Evolutionary significance of the variation in acoustic communication of a cryptic nocturnal primate radiation (Microcebus spp.)

Acoustic phenotypic variation is of major importance for speciation and the evolution of species diversity. Whereas selective and stochastic forces shaping the acoustic divergence of signaling systems are well studied in insects, frogs, and birds, knowledge on the processes driving acoustic phenotypic evolution in mammals is limited. We quantified the acoustic variation of a call type exchanged during agonistic encounters across eight distinct species of the smallest‐bodied nocturnal primate radiation, the Malagasy mouse lemurs. The species live in two different habitats (dry forest vs. humid forest), differ in geographic distance to each other, and belong to four distinct phylogenetic clades within the genus. Genetically defined species were discriminated reliably on the phenotypic level based on their acoustic distinctiveness in a discriminant function analysis. Acoustic variation was explained by genetic distance, whereas differences in morphology, forest type, or geographic distance had no effect. The strong impact of genetics was supported by a correlation between acoustic and genetic distance and the high agreement in branching pattern between the acoustic and molecular phylogenetic trees. In sum, stochastic factors such as genetic drift best explained acoustic diversification in a social communication call of mouse lemurs.     

Two calcium‐binding chaperones from the fat body of the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin‐type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N‐globular domain, a P‐arm domain, and a highly charged C‐terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause‐related expression analysis in the whole body revealed an upregulation of both genes by post‐diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause‐related expression pattern in the entire body for both genes. Fat body‐specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post‐diapause. This study suggests that calcium‐binding chaperones play similar and possibly gender‐specific roles during diapause.     

Ultra‐Conserved Elements and morphology reciprocally illuminate conflicting phylogenetic hypotheses in Chalcididae (Hymenoptera,Chalcidoidea)

Recent technical advances combined with novel computational approaches have promised the acceleration of our understanding of the tree of life. However, when it comes to hyperdiverse and poorly known groups of invertebrates, studies are still scarce. As published phylogenies will be rarely challenged by future taxonomists, careful attention must be paid to potential analytical bias. We present the first molecular phylogenetic hypothesis for the family Chalcididae, a group of parasitoid wasps, with a representative sampling (144 ingroups and seven outgroups) that covers all described subfamilies and tribes, and 82% of the known genera. Analyses of 538 Ultra‐Conserved Elements (UCEs) with supermatrix (RAx ML and IQTREE) and gene tree reconciliation approaches (ASTRAL, ASTRID) resulted in highly supported topologies in overall agreement with morphology but reveal conflicting topologies for some of the deepest nodes. To resolve these conflicts, we explored the phylogenetic tree space with clustering and gene genealogy interrogation methods, analyzed marker and taxon properties that could bias inferences and performed a thorough morphological analysis (130 characters encoded for 40 taxa representative of the diversity). This joint analysis reveals that UCEs enable attainment of resolution between ancestry and convergent/divergent evolution when morphology is not informative enough, but also shows that a systematic exploration of bias with different analytical methods and a careful analysis of morphological features is required to prevent publication of artifactual results. We highlight a GC content bias for maximum‐likelihood approaches, an artifactual mid‐point rooting of the ASTRAL tree and a deleterious effect of high percentage of missing data (>85% missing UCEs) on gene tree reconciliation methods. Based on the results we propose a new classification of the family into eight subfamilies and ten tribes that lay the foundation for future studies on the evolutionary history of Chalcididae.     

Parental care in the Small Tree Finch Camarhynchus parvulus in relation to parasitism and environmental factors

The parental food compensation hypothesis suggests that parents may compensate for the negative effects of parasites on chicks by increased food provisioning. However, this ability differs widely among host species and may also depend on ecological factors such as adverse weather conditions and habitat quality. Although weed management can improve habitat quality, management measures can bring about a temporary decrease in food availability and thus may reduce parents’ ability to provide their nestlings with enough energy. In our study we investigated the interaction of parasitism and weed management, and the influence of climate on feeding rates in a Darwin’s tree finch species, which is negatively impacted by two invasive species. The larvae of the invasive parasitic fly Philornis downsi ingest the blood and body tissues of tree finch nestlings, and the invasive Blackberry Rubus niveus affects one of the main habitats of Darwin’s tree finches. We compared parental food provisioning of the Small Tree Finch Camarhynchus parvulus in parasitized and parasite‐free nests in three different areas, which differed in invasive weed management (no management, short‐term and long‐term management). In a parasite reduction experiment, we investigated whether the Small Tree Finch increases food provisioning rates to nestlings when parasitized and whether this ability depends on weed management conditions and precipitation. Our results provide no evidence that Small Tree Finches can compensate with additional food provisioning when parasitized with P. downsi. However, we found an increase in male effort in the short‐term management area, which might indicate that males compensate for lower food quality with increased provisioning effort. Furthermore, parental food provisioning was lower during rainfall, which provides an explanation for the negative influence of rain on breeding success found in earlier studies. Like other Darwin’s finches, the Small Tree Finch seems to lack the ability to compensate for the negative effects of P. downsi parasitism, which is one explanation for why this invasive parasite has such a devastating effect on this host species.     

Sustainable control of mosquito larvae in the field by the combined actions of the biological insecticide Bti and natural competitors

Integrated management of mosquitoes is becoming increasingly important, particularly in relation to avoiding recolonization of ponds after larvicide treatment. We conducted for the first time field experiments that involved exposing natural populations of the mosquito species Culex pipiens to: a) application of the biological insecticide Bacillus thuringiensis israelensis (Bti), b) the introduction of natural competitors (a crustacean community composed mainly of Daphnia spp.), or c) a combined treatment that involved both introduction of a crustacean community and the application of Bti. The treatment that involved only the introduction of crustaceans had no significant effect on mosquito larval populations, while treatment with Bti alone caused only a significant reduction in the abundance of mosquito larvae in the short‐term (within 3–10 days after treatment). In contrast, the combined treatment rapidly reduced the abundance of mosquito larvae, which remained low throughout the entire observation period of 28 days. Growth of the introduced crustacean communities was favored by the immediate reduction in the abundance of mosquito larvae following Bti administration, thus preventing recolonization of ponds by mosquito larvae at the late period (days 14–28 after treatment). Both competition and the temporal order of establishment of different species are hence important mechanisms for efficient and sustainable mosquito control.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Nematode succession at deep-sea hydrothermal vents after a recent volcanic eruption with the description of two dominant species

Nematodes are very common in the deep sea and are an important component of deep-sea hydrothermal vent communities. In early 2006, the eruption of the underwater volcano at 9°50’N East Pacific Rise wiped out almost the entire faunal communities of the area. This provided us with the opportunity to study nematode primary succession at vents as well as on adjacent seafloor basalt. Nematode abundance and richness were extremely low at all studied sites in late 2006 and 2007, and increased only slightly in 2009. Interestingly, the most abundant species during early succession were also prominent in this area prior to the eruption. Our results show that nematodes are extremely influenced by volcanic eruptions and need a long period of time to colonize the lava-flooded area in greater numbers and richness. We hypothesize that low food availability on the young bare basalt and harsh environmental conditions at early succession vent sites might hinder a more successful nematode establishment. In addition to the newly established active vent sites we also studied an inactive vent site that was not directly hit by the eruption but whose vent fluid had ceased after the eruption. At this inactive and older vent, diversity was also relatively low but was higher than at the younger, newly established sites. In addition to the ecological analyses, we here describe the two most abundant species found at inactive vents, namely Neochromadora aff. poecilosoma De Mann 1893 and Linhomoeus caudipapillosus sp. n.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Munc13-Like skMLCK Variants Cannot Mimic the Unique Calmodulin Binding Mode of Munc13 as Evidenced by Chemical Cross-Linking and Mass Spectrometry

Among the neuronal binding partners of calmodulin (CaM) are Munc13 proteins as essential presynaptic regulators that play a key role in synaptic vesicle priming and are crucial for presynaptic short-term plasticity. Recent NMR structural investigations of a CaM/Munc13-1 peptide complex have revealed an extended structure, which contrasts the compact structures of most classical CaM/target complexes. This unusual binding mode is thought to be related to the presence of an additional hydrophobic anchor residue at position 26 of the CaM binding motif of Munc13-1, resulting in a novel 1-5-8-26 motif. Here, we addressed the question whether the 1-5-8-26 CaM binding motif is a Munc13-related feature or whether it can be induced in other CaM targets by altering the motif''s core residues. For this purpose, we chose skeletal muscle myosin light chain kinase (skMLCK) with a classical 1-5-8-14 CaM binding motif and constructed three skMLCK peptide variants mimicking Munc13-1, in which the hydrophobic anchor amino acid at position 14 was moved to position 26. Chemical cross-linking between CaM and skMLCK peptide variants combined with high-resolution mass spectrometry yielded insights into the peptides'' binding modes. This structural comparison together with complementary binding data from surface plasmon resonance experiments revealed that skMLCK variants with an artificial 1-5-8-26 motif cannot mimic CaM binding of Munc13-1. Apparently, additional features apart from the spacing of the hydrophobic anchor residues are required to define the functional 1-5-8-26 motif of Munc13-1. We conclude that Munc13 proteins display a unique CaM binding behavior to fulfill their role as efficient presynaptic calcium sensors over broad range of Ca²⁺ concentrations.