthropoda 1 Onychophora.
Journal of Limnology, 2013
Little is known about the genetic structure of microscopic animals from mosses and lichens. A few... more Little is known about the genetic structure of microscopic animals from mosses and lichens. A few studies have investigated the geographic variation in tardigrades from mosses, but so far no study has investigated the intra-population or local clonal lineage variation. Echiniscus testudo (Echiniscoidea: Echiniscidae) belongs to a large cosmopolitan genus of terrestrial tardigrades comprising more than 150 species. It is a common tardigrade in mosses in the temperate part of the Northern hemisphere, and is highly tolerant of desiccation and freezing. In a previous study, we reported a maximum of 1.28% sequence variation (uncorrected p-distance) in cytochrome c oxidase subunit I (COI) haplotypes between clonal lineages covering a large geographical area. However, in this previous study we used pooled specimens to constitute a sample, and the genetic diversity from single specimens within a locality therefore remains unknown. Accordingly, the present study investigates the COI sequence variation and haplotype diversity between single specimens of E. testudo collected at three Danish localities, separated by 80 m and 186 km. A total of 10 COI haplotypes were found in the present study (Et2, Et3, Et9,; only three of these were previously reported (Et2, Et3 and Et9). The uncorrected COI sequence diversity ranged between 0-2.07%, with haplotype Et18 having the highest genetic difference. The second most variable haplotypes (Et14, Et15, and Et17) all showed a maximum diversity of 1.19% compared to the other haplotypes. No general pattern of haplotype distribution was evident. Our data suggest that E. testudo has dispersed across the Baltic sea as haplotypes Et3, Et13 and Et14 are present at all three localities. The most likely dispersal mode is passive wind dispersal in the cryptobiotic tun stage. The current study emphasises that numerous sequences from single specimens are needed to describe the genetic diversity within single moss cushions.
Journal of Limnology, 2007
Halobiotus crispae is a marine eutardigrade belonging to Hypsibiidae. A characteristic of this sp... more Halobiotus crispae is a marine eutardigrade belonging to Hypsibiidae. A characteristic of this species is the appearance of seasonal cyclic changes in morphology and physiology, i.e. cyclomorphosis. Halobiotus crispae was originally described from Nipisat Bay, Disko Island, Greenland. The present study investigates the distribution of this species and describes the seasonal appearance of cyclomorphic stages at the southernmost locality, Vellerup Vig in the Isefjord, Denmark. Our sampling data indicate that the distribution of H. crispae is restricted to the Northern Hemisphere where we now have found this species at seven localities. At Vellerup Vig data from sampling cover all seasons of the year and all of the originally described cyclomorphic stages have been found at this locality. However, when comparing the lifecycles of H. crispae at Nipisat Bay and Vellerup Vig, profound differences are found in the time of year, as well as the period in which these stages appear. Noticeably, at Nipisat Bay the pseudosimplex 1 stage is a hibernating stage occurring during the long Arctic winter. In contrast, at Vellerup Vig, this stage appears during the summer. Thus, while pseudosimplex 1 seems to be an adaptation to withstand low temperatures in Greenland, this stage possibly enables the animal to tolerate periods of oxygen depletion and heat stress during the Danish summer. Moreover, a characteristic of the Danish population is the presence of a prolonged pseudosimplex 2 stage. The environmental or endogenous signals underlying the transition between different stages remain unknown. In addition, we report the genetic diversity and phylogenetic position of H. crispae based on the first molecular data obtained from this species. Our molecular data confirm that H. crispae from Greenland and Denmark are in fact the same species. Thus, the observed life cycle changes occur within a species and do not represent life cycle variation between different species. In addition, our molecular data suggest that Halobiotus has evolved within Isohypsibius. Further investigations on the lifecycle of members of the Halobiotus genus as well as other members of the Hypsibiidae is needed in order to establish whether cyclomorphosis is i) a general theme among members of Hypsibiidae or ii) an autapomorphy for Halobiotus.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008
map of tardigrades in active stage, applying proteomic techniques such as high-resolution two-dim... more map of tardigrades in active stage, applying proteomic techniques such as high-resolution two-dimensional polyacrylamide gel electrophoresis and sequencing of peptides with ESI-MS/MS. In the second part of the project our studies will be extended to a comparison of the proteomes of tardigrades in cryptobiotic stage versus those in active stage. With quantification methods like two-dimensional difference gel electrophoresis (2D-DIGE) we will be able to detect differences in protein expression, which are characteristic for active and cryptobiotic stages. The identification and characterisation of proteins, which play important roles in these two stages, could be the first step to understand dynamic processes in cryptobiotic tardigrades.
BMC Developmental Biology, 2010
Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephro... more Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model.
Acta Physiologica, 2011
Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems.... more Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, as do the two major invertebrate model organisms: Caenorhabditis elegans and Drosophila melanogaster. We present a brief description of the tardigrades and highlight species that are currently used as models for physiological and molecular investigations. Tardigrades are uniquely adapted to a range of environmental extremes. Cryptobiosis, currently referred to as a reversible ametabolic state induced by e.g. desiccation, is common especially among limno-terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be extremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below )20°C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation.
Developmental Cell
Highlights d Autophagy regulates steroid production d Autophagosomes sequester and traffic choles... more Highlights d Autophagy regulates steroid production d Autophagosomes sequester and traffic cholesterol substrate for steroid production d The Warts effector microRNA bantam regulates autophagy via the EcR and Tor pathways d Autophagy-dependent steroid regulation adjusts growth to nutrient availability
Journal of Morphology, 2009
The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means... more The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means of fluorescent-coupled phalloidin in combination with confocal laser scanning microscopy and computeraided three-dimensional reconstruction, in addition to light microscopy (Nomarski), scanning electron microscopy, and transmission electron microscopy (TEM). The somatic musculature of H. crispae is composed of structurally independent muscle fibers, which can be divided into a dorsal, ventral, dorsoventral, and a lateral musculature. Moreover, a distinct leg musculature is found. The number and arrangement of muscles differ in each leg. Noticeably, the fourth leg contains much fewer muscles when compared with the other legs. Buccopharyngeal musculature (myoepithelial muscles), intestinal musculature, and cloacal musculature comprise the animal's visceral musculature. TEM of stylet and leg musculature revealed ultrastructural similarities between these two muscle groups. Furthermore, microtubules are found in the epidermal cells of both leg and stylet muscle attachments. This would indicate that the stylet and stylet glands are homologues to the claw and claw glands, respectively. When comparing with previously published data on both heterotardigrade and eutardigrade species, it becomes obvious that eutardigrades possess very similar numbers and arrangement of muscles, yet differ in a number of significant details of their myoanatomy. This study establishes a morphological framework for the use of muscular architecture in elucidating tardigrade phylogeny.
Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships... more Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships of Tardigrada. Here, we present new insight into the morphology of the brain in a marine arthrotardigrade, Acti-narctus doryphorus, based on transmission electron microscopy, supported by scanning electron microscopy, conventional light microscopy as well as confocal laser scanning microscopy. Arthrotardigrades contain a large number of plesiomorphic characters and likely represent ancestral tardigrades. They often have segmented body outlines and each trunk segment, with its paired set of legs, may have up to five sensory appendages. Noticeably, the head carries numerous cephalic appendages that are structurally equivalent to the sensory appendages of the trunk segments. Our data reveal that the brain of A. dory-phorus is partitioned into three paired lobes, and that these lobes exhibit a more pronounced separation as compared to that of eutardigrades. The first brain lobe in A. doryphorus is located anteriodorsally, with the second lobe just below it in an anterioventral position. Both of these two paired lobes are located anterior to the buccal tube. The third pair of brain lobes are situated posterioventrally to the first two lobes, and flank the buccal tube. In addition , A. doryphorus possesses a subpharyngeal ganglion, which is connected with the first of the four ventral trunk ganglia. The first and second brain lobes in A. doryphorus innervate the clavae and cirri of the head. The innerva-tions of these structures indicate a homology between, respectively, the clavae and cirri of A. doryphorus and the temporalia and papilla cephalica of eutardigrades. The third brain lobes innervate the buccal lamella and the stylets as described for eutardigrades. Collectively, these findings suggest that the head region of extant tardi-grades is the result of cephalization of multiple segments. Our results on the brain anatomy of Actinarctus dorypho-rus support the monophyly of Panarthropoda.
Peptides, Jan 17, 2016
Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in ins... more Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways. Finally, alt...
Insect Biochemistry and Molecular Biology, 2015
The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response... more The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism.
Insect Biochemistry and Molecular Biology, 2015
Renal function is essential to maintain homeostasis. This is particularly significant for insects... more Renal function is essential to maintain homeostasis. This is particularly significant for insects that undergo complete metamorphosis; larval mosquitoes must survive a freshwater habitat whereas adults are terrestrial, and mature females must maintain ion and fluid homeostasis after blood feeding. To investigate the physiological adaptations required for successful development to adulthood, we studied the Malpighian tubule transcriptome of Anopheles gambiae using Affymetrix arrays. We assessed transcription under several conditions; as third instar larvae, as adult males fed on sugar, as adult females fed on sugar, and adult females after a blood meal. In addition to providing the most detailed transcriptomic data to date on the Anopheles Malpighian tubules, the data provide unique information on the renal adaptations required for the switch from freshwater to terrestrial habitats, on gender differences, and on the contrast between nectar-feeding and haematophagy. We found clear differences associated with ontogenetic change in lifestyle, gender and diet, particularly in the neuropeptide receptors that control fluid secretion, and the water and ion transporters that impact volume and composition. These data were also combined with transcriptomics from the Drosophila melanogaster tubule, allowing meta-analysis of the genes which underpin tubule function across Diptera. To further investigate renal conservation across species we selected four D. melanogaster genes with orthologues highly enriched in the Anopheles tubules, and generated RNAi knockdown flies. Three of these genes proved essential, showing conservation of critical functions across 150 million years of phylogenetic separation. This extensive data-set is available as an online resource, MozTubules.org, and could potentially be mined for novel insecticide targets that can impact this critical organ in this pest species.
Nature communications, 2015
Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, o... more Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuro...
PLoS ONE, 2013
Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted t... more Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted to handle large fluctuations in physical and chemical parameters. Tardigrades are microscopic ecdysozoans well known for their ability to endure hostile conditions, such as complete desiccation -a phenomenon called anhydrobiosis. During dehydration, anhydrobiotic animals undergo a series of anatomical changes. Whether this reorganization is an essential regulated event mediated by active controlled processes, or merely a passive result of the dehydration process, has not been clearly determined. Here, we investigate parameters pivotal to the formation of the so-called "tun", a state that in tardigrades and rotifers marks the entrance into anhydrobiosis. Estimation of body volume in the eutardigrade Richtersius coronifer reveals an 87 % reduction in volume from the hydrated active state to the dehydrated tun state, underlining the structural stress associated with entering anhydrobiosis. Survival experiments with pharmacological inhibitors of mitochondrial energy production and muscle contractions show that i) mitochondrial energy production is a prerequisite for surviving desiccation, ii) uncoupling the mitochondria abolishes tun formation, and iii) inhibiting the musculature impairs the ability to form viable tuns. We moreover provide a comparative analysis of the structural changes involved in tun formation, using a combination of cytochemistry, confocal laser scanning microscopy and 3D reconstructions as well as scanning electron microscopy. Our data reveal that the musculature mediates a structural reorganization vital for anhydrobiotic survival, and furthermore that maintaining structural integrity is essential for resumption of life following rehydration.
Journal of Experimental Biology, 2012
We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotu... more We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1mmoll -1 ; 81% reduction), the Na + /K + -ATPase inhibitor ouabain (10mmoll -1 ; 21% reduction) and the vacuolar H + -ATPase inhibitor bafilomycin (5mmoll -1 ; 21% reduction), and by the organic anions PAH (10mmoll -1 ; 44% reduction) and probenecid (10mmoll -1 ; 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalization of the Na + /K + -ATPase -subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H + and possibly basal Na + /K + pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades.
Journal of Experimental Biology, 2013
Journal of Experimental Biology, 2009
Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study,... more Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around -20°C, revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350-500 μm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg -1 . Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.
Marine Biology Research, 2013
Tardigrades form an important component of meiofaunal communities across the globe. However, our ... more Tardigrades form an important component of meiofaunal communities across the globe. However, our knowledge on tardigrade ecology is very limited. Here, we report the results of 21 field samplings of the marine tardigrade Halobiotus crispae collected over a period of 74 months at the locality of Vellerup Vig, Denmark, with the aim of providing novel insights into its ecology. Uniquely, H. crispae is characterized by the presence of seasonal cyclic changes in the phenotype of the animal, i.e. cyclomorphosis. Our sampling data include (i) total number of animals extracted, (ii) dominant cyclomorphic stage found, and (iii) important environmental parameters such as temperature, salinity and pH. Our accumulated data constitute a tentative model for the annual fluctuations in animal density, which reveals an annual peak in abundance during the months of February and March. In contrast, tardigrade density appears to decrease in response to increasing temperatures during late spring/early summer. The thermal tolerance of H. crispae was therefore investigated experimentally for the pseudosimplex 2 stage, active stage and pseudosimplex 1 stage, revealing LT min values of 24.68C, 14.28C and 24.78C, LT 50 values of 30.68C, 29.68C and 30.88C, and LT max values of 358C, 35.88C and 358C, respectively. In general, our thermal tolerance data reveal that (i) the entry into the pseudosimplex 1 stage represents an adaptive strategy that enables the survival of the Vellerup Vig population during periods of heat stress, and (ii) temperature is an important factor in limiting the geographic distribution of H. crispae.
Journal of Zoological Systematics and Evolutionary Research, 2011
Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state te... more Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In the anhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in their natural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selected species of limno-terrestrial tardigrades. The primary focus was to assess the effect of cosmic radiation. This was achieved through the RoTaRad (Rotifers, Tardigrades and Radiation) project on the BIOPAN 6 mission, funded by Agenzia Spaziale Italiana under the European Space Agency. To test their tolerance of space environment, tardigrades were sent into low earth orbit, and exposed to cosmic radiation and a microgravity environment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer just after returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmic radiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo were desiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to the applied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in the laboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades in anhydrobiosis are unaffected by these conditions.
Journal of Morphology, 2012
The position of Tardigrada in the animal tree of life is a subject that has received much attenti... more The position of Tardigrada in the animal tree of life is a subject that has received much attention, but still remains controversial. Whereas some think tardigrades should be categorized as cycloneuralians, most authors argue in favor of a phylogenetic position within Panarthropoda as a sister group to Arthropoda or Arthropoda 1 Onychophora. Thus far, neither molecular nor morphological investigations have provided conclusive results as to the tardigrade sister group relationships. In this article, we present a detailed description of the nervous system of the eutardigrade Halobiotus crispae, using immunostainings, confocal laser scanning microscopy, and computer-aided three-dimensional reconstructions supported by transmission electron microscopy. We report details regarding the structure of the brain as well as the ganglia of the ventral nerve cord. In contrast to the newest investigation, we find transverse commissures in the ventral ganglia, and our data suggest that the brain is partitioned into at least three lobes. Additionally, we can confirm the existence of a subpharyngeal ganglion previously called subesophagal ganglion. According to our results, the original suggestion of a brain comprised of at least three parts cannot be rejected, and the data presented supports a sister group relationship of Tardigrada to 1) Arthropoda or 2) Onychophora or 3) Arthropoda 1 Onychophora.
Journal of Limnology, 2013
Little is known about the genetic structure of microscopic animals from mosses and lichens. A few... more Little is known about the genetic structure of microscopic animals from mosses and lichens. A few studies have investigated the geographic variation in tardigrades from mosses, but so far no study has investigated the intra-population or local clonal lineage variation. Echiniscus testudo (Echiniscoidea: Echiniscidae) belongs to a large cosmopolitan genus of terrestrial tardigrades comprising more than 150 species. It is a common tardigrade in mosses in the temperate part of the Northern hemisphere, and is highly tolerant of desiccation and freezing. In a previous study, we reported a maximum of 1.28% sequence variation (uncorrected p-distance) in cytochrome c oxidase subunit I (COI) haplotypes between clonal lineages covering a large geographical area. However, in this previous study we used pooled specimens to constitute a sample, and the genetic diversity from single specimens within a locality therefore remains unknown. Accordingly, the present study investigates the COI sequence variation and haplotype diversity between single specimens of E. testudo collected at three Danish localities, separated by 80 m and 186 km. A total of 10 COI haplotypes were found in the present study (Et2, Et3, Et9,; only three of these were previously reported (Et2, Et3 and Et9). The uncorrected COI sequence diversity ranged between 0-2.07%, with haplotype Et18 having the highest genetic difference. The second most variable haplotypes (Et14, Et15, and Et17) all showed a maximum diversity of 1.19% compared to the other haplotypes. No general pattern of haplotype distribution was evident. Our data suggest that E. testudo has dispersed across the Baltic sea as haplotypes Et3, Et13 and Et14 are present at all three localities. The most likely dispersal mode is passive wind dispersal in the cryptobiotic tun stage. The current study emphasises that numerous sequences from single specimens are needed to describe the genetic diversity within single moss cushions.
Journal of Limnology, 2007
Halobiotus crispae is a marine eutardigrade belonging to Hypsibiidae. A characteristic of this sp... more Halobiotus crispae is a marine eutardigrade belonging to Hypsibiidae. A characteristic of this species is the appearance of seasonal cyclic changes in morphology and physiology, i.e. cyclomorphosis. Halobiotus crispae was originally described from Nipisat Bay, Disko Island, Greenland. The present study investigates the distribution of this species and describes the seasonal appearance of cyclomorphic stages at the southernmost locality, Vellerup Vig in the Isefjord, Denmark. Our sampling data indicate that the distribution of H. crispae is restricted to the Northern Hemisphere where we now have found this species at seven localities. At Vellerup Vig data from sampling cover all seasons of the year and all of the originally described cyclomorphic stages have been found at this locality. However, when comparing the lifecycles of H. crispae at Nipisat Bay and Vellerup Vig, profound differences are found in the time of year, as well as the period in which these stages appear. Noticeably, at Nipisat Bay the pseudosimplex 1 stage is a hibernating stage occurring during the long Arctic winter. In contrast, at Vellerup Vig, this stage appears during the summer. Thus, while pseudosimplex 1 seems to be an adaptation to withstand low temperatures in Greenland, this stage possibly enables the animal to tolerate periods of oxygen depletion and heat stress during the Danish summer. Moreover, a characteristic of the Danish population is the presence of a prolonged pseudosimplex 2 stage. The environmental or endogenous signals underlying the transition between different stages remain unknown. In addition, we report the genetic diversity and phylogenetic position of H. crispae based on the first molecular data obtained from this species. Our molecular data confirm that H. crispae from Greenland and Denmark are in fact the same species. Thus, the observed life cycle changes occur within a species and do not represent life cycle variation between different species. In addition, our molecular data suggest that Halobiotus has evolved within Isohypsibius. Further investigations on the lifecycle of members of the Halobiotus genus as well as other members of the Hypsibiidae is needed in order to establish whether cyclomorphosis is i) a general theme among members of Hypsibiidae or ii) an autapomorphy for Halobiotus.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008
map of tardigrades in active stage, applying proteomic techniques such as high-resolution two-dim... more map of tardigrades in active stage, applying proteomic techniques such as high-resolution two-dimensional polyacrylamide gel electrophoresis and sequencing of peptides with ESI-MS/MS. In the second part of the project our studies will be extended to a comparison of the proteomes of tardigrades in cryptobiotic stage versus those in active stage. With quantification methods like two-dimensional difference gel electrophoresis (2D-DIGE) we will be able to detect differences in protein expression, which are characteristic for active and cryptobiotic stages. The identification and characterisation of proteins, which play important roles in these two stages, could be the first step to understand dynamic processes in cryptobiotic tardigrades.
BMC Developmental Biology, 2010
Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephro... more Background: Three kidney systems appear during vertebrate development: the pronephroi, mesonephroi and metanephroi. The pronephric duct is the first or primary ureter of these kidney systems. Its role as a key player in the induction of nephrogenic mesenchyme is well established. Here we investigate whether the duct is involved in urine modification using larvae of the freshwater amphibian Ambystoma mexicanum (axolotl) as model.
Acta Physiologica, 2011
Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems.... more Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, as do the two major invertebrate model organisms: Caenorhabditis elegans and Drosophila melanogaster. We present a brief description of the tardigrades and highlight species that are currently used as models for physiological and molecular investigations. Tardigrades are uniquely adapted to a range of environmental extremes. Cryptobiosis, currently referred to as a reversible ametabolic state induced by e.g. desiccation, is common especially among limno-terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be extremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below )20°C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation.