Effects of Hibernation Site, Temperature, and Humidity on the Abundance and Survival of Overwintering Culex pipiens pipiens and Anopheles messeae (Diptera: Culicidae).

Knowledge of the hibernation site preferences and the factors which influence winter survival in these hibernation sites may enhance understanding of mosquito population dynamics after winter and how arboviruses persist in temperate regions. Our study quantified the number of adult overwintering mosquitoes in cellars and aboveground constructions and analyzed survival rates in relation to the environmental conditions in these sites. During the winters 2016/2017 and 2018/2019, 149 different constructions in Northwest Germany were sampled for mosquitoes. Mosquitoes were detected in 44% of the cellars and in 33% of the aboveground constructions. Culex p. pipiens Linnaeus was the most abundant species in cellars, whereas high numbers of Anopheles messeae Falleroni were collected from a single barn. Subsequently, an enclosure study was conducted during 2019/2020. Overwintering field-collected Cx. p. pipiens and An. messeae were divided into groups with or without fructose availability, and placed in cages with different man-made hibernations sites, where temperature and relative humidity were recorded hourly. For both species, increasing mean temperatures (5-16°C) but not mean relative humidity (58-94%) were correlated with winter mortality rates of the mosquitoes. The lipid measurements were greater and mortality rates were lower when both species were provided fructose. Larger specimens (determined by wing length) stored more lipids, and in Cx. p pipiens, but not in An. messeae, survival probability of large specimens was significantly greater than for small females. Mosquitoes showed a distinct pattern in the selection of overwintering sites, while temperature was an important driver for survival.

Habitat and microclimate affect the resting site selection of mosquitoes.

Mosquitoes (Diptera: Culicidae) use certain resting sites during their inactive phase. The microclimatic conditions of these resting sites might affect their physiology and vectorial capacity. In this study, we combined a field and a laboratory study to investigate the natural resting site and temperature preferences of mosquitoes. The field study was conducted at a forest close to Oldenburg (Lower Saxony, Germany) from May to October 2018. Mosquitoes were collected in five different natural habitats with seven replicates each. Temperature was recorded hourly at each site. Significantly more mosquitoes were collected in deadwood (predominantly Culiseta morsitans/fumipennis) and shaded herb layer (predominantly Aedes species) compared to unshaded herb layer or broadleaf and coniferous trees. GLMMs revealed resting site habitats as the best predictor to explain the observed preference patterns, but microclimatic conditions are also involved in mosquito resting site selection. Most mosquitoes were collected at resting sites with relatively colder and more stable temperatures. In concert, laboratory choice experiments with a thermal gradient ring demonstrated that Cs. morsitans/fumipennis avoid temperatures over 30 °C. Understanding the small-scaled resting site preferences and the related microclimatic conditions can improve mosquito collection techniques and refine the prediction of mosquito-borne pathogen transmission.

Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany.

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

Chaperone-assisted pilus assembly and bacterial attachment.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

PapD-like chaperones provide the missing information for folding of pilin proteins.

A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.

Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili.

Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone-subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB-HifA periplasmic complexes, whereas mutations at the -14 glycine in HifA had no effect on HifB-HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD-PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure-function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.

PapD-like chaperones and pilus biogenesis.

The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site.

Bacterial pili: molecular mechanisms of pathogenesis.

Gram-negative bacteria produce a diverse array of pili that mediate microbe-microbe and host-pathogen interactions important in the development of disease. The structural and functional characterization of these organelles, particularly their role in triggering signals in both the bacterium and the host upon attachment, has begun to reveal the molecular mechanisms of bacterial diseases.

Structural basis of chaperone function and pilus biogenesis.

Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.

An antibody reactive with domain 4 of the platelet-derived growth factor beta receptor allows BB binding while inhibiting proliferation by impairing receptor dimerization.

A panel of murine monoclonal antibodies was generated against the extracellular domain of the human platelet-derived growth factor (PDGF) beta receptor (PDGFRbeta). These antibodies were assayed for both the ability to inhibit binding of PDGF BB to PDGFRbeta+ cells as well as the capacity to inhibit PDGF BB-mediated mitogenesis. As expected, all antibodies that could prevent PDGF BB binding also inhibited mitogenesis. However one antibody (M4TS.11), with no detectable ability to inhibit PDGF BB binding, was a potent inhibitor of proliferation induced by PDGF BB. Further characterization indicated that M4TS.11 impaired PDGFRbeta dimerization, revealing the mechanism by which it prevented PDGF BB-mediated mitogenesis. Using domain deletion mutants of the extracellular portion of PDGFRbeta, the determinant recognized by this antibody was localized to the fourth extracellular domain of PDGFRbeta, indicating that this domain, which is not involved in ligand binding, actively participates in receptor dimerization and signal transduction. The M4TS.11 antibody could also inhibit PDGF BB-mediated proliferation of responsive cells from both the baboon and the rabbit, indicating the determinant recognized by the antibody is not limited to humans and making it possible to use this antibody to evaluate the therapeutic benefit of interfering with PDGF in animal models of human disease.