Christian Melaun

First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids in Germany.

Mosquitoes and other arthropods may transmit medically important pathogens, in particular viruses such as West Nile virus. The presence of suitable hosts and competent vectors for those zoonotic viruses is essential for an enzootic transmission, which is a prerequisite for epidemics. To establish reliable risk projections, it is an urgent need for an exact identification of mosquito species, which is especially challenging in the case of sibling species, such as Culex. pipiens pipiens biotypes pipiens and molestus. To facilitate detection of different Culex pipiens forms and their hybrids we established a multiplex real-time PCR. Culex pipiens samples were obtained by egg raft collection and rearing until imago stage or adult sampling using CO2 baited traps and gravid traps. In total, we tested more than 16,500 samples collected all over Germany in the years 2011 and 2012. The predominant species in Germany are Culex pipiens pipiens biotype pipiens and Culex. torrentium, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes at sites where both species occur sympatrically. This report of a potentially important bridge vector for West Nile virus might have major impact in the risk projections for West Nile virus in Germany.

RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinsons disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3β4 nAChRs found to date and is also active at α3β2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3β4 nAChRs.

The use of morphometric wing characters to discriminate female Culex pipiens and Culex torrentium

ABSTRACT: The reliability of the length of wing radial vein r2/3 as a character for the morphological discrimination of the two potential arbovirus vectors Culex pipiens s.s. and Cx. torrentium from Germany was reassessed, after this character had been neglected for more than 40 years. Additionally, multivariate morphometric analyses were applied to evaluate wing shape variation between both species. Although high-throughput molecular tools are now available to differentiate the two species, a simple, low-cost routine alternative may be useful in the absence of a molecular laboratory, such as under semi-field conditions. A thin-plate splines transformation confirmed that primarily the shrinkage of vein r2/3 is responsible for the wing differences between the two species. In the bivariate analysis, the r2/3/r3 indices of Cx. pipiens s.s. and Cx. torrentium were 0.185 and 0.289, respectively, resulting in a correct classification of more than 91% of all tested specimens. Using the absolute length of vein r2/3 alone still allowed for more than 90% accurate discrimination. Furthermore, classification accuracy of linear discriminant analysis exceeded 97%.

Modeling of the putative distribution of the arbovirus vector Ochlerotatus japonicus japonicus (Diptera: Culicidae) in Germany

Today, international travel and global freight transportation are increasing and have a direct influence on the introduction and establishment of non-native mosquito species as well as on the spread of arthropod (mosquito)-borne diseases inside Europe. One of the mosquito species that has become invasive in many areas is the Asian rock pool or bush mosquito Ochlerotatus japonicus japonicus (synonyms: Aedes japonicus japonicus or Hulecoeteomyia japonica japonica). This species was detected in Germany in 2008 for the first time. Until today, three different Oc. j. japonicus populations have been documented. Laboratory studies have shown that Oc. j. japonicus can act as a vector for a variety of disease agents. Thus, the knowledge on its current distribution is essential for different measurements. In the present study, ecological niche models were used to estimate the potential distribution of Oc. j. japonicus in Germany. The aim was to detect areas within Germany that could potentially function as habitats for this species. According to our model, areas in western, southern, and central Germany offer suitable conditions for the mosquito and may therefore be at risk for an invasion of the species. We strongly suggest that those areas should be monitored more intensively in the future. For this purpose, it would also be essential to search for possible dispersal routes as well as for natural barriers.

New record of the suspected leishmaniasis vector Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Psychodidae: Phlebotominae) — the northernmost phlebotomine sandfly occurrence in the Palearctic region

Although being typical Mediterranean faunal elements, phlebotomine sandflies have also been recorded in central Europe for several countries including Germany, where two species, Phlebotomus mascittii and Phlebotomus perniciosus, occur. In Europe, P. mascittii is the northernmostly distributed phlebotomine species. While P. perniciosus is a proven vector of leishmaniasis as well as various sandfly fever causing phleboviruses, the situation for P. mascittii is different. For this species, vector competence could not be proven yet, but is strongly suspected. During an entomological survey in July 2013, one female sandfly was caught in Giessen in the German state of Hesse. Adjacent to the collection site, different potential habitats could be found. Morphological examination of the cibarium, pharynx, and genitalia revealed the specimen as P. mascittii. This is the first reported occurrence for Hesse, and not only the northernmost documented occurrence for P. mascittii, but also of the whole subfamily in the Palearctic region. New records of proven or suspected vectors are of medical relevance because of potential Leishmania and/or Phlebovirus transmission and the awareness therefore in the public.

Venomous Secretions from Marine Snails of the Terebridae Family Target Acetylcholine Receptors

Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland extracts from three species of the superfamily Terebridae. By 2-electrode voltage-clamp technique the gland extracts were tested on Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) of rat neuronal (α3β2, α3β4, α4β2, α4β4, α7) and muscle subtypes (α1β1γδ), and expressing potassium (Kv1.2 and Kv1.3) and sodium channels (Nav1.2, 1.3, 1.4, 1.6). The extracts were shown to exhibit remarkably high inhibitory activities on almost all nAChRs tested, in particular on the α7 subtype suggesting the presence of peptides of the A-superfamily from the venom of Conus species. In contrast, no effects on the potassium and sodium channels tested were observed. The venoms of terebrid snails may offer an additional source of novel biologically active peptides.

Functional Hypervariability and Gene Diversity of Cardioactive Neuropeptides

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Diversity of Culex torrentium Martini, 1925 — a potential vector of arboviruses and filaria in Europe

Culex torrentium is one of the most common mosquito species in Germany. Due to its sympatric occurrence as well as its similar morphological and ecological characteristics, it has often been confused with another common species, Culex pipiens. Both species are known to be potential vectors for different arboviruses (not only in Germany) with C. torrentium being a possible vector for Sindbis or Ockelbo virus. In our study, we analyzed the genetic variability in a 658 bp fragment of the cytochrome c oxidase subunit I gene (coxI) of C. torrentium, from nine localities in the Frankfurt/Rhine-Main Metropolitan Region. The results of our genetic survey indicate a higher genetic diversity in this gene region for C. torrentium than for the morphologically similar C. pipiens. Our findings may explain the difficulties in the past to find morphological characteristics that apply to all populations of C. torrentium, when attempting to separate them clearly from C. pipiens, by any other criteria than male genitalia. Being ornithophilic, possible hybrids between C. torrentium and the humanophilic C. pipiens biotype molestus, could potentially serve as important vectors for zoonotic diseases. Therefore, we recommend that greater emphasis is placed on the ecological characteristics, population structure, and the taxonomy of this often neglected species, in the future.

Identification of sympatric cryptic species of Aedes albopictus subgroup in Vietnam: new perspectives in phylosymbiosis of insect vector

BackgroundThe Aedes (Stegomyia) albopictus subgroup includes 11 cryptic species of which Ae. albopictus is the most widely distributed. Its global expansion associated with a documented vector competence for several emerging arboviruses raise obvious concerns in the recently colonized regions. While several studies have provided important insights regarding medical importance of Ae. albopicus, the investigations of the other sibling species are scarce. In Asia, indigenous populations within the Ae. albopictus subgroup can be found in sympatry. In the present study, we aimed to describe and compare molecular, morphological and bacterial symbionts composition among sympatric individuals from the Ae. albopictus subgroup inhabiting a Vietnamese protected area.ResultsBased on morphological structure of the cibarial armarture, we identified a cryptic species in the forest park at Bù Gia Mập in the south-eastern region of Vietnam. Analysis of nuclear (ITS1-5.8S-ITS2) and mitochondrial (cox1, nad5) markers confirmed the divergence between the cryptic species and Ae. albopictus. Analysis of midgut bacterial microbiota revealed a strong similarity among the two species with a notable difference; contrary to Ae. albopictus, the cryptic species did not harbour any Wolbachia infection.ConclusionsThese results could reflect either a recent invasion of Wolbachia in Ae. albopictus or alternatively a loss of this symbiont in the cryptic species. We argue that neglected species of the Ae. albopictus subgroup are of main importance in order to estimate variation of host-symbionts interactions across evolution.

Bats as Potential Reservoir Hosts for Vector-Borne Diseases

Bats are the only mammals with the capacity of powered flight. Nearly 1,000 species can be found all over the world except in the northern and southern polar areas. They perform important ecosystem services such as control of insects, reseeding of cut forests and pollination of plants, which provide food for humans and animals. On the other side, they are also recognized to be natural reservoir hosts of a large variety of zoonotic diseases with the ability to cross species barriers. To date, more than 80 virus species of different groups and various parasites, which can cause several diseases have been isolated or detected in bats. Especially their high population density and gregarious roosting behaviour increase the likelihood of intra- and inter-species transmission of infections. Another important factor, which enables pathogens to spread long distances, is the migratory habit of some bat species, resulting in a great dispersal capacity. The transmission of pathogens from bats to humans or other animals occurs by direct contact with infected animals, their blood and tissue or through vector species. One of the most important vector groups are insects. With more than a million described species, they are the most diverse group of animals. Especially haematophagous groups such as Cimicidae, Culicidae or Phlebotominae are known as vectors for a variety of diseases. These include bacteria, protozoan and metazoan parasites as well as viruses. We focused on blood-feeding insects, because the presence of certain viruses in them as well as in bats comprises a potential virus transmission from bats to humans through mosquitoes or other blood-feeding insects. For this chapter, we could find 20 viruses from four different families and two parasitic pathogens detected in all three groups of haematophagous insects.