Right phenotype, wrong place: predator-induced plasticity is costly in a mismatched environment.

Like many animals, tadpoles often produce different, predator-specific phenotypes when exposed to risk of predation. It is generally assumed that such plasticity enhances survival in the presence of the predator and is costly elsewhere, but evidence remains surprisingly scarce. We measured (1) the survival trade-off of opposing phenotypes developed by Dendropsophus ebraccatus tadpoles when exposed to different predators and (2) which specific aspects of morphology drive any potential survival benefit or cost. Tadpoles developed predator-specific phenotypes after being reared with caged fish or dragonfly predators for two weeks. In 24 h predation trials with either a fish or a dragonfly, survival was highest in the groups with their matched predator, and lowest among with those the mismatched predator, with predator-naive controls being relatively intermediate. Then, using a large group of phenotypically variable predator-naive tadpoles, we found that increased survival rates are directly related to the morphological changes that are induced by each predator. This demonstrates that induced phenotypes are indeed adaptive and the product of natural selection. Furthermore, our data provide clear evidence of an environmental cost for phenotypic plasticity in a heterogeneous environment. Such costs are fundamental for understanding the evolution and maintenance of inducible phenotypes.

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

Survey of trematodes in intertidal snails from Patagonia, Argentina: new larval forms and diversity assessment.

Larval trematodes are the main parasites of snails, and they play a crucial role because they usually castrate their snail hosts and can thus alter their population and community dynamics. This study involved a survey of seven gastropod species (Crepipatella dilatata, Fissurella radiosa, Nacella magellanica, Pareuthria fuscata, Siphonaria lessonii, S. lateralis and Trophon geversianus) parasitized by 12 trematode species (one hemiurid, one gymnophallid, two lepocreadiids, two microphallids, one notocotylid, two renicolids, one philophtalmid, one schistosomatid and one zoogonid) from southern Patagonia (47°S, 65°W), Argentina. Only F. radiosa was free of parasites. The study included the description of five new larvae, based on morphological and molecular information, and a comparison of the parasite diversity with that of a northern locality (42°S, 64°W), characterized by a lower mollusc diversity. Species richness and diversity of parasites were higher in the southern site. This suggests a correlation between the level of parasitism and the diversity of molluscs (first intermediate hosts), which is higher at the high-latitude site and seems to attract shorebirds, which disperse the digenean eggs and facilitate the completion of their life cycles. These results support the notion that parasitism is influenced by large-scale factors such as biogeographical patterns, and small-scale factors such as diversity or abundance of intermediate and definitive hosts.

Assessment of multiple stressors on the growth of larval green sturgeon Acipenser medirostris: implications for recruitment of early life-history stages.

Early developmental stages of fishes are particularly sensitive to changes in environmental variables that affect physiological processes such as metabolism and growth. Both temperature and food availability have significant effects on the growth and survival of larval and juvenile fishes. As climate change and anthropogenic disturbances influence sensitive rearing environments of fishes it is unlikely that they will experience changes in temperature or food availability in isolation. Therefore, it is critical that we determine the effects of each of these potential stressors on larval growth and development, as well as understand the additive, synergistic or antagonistic effects of both. We reared threatened green sturgeon Acipenser medirostris (initial age ca. 32 days post hatch) at four temperatures (11, 13, 16 and 19°C) and two food availability rates (100% and 40% of optimal) to assess the effects of these stressors and their interactions on larval growth. We compared the overall size (fork length, total length and mass), growth rates (cm day-1 and g day-1 ) and relative condition factor of these larval and juvenile fish at 3 week intervals for up to 12 weeks. Our results indicated that temperature and food availability both had significant effects on growth and condition and that there was a significant interaction between the two. Fish reared with limited food availability exhibited similar patterns in growth rates to those reared with elevated food rates, but the effects of temperature were greatly attenuated when fish were food-limited. Also, the effects of temperature on condition were reversed when fish were reared with restricted food, such that fish reared at 19°C exhibited the highest relative condition when fed optimally, but the lowest relative condition when food was limited. These data are critical for the development of relevant bioenergetics models, which are needed to link the survival of larval sturgeons with historic environmental regimes, pinpoint temperature ranges for optimal survival and help target future restoration sites that will be important for the recovery of sturgeon populations.

Noise-Robust Modes of the Retinal Population Code Have the Geometry of "Ridges" and Correspond to Neuronal Communities.

An appealing new principle for neural population codes is that correlations among neurons organize neural activity patterns into a discrete set of clusters, which can each be viewed as a noise-robust population codeword. Previous studies assumed that these codewords corresponded geometrically with local peaks in the probability landscape of neural population responses. Here, we analyze multiple data sets of the responses of approximately 150 retinal ganglion cells and show that local probability peaks are absent under broad, nonrepeated stimulus ensembles, which are characteristic of natural behavior. However, we find that neural activity still forms noise-robust clusters in this regime, albeit clusters with a different geometry. We start by defining a soft local maximum, which is a local probability maximum when constrained to a fixed spike count. Next, we show that soft local maxima are robustly present and can, moreover, be linked across different spike count levels in the probability landscape to form a ridge. We found that these ridges comprise combinations of spiking and silence in the neural population such that all of the spiking neurons are members of the same neuronal community, a notion from network theory. We argue that a neuronal community shares many of the properties of Donald Hebb's classic cell assembly and show that a simple, biologically plausible decoding algorithm can recognize the presence of a specific neuronal community.

Toxicity assessment of 7 anticancer compounds in zebrafish.

Toxicity is one of the major reasons for failure in drug development. Zebrafish, as an ideal vertebrate model, could also be used to evaluate drug toxicity. In this study, we aimed to show the predictability and highlight novel findings of toxicity in zebrafish model. Seven anticancer compounds, including triptolide (TP), gambogic acid (GA), mycophenolic acid (MPA), curcumin, auranofin, thalidomide, and taxol, were assessed in zebrafish for their toxicity. Three compounds (GA, TP, and taxol) showed highest acute lethality, with 50% lethal concentration ≈ 1 µmol/L. Missing tails, severe pericardial edema, and enlarged yolk sacs were observed in MPA-treated embryos. The development of pectoral fins was severely disturbed in thalidomide-, GA-, and TP-treated embryos. Bradycardia was observed in MPA- and thalidomide-treated groups. Our findings suggested that the zebrafish are a good model for toxicity assessment of anticancer compounds.

Low-cost silicone imaging casts for zebrafish embryos and larvae.

Due to their size and optical clarity, zebrafish embryos have long been appreciated for their usefulness in time-lapse confocal microscopy. Current methods of mounting zebrafish embryos and larvae for imaging consist mainly of mounting in low percentage, low melting temperature agarose in a Petri dish. Whereas imaging methods have advanced greatly over the last two decades, the methods for mounting embryos have not changed significantly. In this article, we describe the development and use of 3D printed plastic molds. These molds can be used to create silicone casts and allow embryos and larvae to be mounted with a consistent and reproducible angle, and position in X, Y, and Z. These molds are made on a 3D printer and can be easily and cheaply reproduced by anyone with access to a 3D printer, making this method accessible to the entire zebrafish community. Molds can be reused to create additional casts, which can be reused after imaging. These casts are compatible with any upright microscope and can be adapted for use on an inverted microscope, taking the working distance of the objective used into account. This technique should prove to be useful to any researcher imaging zebrafish embryos.

A new treefrog (Hylidae: Litoria) from Kroombit Tops, east Australia, and an assessment of conservation status.

The Litoria phyllochroa species-group are small hylid frogs that occur in wet forests of south-east Australia. This group has had a long history of taxonomic confusion and has received little attention in the last decade. A population of this species-group at Kroombit Tops, several hundred kilometers north of all other populations, has been recognised for some time as being genetically highly distinct. Here we describe this population as a new species, L. kroombitensis sp. nov. This species is most similar to L. barringtonensis and L. pearsoniana but is readily distinguished based on differences in morphology, colour pattern, mating call and genetics. Litoria kroombitensis sp. nov. is restricted to Kroombit Tops, an isolated area of wet forest in south-east Queensland. The species inhabits slow and intermittently flowing streams in rainforest and adjoining wet sclerophyll forest. The tadpole of L. kroombitensis sp. nov., described herein, is similar in morphology and behaviour to the tadpoles of other species within the Litoria phyllochroa species-group, in particular L. pearsoniana. Li toria kroombitensis sp. nov. has a very small distribution, with all records coming from the headwaters of five streams, Extensive surveys since the mid-1990s have revealed population declines, attributable to amphibian chytrid fungus (Batrachochytrium dendrobatidis). Other threats include degradation of riparian habitat due to invasive weeds, feral pigs and livestock, and fire. Further, the extent of wet forest habitats at Kroombit Tops is likely to be reduced by climate change impacts. Litoria kroombitensis sp. nov. meets IUCN Red List criteria for critically endangered CR B lab (i-v) due to its small geographic range, naturally fragmented distribution, and observed and projected decline in populations. In this paper we also assess the validity of the names L. barringtonensis, L. pearsoniana and L. piperata. We conclude that the names L. barringtonensis and L. pearsoniana are valid but the validity of L. piperata requires further investigation.

Molecular phylogeny and host specificity of the larval Eustrongylides (Nematoda: Dioctophmidae) from freshwater fish in China.

The nematodes Eustrongylides spp. collected from different fish species in China were examined for their intra- and interspecific evolutionary variations using the molecular markers mitochondrial cytochrome oxidase c subunit 1 (COI) gene and internal transcribed spacer (ITS) rDNA regions. The phylogenetic analysis indicated that Eustrongylides species are divided into 3 well-supported clades. The ITS divergence between the clades suggested that clades 2 and 3 might represent the same species, whereas clade 1 represent another cryptic species. The host specificity of these nematodes was analyzed according to prevalence data, host range, and phylogenetic information. Clade 1 was found in 4 fish species, i.e., Odontobutis obscurus, Silurus asotus, Culter mongolicus, and Acanthogobius flavimanus, but was predominant in the 2 perciform species, O. obscurus and A. flavimanus. Clade 2 was found in 3 fish species, Monopterus albus, Channa argus, and Channa asiatica, but was predominant in M. albus, reported to feed primarily on oligochaetes, the first intermediate host of Eustrongylides sp. Clade 3 was found in 9 species, but its low prevalence suggests accidental infection in all species. Although the larval nematode presented low host specificity, it exhibited some host preference.

High stability and no fitness costs of the resistance of codling moth to Cydia pomonella granulovirus (CpGV-M).

Resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV-M) was previously observed in field populations of codling moth (CM, C. pomonella) in South-West Germany. Incidental observations in a laboratory reared field colony (CpR) indicated that this resistance is rather stable, even in genetically heterogeneous CM colonies consisting of both susceptible and resistant individuals. To test this hypothesis, the resistance level of CpR that was 1000times less susceptible to CpGV-M was followed for more than 60 generations of rearing. Even without virus selection pressure, the high level of resistance, expressed as median lethal concentration, remained stable for more than 30 generations and declined only by a factor of 10 after 60 generations. When cohorts of the F32 and F56 generations of the same colony were selected to CpGV-M for five and two generations, respectively, the resistance level increased to factor of >1,000,000 compared to a susceptible control colony. Laboratory reared colonies of CpR, did not exhibit any measurable fitness costs under laboratory conditions in terms of fecundity and fertility. Resistance testing of seven selected codling moth field populations collected between 2003 and 2008 in commercial orchards in Germany that were repeatedly sprayed with CpGV products gave evidence of different levels of resistance and a more than 20-fold increase of the resistance in 1-3 years when selection by CpGV-M was continued. A maximum 1,000,000-fold level of resistance to CpGV-M that could be induced in the laboratory under virus pressure had been also observed in one field population. The high stability of resistance observed in the genetically heterogenous colony CpR indicates that resistance to CpGV-M is not very costly.

Ancestral state reconstruction for Dendroctonus bark beetles: evolution of a tree killer.

While most bark beetles attack only dead or weakened trees, many species in the genus Dendroctonus have the ability to kill healthy conifers through mass attack of the host tree, and can exhibit devastating outbreaks. Other species in this group are able to successfully colonize trees in small numbers without killing the host. We reconstruct the evolution of these ecological and life history traits, first classifying the extant Dendroctonus species by attack type (mass or few), outbreaks (yes or no), host genus (Pinus and others), location of attacks on the tree (bole, base, etc.), whether the host is killed (yes or no), and if the larvae are gregarious or have individual galleries (yes or no). We then estimated a molecular phylogeny for a data set of cytochrome oxidase I sequences sampled from nearly all Dendroctonus species, and used this phylogeny to reconstruct the ancestral state at various nodes on the tree, employing maximum parsimony, maximum likelihood, and Bayesian methods. Our reconstructions suggest that extant Dendroctonus species likely evolved from an ancestor that killed host pines through mass attack of the bole, had individual larvae, and exhibited outbreaks. The ability to colonize a host tree in small numbers (as well as gregarious larvae and attacks at the tree base) apparently evolved later, possibly as two separate events in different clades. It is likely that tree mortality and outbreaks have been continuing features of the interaction between conifers and Dendroctonus bark beetles.

"Vacuum-assisted staining": a simple and efficient method for screening in Drosophila.

The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio).

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Morphological assessment and molecular phylogenetics of the Funestus and Minimus groups of Anopheles (Cellia).

A morphological comparison and molecular study of the Afrotropical Funestus and Afro-Oriental Minimus groups within the Myzomyia series of Anopheles (Cellia) was conducted to determine their phylogenetic affinities. Relationships were investigated using morphological characters and ribosomal (D3) and mitochondrial (COII) nucleotide sequences. Cross-identification of specimens from one group by using keys for the other group confirmed their morphological similarity, i.e., members of one group shared the key characters with members of the other group. Molecular analyses recognized five clades, not strictly related to geographical distribution: the Aconitus, Culicifacies, Funestus, Minimus, and Rivulorum subgroups. Morphological observations were congruent with the results of molecular analyses. Anopheles leesoni, an Afrotropical species, is closely related to the Oriental Minimus complex, and these taxa share a close relationship with the Fluviatilis complex that occurs from the Arabian Peninsula through India. The immature and adult stages of An. rivulorum in Africa bear morphological characters that distinguish this species from members of the Afrotropical Funestus subgroup. A composite scheme of classification based on the results and previously published information is proposed for the two groups. It is noted that An. fluviatilis species S is conspecific with An. minimus species C.

Costs and limits of dosage response to predation risk: to what extent can tadpoles invest in anti-predator morphology?

Inducible defences have long been considered as a polyphenism opposing defended and undefended morphs. However, in nature, preys are exposed to various levels of predation risk and scale their investment in defence to actual predation risk. Still, among the traits that are involved in the defence, some are specific to one predator type while others act as a more generalised defence. The existence of defence costs could prevent an individual investing in all these traits simultaneously. In this study, we investigate the impact of an increasing level of predator density (stickleback, Gasterosteus aculeatus) on the expression of morphological inducible defences in tadpoles of Rana dalmatina. In this species, investment in tail length and tail muscle is a stickleback-specific response while increased tail fin depth is a more general defence. As expected, we found a relationship between investment in defence and level of risk through the responses of tail fin depth and tail length. We also found an exponential increase of defence cost, notably expressed by convex decrease of growth and developmental rates. We found a relative independence of investment in the different traits that compose the defence, revealing a high potential for fine tuning the expression of defended phenotypes with respect to local ecological conditions.

The cost of resistance to Bacillus thuringiensis varies with the host plant of Trichoplusia ni.

Selection for resistance to insecticides, diseases and parasitoids is assumed to be costly and often requires tradeoffs with reproductive fitness. The costs of resistance, however, are often difficult to measure. Cabbage looper, Trichoplusia ni, a generalist Lepidopteran herbivore, has become highly resistant following the extensive use of the microbial insecticide, Bacillus thuringiensis kurstaki (Bt) in vegetable greenhouses. We compared the growth rate, pupal size and survival of resistant, susceptible and hybrid T. ni larvae fed on tomato, bell pepper and cucumber. Performance was best on cucumber and worst on pepper, and the magnitude of fitness costs associated with Bt resistance increased with declining host plant suitability. This supports the hypothesis that in this system, resistance costs are condition dependent and are greatest in the most stressful environment. Management strategies that rely on the presence of fitness costs to reduce the frequency of resistance genes must consider this variation and should be more successful on crops that are less suitable food plants. In general, condition dependence should be considered in studies designed to measure the costs of resistance.

Unraveling the evolutionary radiation of the thoracican barnacles using molecular and morphological evidence: a comparison of several divergence time estimation approaches.

The Thoracica includes the ordinary barnacles found along the sea shore and is the most diverse and well-studied superorder of Cirripedia. However, although the literature abounds with scenarios explaining the evolution of these barnacles, very few studies have attempted to test these hypotheses in a phylogenetic context. The few attempts at phylogenetic analyses have suffered from a lack of phylogenetic signal and small numbers of taxa. We collected DNA sequences from the nuclear 18S, 28S, and histone H3 genes and the mitochondrial 12S and 16S genes (4,871 bp total) and data for 37 adult and 53 larval morphological characters from 43 taxa representing all the extant thoracican suborders (except the monospecific Brachylepadomorpha). Four Rhizocephala (highly modified parasitic barnacles) taxa and a Rhizocephala + Acrothoracica (burrowing barnacles) hypothetical ancestor were used as the outgroup for the molecular and morphological analyses, respectively. We analyzed these data separately and combined using maximum likelihood (ML) under "hill-climbing" and genetic algorithm heuristic searches, maximum parsimony procedures, and Bayesian inference coupled with Markov chain Monte Carlo techniques under mixed and homogeneous models of nucleotide substitution. The resulting phylogenetic trees answered key questions in barnacle evolution. The four-plated Iblomorpha were shown as the most primitive thoracican, and the plateless Heteralepadomorpha were placed as the sister group of the Lepadomorpha. These relationships suggest for the first time in an invertebrate that exoskeleton biomineralization may have evolved from phosphatic to calcitic. Sessilia (nonpedunculate) barnacles were depicted as monophyletic and appear to have evolved from a stalked (pedunculate) multiplated (5+) scalpelloidlike ancestor rather than a five-plated lepadomorphan ancestor. The Balanomorpha (symmetric sessile barnacles) appear to have the following relationship: (Chthamaloidea(Coronuloidea(Tetraclitoidea, Balanoidea))). Thoracican divergence times were estimated under ML-based local clock, Bayesian, and penalized likelihood approaches using an 18S data set and three calibration points: Heteralepadomorpha = 530 million years ago (MYA), Scalpellomorpha = 340 MYA, and Verrucomorpha = 120 MYA. Estimated dates varied considerably within and between approaches depending on the calibration point. Highly parameterized local clock models that assume independent rates (r > or = 15) for confamilial or congeneric species generated the most congruent estimates among calibrations and agreed more closely with the barnacle fossil record. Reasonable estimates were also obtained under the Bayesian procedure of Kishino et al. (2001, Mol. Biol. Evol. 18:352-361) but using multiple calibrations. Most of the dates estimated under the Bayesian procedure of Aris-Brosou and Yang (2002, Syst. Biol. 51:703-714) and the penalized likelihood method using single and/or multiple calibrations were inconsistent among calibrations and did not fit the fossil record.

Caste morphology and development in Termitogeton nr. planus (Insecta, Isoptera, Rhinotermitidae).

The termite family Rhinotermitidae displays a wide diversity in its patterns of social organization in castes. The genus Termitogeton probably branched off early in the evolution of this family. We studied the developmental pathways of a putative undescribed species from New Guinea, Termitogeton nr planus. The development begins with two white inactive instars (larvae), the second of which possesses small wing buds. These are followed by a relatively homogenous group of active immature stages (pseudergates) among which a biometric study revealed the presence of four instars. The first of these instars possesses wing buds that regress at subsequent molts. The external morphology of older instars resembles that of higher termite workers. Older pseudergates can differentiate into presoldiers and then into soldiers, but they are also able to molt into a unique stage with long wing pads (nymph), preceding the imago. Colony maturity can be reached with about 10(3) individuals. The sex ratio is near 1:1 in all castes except soldiers, among which females are more numerous than males. The caste pattern of T. nr. planus is reminiscent of those of the Kalotermitidae and Termopsidae, and of Prorhinotermes among the Rhinotermitidae. None of these taxa possess a true worker caste, permanently diverted from imaginal development: social tasks are done by unspecialized immatures that retain a full array of developmental options, including that of proceeding to the imago. The most remarkable trait of T. nr. planus is the presence in all second-instar individuals of wing buds that later regress to reappear in the single nymphal stage. We suggest that the traditional definitions of the terms larvae, nymphs, and pseudergates should be revised because they cannot be satisfactorily applied to the castes of Termitogeton.