Cryptic invasion of Italian pool frogs (<Emphasis Type="Italic">Pelophylax bergeri</Emphasis>) across Western Europe unraveled by multilocus phylogeography

Human introductions of exotic amphibians can have catastrophic effects on native species. However, they usually remain unnoticed without genetic tools when species are difficult to distinguish morphologically. In Western Europe, pool frogs (Pelophylax sp.) make a worrisome case: recent genetic data showed the presence of Italian (Pelophylax bergeri) mtDNA haplotypes within the range of the threatened European Pelophylax lessonae, two morphologically similar taxa. Here we conduct a multilocus phylogeographic and population genetic survey of European and Italian pool frogs (combining present and historic samples), to investigate the origin(s) and consequences of potential introductions. Results are unequivocal: we show that the alien P. bergeri have extensively invaded France and north-Alpine Switzerland, and have also deeply introgressed with P. lessonae, which has led to the complete replacement of most populations. Alien specimens have probably been translocated multiple times from Central Italy at least prior to the 1960s. Based on our dense sampling, only two areas, north and south of the Alps still host native pool frogs in Switzerland, the Joux Valley near the French border and the canton of Ticino, respectively. Importantly, we show that these last P. lessonae populations are remnants of a private genetic diversity specific to Western Europe, which vanished during the P. bergeri’s invasion. Our study emphasizes the risk of genetic pollution during invasion by human-introduced taxa and brings alarming concern regarding uncontrolled amphibian translocations. Moreover, it demonstrates the necessity for genetic surveys to detect and monitor these invasions, especially where species determination is problematic.     

<Emphasis Type="Italic">Parapharyngodon hugoi</Emphasis> n. sp., a new nematode (Oxyuroidea: Pharyngodonidae) of the tree frog <Emphasis Type="Italic">Trachycephalus typhonius</Emphasis> (Linnaeus) from the Brazilian Pantanal,including a key to the congeners from amphibians of the American continent

A new nematode Parapharyngodon hugoi n. sp. (Oxyuroidea: Pharyngodonidae) is described parasitising the large intestine of the tree frog Trachycephalus typhonius (Linnaeus) (Anura: Hylidae) from the wetlands of Pantanal, State of Mato Grosso do Sul, Brazil. The new species exhibits a unique structure of the posterior cloacal lip in males, which is supported by a rigid V-shaped structure. Parapharyngodon hylidae parasitic in hylid frogs, including T. typhonius, from Mexico, is the most similar congener to P. hugoi n. sp. but is distinguished from the new species by the presence of a gubernaculum (vs absence), by the lateral alae in males ending far anterior to cloacal opening (vs near to it) and because in gravid females the ovaries encircle the oesophageal corpus. Additionally, the new species differs from its congeners as well as from species of Thelandros Wedl, 1862, a very closely related genus, by the combination of features such as spicule length, number of caudal papillae, morphology of the anterior cloacal lip, which is echinate, and position of ovaries. The geographical distribution of hosts seems to play an important role in the speciation process of Parapharyngodon spp.; however, due the lack of molecular data this issue along with the validity of both Thelandros and Parapharyngodon are still questions to be solved in the future, after improvement of the genetic database. A key to the species of Parapharyngodon parasitic in amphibians from the American continent is provided.     

<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and the Decline and Survival of the Relict Leopard Frog

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.     

Functional analysis for gut microbes of the brown tree frog (<Emphasis Type="Italic">Polypedates megacephalus</Emphasis>) in artificial hibernation

Background

Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.

Methods

In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.

Results

Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.

Conclusions

We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

     

Variation in the Presence of Anti-<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Bacteria of Amphibians Across Life Stages and Elevations in Ecuador

Amphibian populations are decreasing worldwide due to a variety of factors. In South America, the chytrid fungus Batrachochytrium dendrobatidis (Bd) is linked to many population declines. The pathogenic effect of Bd on amphibians can be inhibited by specific bacteria present on host skin. This symbiotic association allows some amphibians to resist the development of the disease chytridiomycosis. Here, we aimed (1) to determine for the first time if specific anti-Bd bacteria are present on amphibians in the Andes of Ecuador, (2) to monitor anti-Bd bacteria across developmental stages in a focal amphibian, the Andean marsupial tree frog, Gastrotheca riobambae, that deposits larvae in aquatic habitats, and (3) to compare the Bd presence associated with host assemblages including 10 species at sites ranging in biogeography from Amazonian rainforest (450 masl) to Andes montane rainforest (3200 masl). We sampled and identified skin-associated bacteria of frogs in the field using swabs and a novel methodology of aerobic counting plates, and a combination of morphological, biochemical, and molecular identification techniques. The following anti-Bd bacteria were identified and found to be shared among several hosts at high-elevation sites where Bd was present at a prevalence of 32.5%: Janthinobacterium lividum, Pseudomonas fluorescens, and Serratia sp. Bd were detected in Gastrotheca spp. and not detected in the lowlands (sites below 1000 masl). In G. riobambae, recognized Bd-resistant bacteria start to be present at the metamorphic stage. Overall bacterial abundance was significantly higher post-metamorphosis and on species sampled at lower elevations. Further metagenomic studies are needed to evaluate the roles of host identity, life-history stage, and biogeography of the microbiota and their function in disease resistance.     

Risk reduction strategies in <Emphasis Type="Italic">Branchipus schaefferi</Emphasis> (Crustacea: Anostraca: Branchiopoda) as adaptation to a variable environment

The effect of long-term changes in total precipitation on physical and chemical parameters of the water and the structure of phytoplankton community during a year were studied in a deep hard water lake. With respect to total precipitation, two different periods were distinguished: dry and wet. In the wet period, the water level rose and caused an increase in the water colour and a decrease in the electrolytic conductivity and concentration of nutrients. These changes were reflected in the composition and amount of phytoplankton. Certain phytoplankton groups, e.g. Cryptophyceae, Dinophyceae, Chrysophyceae, Bacillariophyceae and Chlorophyta/Charophyta, were positively affected by the environmental changes; instead, these effects were not observed in Cyanobacteria and Euglenophyta. The development of flagellates, such as Cryptomonas curvata, Plagioselmis nannoplanctica and Ceratium hirundinella, was enhanced during the wet period, whereas the dry period favoured non-flagellates, such as Planktothrix agardhii, Planktothrix rubescens and Limnothrix planctonica. Hence, the long-term variability in total precipitation can be a switch from non-flagellate- to flagellate-dominated phytoplankton in lakes. Moreover, a short time of ice cover duration in winter linked with the wet period promoted phytoplankton groups typical for spring and autumn, e.g. Cryptophyceae and Bacillariophyceae.     

Taxonomy and First Records of Two <Emphasis Type="Italic">Megaselia</Emphasis> Rondani Species (Diptera: Phoridae) Preying upon Eggs of <Emphasis Type="Italic">Phyllomedusa iheringii</Emphasis> Boulenger (Anura: Phyllomedusidae)

Non-aquatic reproductive modes have evolved among frogs possibly favored by some advantages such as the avoidance of aquatic predators. These reproductive modes, however, make the egg clutches susceptible to terrestrial predators, among which Diptera larvae are some of the most harmful. The present work reports the predation by phorid flies of 22 egg clutches of Phyllomedusa iheringii Boulenger in the South of Brazil. Phorid specimens were identified as Megaselia bruchiana (Borgmeier & Schmitz) and Megaselia necrophaga (Enderlein), species that were reported previously to be associated with ants and dead beetles, respectively. Frog-feeding in these species is hypothesized to be use of an alternative seasonal food source. We amend the diagnoses of both Megaselia species and provide new illustrations to facilitate their identification. We also describe the male of M. bruchiana for the first time and associate males with females of both species.     

A new species of <Emphasis Type="Italic">Kiluluma</Emphasis> Skrjabin, 1916 (Nematoda: Strongyloidea) from the white rhinoceros, <Emphasis Type="Italic">Ceratotherium simum</Emphasis> (Burchell) from South Africa

A new species of Kiluluma Skrjabin, 1916, Kiluluma ornata n. sp., is described from the intestine of the white rhinoceros Ceratotherium simum (Burchell) from South Africa. The new species is virtually identical with a species described as Kiluluma sp., but not named due to a paucity of material then available. The new species most closely resembles K. solitaria Thapar, 1924 and K. ceratotherii Beveridge & Jabbar, 2013, in the possession of an undulating anterior margin of the buccal capsule but differs in the leaf crown elements which have bulbous, lip-like expansions, which are lacking in K. solitaria and K. ceratotherii.     

A new Species of <Emphasis Type="Italic">Ceratozamia</Emphasis> (Zamiaceae) from the Sierra Norte of Puebla,Mexico

Recent collections of Ceratozamia from the Sierra Norte of Puebla, Mexico, point to a new undescribed species, which was previously subsumed under the concept of C. mexicana. Identification of this new species resulted from a review of three associated species: C. mexicana, C. tenuis, and C. delucana. Here we describe Ceratozamia totonacorum and provide information regarding its distribution and natural history. The Ceratozamia species from the Sierra Madre Oriental, Mexico, are circumscribed using morphological characters, including a detailed evaluation of reproductive structures such as the apex, position, color and shape of the ovulate strobilus.     

Autecology of a new species of carnivorous marsupial,the endangered black-tailed dusky antechinus (<Emphasis Type="Italic">Antechinus arktos</Emphasis>), compared to a sympatric congener,the brown antechinus (<Emphasis Type="Italic">Antechinus stuartii</Emphasis>)

The carnivorous marsupial Antechinus are one of the few mammal genera known to exhibit the phenomenon of semelparous reproduction, where all males die at the end of a frenetic annual mating period. The genus Antechinus has recently been revised, seeing four new species named and one existing subspecies raised to species status. Here, we present the first ecological assessment of one new species, the endangered black-tailed dusky antechinus (Antechinus arktos), based on a 2-year mark-recapture study of two proximate sites within the cloud forest of Springbrook National Park, south-east Queensland, Australia. We also present comparative ecological data from a sympatric congener, Antechinus stuartii. In total, 103 A. arktos (49 male; 54 female) and 2125 A. stuartii (1229 male; 896 female) captures were made over 16,630 trap nights at the two sites. The occurrence and synchrony of reproductive events observed in A. arktos closely parallel the semelparous reproductive strategy exhibited by all congeners studied to date. The A. arktos populations mate during mid-September, with birth of up to six young occurring during mid-October. The low abundance and fecundity, together with a limited and apparently retracting distribution, suggest A. arktos is under threat of climate-induced extinction in the coming decades.     

Influence of Trichomes in Strawberry Cultivars on the Feeding Behavior of <Emphasis Type="Italic">Chaetosiphon fragaefolii</Emphasis> (Cockerell) (Hemiptera: Aphididae)

Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae) is the predominant aphid in strawberry (Fragaria × ananassa Duchesne) production systems in Brazil. This pest species directly damages the plants and is also responsible for spreading viruses. Further, C. fragaefolii often renders strawberry cultivation unviable, because of its high reproductive rate, as well as the large number of individuals generated through parthenogenesis. The present study aimed to (1) evaluate the feeding behavior of C. fragaefolii in four strawberry cultivars (Albion, Aromas, Camarosa, and San Andreas) and (2) identify the resistance factors associated with the number and type of trichomes in the cultivars, and also its effect on the feeding behavior of C. fragaefolii, using the electrical penetration graph (EPG) technique. The results revealed an intrinsic relationship between the number of trichomes on the cultivar and feeding behavior of C. fragaefolii. A higher number of trichomes, both tector and glandular, was observed in Albion compared to that of other cultivars, resulting in a longer no probing (Np) period per insect, and a longer Np phase. A relatively short phloem phase and ingestion time of the phloem sieve elements were also observed in Albion. These results suggest that the trichomes act as a physical barrier creating difficulties for C. fragaefolii to feed, thereby altering its feeding behavior in the four cultivars studied.     

Recent Emergence of a Chytrid Fungal Pathogen in California Cascades Frogs (<Emphasis Type="Italic">Rana cascadae</Emphasis>)

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has been associated with global amphibian declines, but it is often difficult to discern the relative importance of Bd as a causal agent in declines that have already occurred. Retrospective analyses of museum specimens have allowed researchers to associate the timing of Bd arrival with the timing of past amphibian declines. Cascades frogs (Rana cascadae) have experienced dramatic declines in northern California, but it is not clear whether the onset of these declines corresponds to the arrival of Bd. We used quantitative real-time PCR assays of samples collected from museum specimens to determine historical Bd prevalence in the northern California range of Cascades frogs. We detected Bd in 13 of 364 (3.5%) Cascades frog specimens collected between 1907 and 2003, with the first positive result from 1978. A Bayesian analysis suggested that Bd arrived in the region between 1973 and 1978, which corresponds well with the first observations of declines in the 1980s.     

Life table demography of <Emphasis Type="Italic">Asplanchna brightwellii</Emphasis> Gosse, 1850 fed with five different prey items

We conducted life table experiments on the freshwater rotifer Asplanchna brightwellii to analyze its demography when fed with prey items from several taxonomic groups (cladocerans, protozoans, and rotifers) and under two different temperature regimes (20 and 25°C); the aim of the study was to determine the preferred prey for A. brightwellii in terms of fitness (evaluated as reproductive success) among five cladoceran, protozoan, and rotifer preys, and to test which temperature (20 or 25°C) is better for life table parameters of Asplanchna. Our analysis identified Brachionus calyciflorus as the preferred prey for A. brightwellii based on life table statistics, ingestion rate and electivity indices. The greatest values for net reproductive rate and intrinsic growth rate were achieved when A. brightwellii was fed B. calyciflorus. Greater reproductive values (R _o and r) were found at 25°C than at 20°C for A. brightwellii across the five prey species. We found significant differences in the ingestion rate and electivity index among zooplanktonic and benthic preys. The influence of temperature, the cost of predation, and how prey selection by A. brightwellii is influenced by: biomass, size, and swimming speed; they are discussed hoping to gain a better understanding of trophic transfers in zooplankton communities.     

<Emphasis Type="Italic">Batrachochytrium salamandrivorans</Emphasis> and the Risk of a Second Amphibian Pandemic

Amphibians are experiencing devastating population declines globally. A major driver is chytridiomycosis, an emerging infectious disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Bd was described in 1999 and has been linked with declines since the 1970s, while Bsal is a more recently discovered pathogen that was described in 2013. It is hypothesized that Bsal originated in Asia and spread via international trade to Europe, where it has been linked to salamander die-offs. Trade in live amphibians thus represents a significant threat to global biodiversity in amphibians. We review the current state of knowledge regarding Bsal and describe the risk of Bsal spread. We discuss regional responses to Bsal and barriers that impede a rapid, coordinated global effort. The discovery of a second deadly emerging chytrid fungal pathogen in amphibians poses an opportunity for scientists, conservationists, and governments to improve global biosecurity and further protect humans and wildlife from a growing number of emerging infectious diseases.     

The Structure and Density of Aggregations of the Hermit Crab <Emphasis Type="Italic">Pagurus proximus</Emphasis> Komai, 2000 in Epibiosis of the Brown Alga <Emphasis Type="Italic">Costaria costata</Emphasis> (C. Agardh) De A. Saunders, 1895 in the Sublittoral Zone of Vostok Bay,Sea of Japan

A study of epibiosis of the brown alga Costaria costata in the sublittoral zone of Vostok Bay, Sea of Japan during the spring and summer of 2016 showed that hermit crabs of the following four species accounted for approximately 85% of the total biomass: Pagurus proximus, P. minutus, P. brachiomastus, and P. middendorffii. Of these, the most abundant one (96.5% of the total number of hermit crabs) was P. proximus, the mean aggregation density of which on C. costata (429 ± 221 ind./m²) was higher by an order of magnitude than that in the adjacent area of the bouldery bottom. The size ranges of males and females of this species in aggregations on boulders and those on C. costata were similar, with a predominance of larger individuals on algae. In the size–frequency distribution, three groups of individuals corresponding to three size–age cohorts were differentiated. The male-to-female ratio was 1 : 1.7. Hermit crab aggregations on C. costata are ephemeral; their existence is determined by the duration of the period of algae development, that is, from late spring to the middle of the summer.     

On the Biology of the Colombian Fluted Scale, <Emphasis Type="Italic">Crypticerya multicicatrices</Emphasis> Kondo &amp; Unruh (Hemiptera: Monophlebidae)

The Colombian fluted scale Crypticerya multicicatrices Kondo & Unruh (Hemiptera: Monophlebidae) is a polyphagous insect pest native to continental Colombia and reported on 148 species, mainly palms (Arecaceae); mango, Mangifera indica (Anacardiaceae); and leguminous trees and shrubs (Fabaceae). The biology and ecological parameters of C. multicicatrices were investigated using as a host plant Caesalpinia pluviosa var. peltophoroides (Fabaceae) under two environmental conditions: semifield (max temp., 31.5?±?3.0°C; max. RH, 78.9?±?6.0%) and a glass house (max. temp., 37.1?±?5.1°C, max. RH, 67.0?±?6.2%) in the premises of Corpoica, Palmira Research Station. The duration of the different developmental stages was highly different under both temperature conditions. First- and third-instar nymphs, the period of ovisac growth of the adult, and the duration of the life cycle were significantly higher in the semifield conditions compared to the glass house conditions. In contrast, the second-instar nymph stage was shorter in the semifield conditions. The duration of the fourth-instar stage (adult) and the reproductive period were the same in both environments. As for the evaluated ecological parameters, a great difference was found between the two environmental conditions, where the net reproductive rate (R _o) and mean generation time (T) were significantly higher in the semifield conditions than in the glass house conditions. The obtained biological and ecological information is an essential tool for finding strategies to control this insect pest. This is the first detailed study on the biology of a species in the genus Crypticerya Cockerell.     

Association analysis of the SNP (rs345476947) in the <Emphasis Type="Italic">FUT2</Emphasis> gene with the production and reproductive traits in pigs

The FUT2 gene was considered as an important candidate for pathogenic infections, while the potential associations between this gene and the production and reproductive traits of pigs have not been explored. In this study, we detected the genetic variants of porcine FUT2 gene and analyzed the associations of the polymorphisms with FUT2 mRNA expression and production and reproductive traits (age at 100 kg, backfat thickness at 100 kg, eye muscle thickness, the number of newborn piglets, the number of weaned piglets, and birth weight) in 100 Large White sows. One single nucleotide polymorphism (SNP) (rs345476947, C→T) in the intron of FUT2 and three genotypes (TT, CT and CC) were determined. Association analysis revealed significant associations between this SNP with the number of newborn piglets and weaned piglets. Furthermore, individuals with the TT genotype had significantly higher numbers of newborn piglets and weaned piglets than those with the CC genotype (P?<?0.05). Quantitative PCR analysis showed that FUT2 expression in individuals with CC genotype was significantly higher than those with TT and CT genotypes in the liver and lymph gland (P?<?0.05) and higher than that of CT in the spleen, kidney, and duodenum (P?<?0.05). These findings indicated that the TT genotype may be a favorable genotype for the reproductive traits of pigs. Our study revealed the genetic variants of the FUT2 gene and identified a promising candidate SNP (rs345476947) associated with the reproductive traits, which has the potential to be applied in selective breeding of pigs.