UV radiation affects antipredatory defense traits in Daphnia pulex

In aquatic environments, prey perceive predator threats by chemical cues called kairomones, which can induce changes in their morphology, life histories, and behavior. Predator‐induced defenses have allowed for prey, such as Daphnia pulex, to avert capture by common invertebrate predators, such as Chaoborus sp. larvae. However, the influence of additional stressors, such as ultraviolet radiation (UVR), on the Daphnia–Chaoborus interaction is not settled as UVR may for instance deactivate the kairomone. In laboratory experiments, we investigated the combined effect of kairomones and UVR at ecologically relevant levels on induced morphological defenses of two D. pulex clones. We found that kairomones were not deactivated by UVR exposure. Instead, UVR exposure suppressed induced morphological defense traits of D. pulex juveniles under predation threat by generally decreasing the number of neckteeth and especially by decreasing the size of the pedestal beneath the neckteeth. UVR exposure also decreased the body length, body width, and tail spine length of juveniles, likely additionally increasing the vulnerability to Chaoborus predation. Our results suggest potential detrimental effects on fitness and survival of D. pulex subject to UVR stress, with consequences on community composition and food web structure in clear and shallow water bodies.     

Complex effects of a kairomone of Chaoborus and an insecticide on Daphnia pulex

Daphnia pulex were reared in Chaoborus-conditioned water containingthe insecticide carbaryl, and their life history parametersand morphologies were investigated. The insecticide inhibitedthe animals' growth and reproduction and delayed their maturationtime more intensely in the chaoborus-conditioned water thanin the control Chaoborus-free water, indicating that a kairomoneof Chaoborus made the Daphnia more sensitive to the insecticide.The Chaoborus conditioned water induced neckteeth formationof D.pulex in instars 1–2 and elongated the intermoultingperiod of juveniles. The moulting to the spined morphs and elongationin duration of juvenile stages seemed to increase the risk ofdamage from the insecticide. The potential population growthrate of D.pulex in treatments was estimated as a possible fitnessindicator of the animals. It was reduced synergistically bythe kairomone of Chaoborus and the insecticide. Some individualskept neckteeth until the third or fourth instar stage when theywere exposed to sublethal concentrations of the insecticidein the Chaoborus-conditioned water. This was considered as aresult of synergistic effects of both the kairomone and theinsecticide. Insecticides may be a factor inducing further developmentof protuberant structures in cyclomorphic Daphnia in naturalwater bodies.     

Genetic Diversity of Daphnia pulex in the Middle and Lower Reaches of the Yangtze River

Increased human activities and environmental changes may lead to genetic diversity variations of Cladocerans in water. Daphnia pulex are distributed throughout the world and often regarded as a model organism. The 16S rDNA, cytochrome c oxidase subunit I (COI), and 18S genes were used as molecular marks. The genetic diversity and phylogeny of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River were studied. For 16S rDNA, COI gene, and 18S gene, the A+T content (65.4%, 58.4%, and 54.6%) was significantly higher than the G+C content (34.6%, 41.6% and 45.4%). This result was consistent with higher A and T contents among invertebrates. Based on the genetic distances of 16S rDNA and COI genes, the genetic differences of D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River in China was minimal (0%–0.8% for 16S rDNA and 0%–1.5% for COI gene). However, D. pulex evolved into two branches in the phylogenetic trees, which coincided with its geographical distribution. Compared with D. pulex from other countries, the average genetic distance of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River reached 9.1%–10.5%, thereby indicating that D. pulex may have evolved into different subspecies.     

Predator-induced defences in <Emphasis Type="Italic">Daphnia pulex</Emphasis>: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

Background  

The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.     

Embryology of Chaoborus-induced spines in Daphnia pulex

Daphnia pulex (Crustacea: Cladocera) embryos were found to be sensitive to a chemical cue (kairomone) in an extract of the predator Chaoborus americanus (Insecta:Diptera). Sensitivity of embryos to the kairomone remains throughout embryonic development. Apparently declining sensitivity as development proceeds may be due to the amount of time the embryos are exposed to the kairomone. Male embryos were also found to be sensitive to the kairomone. The smallest eggs within a brood produced small offspring, which showed the antipredator morphology to a significantly lower degree than largest eggs. The production of the neckteeth is described, at the developmental stage in the maturation of the Daphnia coinciding approximately with the escape of the embryos from the brood chamber.     

Social Experience Is Sufficient to Modulate Sleep Need of Drosophila without Increasing Wakefulness

Organisms quickly learn about their surroundings and display synaptic plasticity which is thought to be critical for their survival. For example, fruit flies Drosophila melanogaster exposed to highly enriched social environment are found to show increased synaptic connections and a corresponding increase in sleep. Here we asked if social environment comprising a pair of same-sex individuals could enhance sleep in the participating individuals. To study this, we maintained individuals of D. melanogaster in same-sex pairs for a period of 1 to 4 days, and after separation, monitored sleep of the previously socialized and solitary individuals under similar conditions. Males maintained in pairs for 3 or more days were found to sleep significantly more during daytime and showed a tendency to fall asleep sooner as compared to solitary controls (both measures together are henceforth referred to as “sleep-enhancement”). This sleep phenotype is not strain-specific as it is observed in males from three different “wild type” strains of D. melanogaster. Previous studies on social interaction mediated sleep-enhancement presumed ‘waking experience’ during the interaction to be the primary underlying cause; however, we found sleep-enhancement to occur without any significant increase in wakefulness. Furthermore, while sleep-enhancement due to group-wise social interaction requires Pigment Dispersing Factor (PDF) positive neurons; PDF positive and CRYPTOCHROME (CRY) positive circadian clock neurons and the core circadian clock genes are not required for sleep-enhancement to occur when males interact in pairs. Pair-wise social interaction mediated sleep-enhancement requires dopamine and olfactory signaling, while visual and gustatory signaling systems seem to be dispensable. These results suggest that socialization alone (without any change in wakefulness) is sufficient to cause sleep-enhancement in fruit fly D. melanogaster males, and that its neuronal control is context-specific.     

Morphological defenses induced in situ by the invertebrate predator Chaoborus : comparison of responses between Daphnia pulex and D. rosea

The presence of plankton predators may induce altered morphology in their potential prey. To date, the mechanism of induction and adaptive value of such defensive responses have been examined in the laboratory. This study investigated the morphological defense structures induced by the invertebrate predator Chaoborus in two coexisting Daphnia species, D. pulex and D. rosea, in the field. In Piscivore Lake (Gr?fenhain, Germany), continuous and intense biomanipulation had led to near elimination of planktivorous fish and greatly increased abundances of Chaoborus (up to >10 larvae l^–1). Here, the density of Chaoborus was manipulated within the lake by an enclosure/exclosure setup and resulting morphological responses of Daphnia spp. were investigated in situ. Three replicate enclosures (4.6 m³) contained no Chaoborus (predator exclusion bags), whereas Chaoborus entered three others at ambient densities (predator enclosures). In both species of Daphnia, formation of neckteeth and elongation of the tail spine were recorded in the predator enclosures, but not in the predator exclusion treatments. Additionally, D. rosea responded to predator inclusion with an increase of the size at first reproduction. Despite the induced defense structures, the presence of Chaoborus caused increased mortality of both Daphnia species. In addition, Chaoborus affected the coexistence of the two populations of Daphnia by causing higher relative mortality in D. rosea. Neckteeth formation was always more pronounced in D. pulex than in D. rosea of the same size. Neckteeth were induced specifically in vulnerably sized juvenile instars of D. pulex, but were not found in all vulnerable instars of D. rosea. In D. rosea, neckteeth were few or absent in the ephippial hatchlings, and neckteeth formation ceased before juveniles reached a body size outside the range that larger larval stages of Chaoborus could ingest. This study provides the first experimental demonstration in the field of the inducibility of morphological defense structures in Daphnia at ambient densities of Chaoborus larvae, and quantifies these in situ responses. This expands on earlier observations of a correlation between predator density in the field and the expression of neckteeth in Daphnia. The term ”maximum size for neckteeth formation” (MSNF) is defined as the limit in body size above which no production of neckteeth was evident. This limit was used to distinguish the size classes of Daphnia that show a sensitive response to Chaoborus kairomone. This new term may be used for further comparisons among species and among different types of predator-induced responses as well as for the evaluation of the adaptive value of defense structures. Received: 10 April 1999 / Accepted: 6 April 2000     

Juvenile hormone pathway in honey bee larvae: A source of possible signal molecules for the reproductive behavior of Varroa destructor

The parasitic mite Varroa destructor devastates honey bee (Apis mellifera) colonies around the world. Entering a brood cell shortly before capping, the Varroa mother feeds on the honey bee larvae. The hormones 20‐hydroxyecdysone (20E) and juvenile hormone (JH), acquired from the host, have been considered to play a key role in initiating Varroa''s reproductive cycle. This study focuses on differential expression of the genes involved in the biosynthesis of JH and ecdysone at six time points during the first 30 hr after cell capping in both drone and worker larvae of A. mellifera. This time frame, covering the conclusion of the honey bee brood cell invasion and the start of Varroa''s ovogenesis, is critical to the successful initiation of a reproductive cycle. Our findings support a later activation of the ecdysteroid cascade in honey bee drones compared to worker larvae, which could account for the increased egg production of Varroa in A. mellifera drone cells. The JH pathway was generally downregulated confirming its activity is antagonistic to the ecdysteroid pathway during the larva development. Nevertheless, the genes involved in JH synthesis revealed an increased expression in drones. The upregulation of jhamt gene involved in methyl farnesoate (MF) synthesis came into attention since the MF is not only a precursor of JH but it is also an insect pheromone in its own right as well as JH‐like hormone in Acari. This could indicate a possible kairomone effect of MF for attracting the mites into the drone brood cells, along with its potential involvement in ovogenesis after the cell capping, stimulating Varroa''s initiation of egg laying.     

Fingerprinting of Campylobacter jejuni by Using Resolution-Optimized Binary Gene Targets Derived from Comparative Genome Hybridization Studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with “Minimum SNPs” software. “Minimum SNPs” selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D = 1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

Chaoborus crystallinus predation on Daphnia pulex: can induced morphological changes balance effects of body size on vulnerability?

Juvenile Daphnia pulex form neckteeth in reponse to chemicals released by predatory Chaoborus crystallinus larvae. Formation of neckteeth is strongest in the second instar followed by the third instar, whereas only small neckteeth are found in the first and fourth instar of experimental clones. Predation experiments showed that body-size-dependent vulnerability of animals without neckteeth to fourth instar C. crystallinus larvae matched the pattern of neckteeth formation over the four juvenile instars. Predation experiments on D. pulex of the same clone with neckteeth showed that vulnerability to C. crystallinus predation is reduced, and that the induced protection is correlated with the degree of neckteeth formation. The pattern of neckteeth formation in successive instars is probably adaptive, and it can be concluded that neckteeth are formed to different degrees in successive instars as an evolutionary compromise to balance prediation risk and protective costs.     

Helicobacter Pylori's Plasticity Zones Are Novel Transposable Elements

Background

Genes present in only certain strains of a bacterial species can strongly affect cellular phenotypes and evolutionary potentials. One segment that seemed particularly rich in strain-specific genes was found by comparing the first two sequenced Helicobacter pylori genomes (strains 26695 and J99) and was named a “plasticity zone”.

Principal Findings

We studied the nature and evolution of plasticity zones by sequencing them in five more Helicobacter strains, determining their locations in additional strains, and identifying them in recently released genome sequences. They occurred as discrete units, inserted at numerous chromosomal sites, and were usually flanked by direct repeats of 5′AAGAATG, a sequence generally also present in one copy at unoccupied sites in other strains. This showed that plasticity zones are transposable elements, to be called TnPZs. Each full length TnPZ contained a cluster of type IV protein secretion genes (tfs3), a tyrosine recombinase family gene (“xerT”), and a large (≥2800 codon) orf encoding a protein with helicase and DNA methylase domains, plus additional orfs with no homology to genes of known function. Several TnPZ types were found that differed in gene arrangement or DNA sequence. Our analysis also indicated that the first-identified plasticity zones (in strains 26695 and J99) are complex mosaics of TnPZ remnants, formed by multiple TnPZ insertions, and spontaneous and transposable element mediated deletions. Tests using laboratory-generated deletions showed that TnPZs are not essential for viability, but identified one TnPZ that contributed quantitatively to bacterial growth during mouse infection and another that affected synthesis of proinflammatory cytokines in cell culture.

Conclusions

We propose that plasticity zone genes are contained in conjugative transposons (TnPZs) or remnants of them, that TnPZ insertion is mediated by XerT recombinase, and that some TnPZ genes affect bacterial phenotypes and fitness.     

Morphological responses of four species of cyclomorphic Daphnia to a short-term exposure to the insecticide carbaryl

Four species of cyclomorphic Daphnia (D.pulex, D.galeata mendotae,D.retrocurva, D.lumholtzi) were exposed to the insecticide carbarylfor a short term (8–4 h) from the final embryonic stageto the first instar. Daphnia pulex formed neckteeth, and theremaining three Daphnia species developed high helmets and longtailspines. The results suggest that the development of suchprotuberant structures (anti-predator morphologies) in responseto the insecticide exposure is a general phenomenon in Daphnia,and that stimuli on the nervous system of Daphnia may inducethe morphological changes, which originally evolved as a responseto predator kairomone. Two clones of D.pulcx were examined anda clone which was more sensitive to the predator Chaobonts kairomonethan another developed more marked neckteeth in response tocarbaryl, suggesting that the sensitivity in morphological responseto the insecticide may be related to the sensitivity to thekairomone. ¹Present and permanent address: Regional Environment Division,National Institute for Environmental Studies Onogawa, Tsukuba,Ibaraki 305, Japan     

Analysis of human clinical and environmental Leptospira to elucidate the eco-epidemiology of leptospirosis in Yaeyama,subtropical Japan

BackgroundLeptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama region in Okinawa, subtropical Japan. Species of the P1 subclade “virulent” group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region.FindingsPolymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade “virulent” subgroup and 11 environmental soil isolates of the P1subclade “low virulent” subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal.ConclusionOur study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance.     

Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment

Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “Candidatus Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee, Bombus impatiens. This symbiont is a member of the recently described bacterial order Orbales, which has been collected from the guts of diverse insect species; however, “Ca. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “Ca. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome o, and most genes in the tricarboxylic acid (TCA) cycle. “Ca. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome bd, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory B. impatiens revealed that “Ca. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.     

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

Waterborne metals impair inducible defences in Daphnia pulex: morphology, life-history traits and encounters with predators

1.  Inducible defences may be temporary and favoured where predation is intermittent and have been demonstrated in several invertebrates and vertebrates when prey detect chemical cues (kairomones) released by predators. Daphnia pulex (a water flea) exposed to Chaoborus (midge larvae) kairomones produce small neckteeth on the dorsal surface of the head as a defence against this gape-limited predator and survive better in the presence of Chaoborus . Recent studies have shown that waterborne copper (Cu) impairs the induction of neckteeth which could lead to lower survival.
2.  Here, we examined the effects of Cu on morphological changes and shifts in life-history traits in D. pulex exposed to kairomone from Chaoborus americanus . We exposed D. pulex mothers to chemical cues of C. americanus fed on either D. pulex neonates or on brine shrimp Artemia salina , the same Chaoborus cues combined with an environmentally relevant concentration of copper (10 μg L⁻¹), or dechlorinated tap water. We examined several morphological characteristics of neonates and life-historical characteristics of adults as well as assessing survival of neonates by staging encounters with predators.
3.  Neonates from mothers exposed to kairomone plus copper had fewer and shorter neckteeth than neonates from mothers exposed to kairomone alone. Moreover, neonates exposed to Cu had lower survival during encounters with predators than neonates exposed to kairomone without Cu.
4.  Adult female Daphnia exposed to kairomones released more neonates within the first 24 h of brood release and emptied their brood pouches quicker than mothers not exposed to kairomones, irrespective of the presence of Cu.
5.  Impairment by metals of morphological defences in zooplankton could lead to a decline in population density and alter community structure.