Disentangling host,pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogen''s distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.     

Fluorescent histochemical studies on the effects of 6-hydroxydopamine on adrenaline-containing nerves in the toad

Summary Fluorescence histochemistry has been used to study the effects of 6-hydroxydopamine (6-OHDA) (100 mg/kg injected into the dorsal lymph sac) on adrenaline-containing nerves in the large intestine, mesentery, lung, bladder and heart atria of the toad Bufo marinus. A gradual decrease both in fluorescence intensity and in number of detectable fibres during the first 4 hours after 6-OHDA was accompanied by a build-up of fluorescence in the nonterminal regions. These phenomena have been discussed in relation to the time course of the degeneration produced by 6-OHDA in noradrenergic nerves of higher vertebrates. Almost complete chemical sympathectomy was seen after one day, and it was not till 13 days that regenerating nerve fibres were seen in any organ. In the large intestine, however, re-innervation was slower, being first noted after 39 days. The time course of regeneration has been compared with that following sympathectomy in various mammalian organs.     

Bottom‐up effects of fungicides on tadpoles of the European common frog (Rana temporaria)

Biodiversity is under pressure worldwide, with amphibians being particularly threatened. Stressors related to human activity, such as chemicals, are contributing to this decline. It remains, however, unclear whether chemicals exhibiting a fungicidal activity could indirectly affect tadpoles that depend on microbially conditioned leaf litter as food source. The indirect effect of fungicides (sum concentration of a fungicide mixture composed of azoxystrobin, carbendazim, cyprodinil, quinoxyfen, and tebuconazole: 100 µg/L) on tadpoles was assessed relative to leaf litter colonized by microbes in absence of fungicides (control) and a worst‐case scenario, that is leached leaf litter without microbial colonization. The quality of leaf litter as food for tadpoles of the European common frog (Rana temporaria) was characterized through neutral lipid fatty acid profiles and microbial sum parameters and verified by sublethal responses in tadpoles (i.e., feeding rate, feces production, growth, and fatty acid composition). Fungicides changed the nutritious quality of leaf litter likely through alterations in leaves’ neutral lipid fatty acid profiles (i.e., changes in some physiologically important highly unsaturated fatty acids reached more than 200%) in combination with a potential adsorption onto leaves during conditioning. These changes were reflected by differences in the development of tadpoles ultimately resulting in an earlier start of metamorphosis. Our data provide a first indication that fungicides potentially affect tadpole development indirectly through bottom‐up effects. This pathway is so far not addressed in fungicide environmental risk assessment and merits further attention.     

Critical,pupal and adult weights in the size related metamorphosis of the black lyre leafroller ‘Cnephasia’ jactatana

Adult reproductive performance is linked to the period of feeding done by the final instar larva after attainment of a larval critical weight (L_CW). The highest weight attained by a final instar larva is referred to as the larval maximum weight (L_MW) and is the onset of the pre-pupal period. The relationships between L_CW, pupal weight (P_W) and adult weight (A_W) are described as functions of the L_MW. In the leafroller Cnephasia jactatana (Walker) (Lepidoptera: Tortricidae) L_CW was dependent on larval size and was approximately 75% of the mean L_MW. L_CW was about 29 mg and 36 mg for male and female larvae of 1.18 mm and 1.20 mm head-capsule width, respectively. Over three successive generations of laboratory rearing, P_W was approximately 30% and 25% lower than the L_MW for males and females, respectively. A_W was consistently about 50% and 40% lower than the P_W for males and females, respectively. The decrease in weight from L_MW to P_W was named as the constant D_P and found to be 0.3 for males and 0.25 for females. The total decrease from L_MW to A_W was the constant D_A and was 0.6 for males and 0.5 for females. The duration of the latent feeding period was positively correlated to P_W and A_W. L_CW may be used to derive quality indices that describe and predict pupal and adult performance.

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Résumé Les performances reproductives sont liées à la période d'alimentation du dernier stade larvaire après l'obtention d'un poids critique (L_CW). Le poids maximal atteint au dernier stade larvaire est défini comme le poids larvaire maximal (L_MW), il correspond au début de la période prénymphale. Les relations entre poids de la chrysalide (P_W) et poids de l'audulte A_W) sont présentées comme des fonctions de L_MW). Chez Cnephasia jactatana Walker (Lep. Tortricidae), L_CW dépend de la taille de la chenille et correspond à environ 75% de la valeur moyenne de L_MW. L_CW est respectivement de 29 mg et 36 mg pour les chenilles mâles et femelles dont les capsules céphaliques ont 1,18 et 1,20 mm. Pour 3 générations successives, P_W est environ 30% et 25% inférieur à L_MW des mâles et des femelles. La régression du poids de L_MW à P_W est désignée comme la constante D_P et vaut 0,3 pour les mâles et 0,25 pour les femelles. La régression de L_MW à A_W est désignée comme la régression D_M et vaut 0,6 pour les mâles et 0,5 pour les femelles. La durée de la période d'alimentation latente est liée positivement à P_W et A_W. L_CW peut être utilisé comme indice dérivé décrivant et prédisant les potentialités nymphales et imaginales.

     

Why the term “larva” is ambiguous,or what makes a larva?

The term “larva” is used for many different metazoans. Although this implies a uniform meaning, the term has in fact been used to address immatures with very different characteristics. For providing more precise reference how the term larva is applied in a specific context, I outline here different criteria that have been used to identify an immature as a larva. These include larvae that (a) differ morphologically from their adult (morpho-larva s. l.); (b) differ morphologically from their adult and additionally possess structures that become reduced during ontogeny (morpho-larva s. str.); (c) have a different ecological niche than their adult (eco-larva s. l.); (d) have a different ecological niche than their adult and additionally fulfil a dispersal function (eco-larva s. str.); (e) transform by a metamorphosis to the non-larval immature or adult (metamorph-larva); (f) differ from the adult by having evolved new structures in the early stages (apo-larva); (g) differ from the adult as the adult has evolved new structures (plesio-larva). The differentiation of these criteria will provide a more precise reference reducing possible misunderstanding and allowing a more precise communication.     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways.