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.

Activation of Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii.

The capability of regenerating posterior segments and pygidial structures is ancestral for annelids and has been lost only a few times within this phylum. As one of the three major segmented taxa, annelids enable us to monitor reconstruction of lost tissues and organs. During regeneration, regional identities have to be imprinted onto the newly formed segments. In this study, we show spatial and temporal localization of expression of nine Hox genes during caudal regeneration of the polychaete annelid Platynereis dumerilii. Hox genes are homeodomain genes encoding transcriptional regulators of axial patterning in bilaterian animals during development. We demonstrate that five Platynereis Hox genes belonging to paralog groups (PG) 1, 4, 5, 6, and 9-14 are expressed in domains of the regenerating nervous system consistent with providing positional information along the anteroposterior axis of the regenerate. We report that expression in regenerating neuromeres is limited to varying subsets of perikarya, called gangliosomes. Four of nine genes analyzed do not appear to be involved in axial patterning. Two genes, Pdu-Hox2 and Pdu-Hox3, are predominantly expressed in the growth zone region. For some Hox genes expression in newly formed coelomic epithelia can be observed. Platynereis Hox genes do not exhibit temporal or spatial colinearity. Although there are some similarities to previously reported expression patterns during larval and postlarval development in Nereididae (Kulakova et al. 2007), expression patterns observed during caudal regeneration also show unique patterns.

Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation.

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.

Formation of body appendages during caudal regeneration in Platynereis dumerilii: adaptation of conserved molecular toolsets.

BACKGROUND: Platynereis and other polychaete annelids with homonomous segmentation are regarded to closely resemble ancestral forms of bilateria. The head region comprises the prostomium, the peristomium, a variable number of cephalized body segments and several appendages, like cirri, antennae and palps. The trunk of such polychaetes shows numerous, nearly identical segments. Each segment bears a parapodium with species-specific morphology on either side. The posterior end of the trunk features a segment proliferation zone and a terminal pygidium with the anus and anal cirri. The removal of a substantial part of the posterior trunk is by no means lethal. Cells at the site of injury dedifferentiate and proliferate forming a blastema to regenerate both the pygidium and the proliferation zone. The pygidium forms new anal cirri, and the proliferation zone generates new segments at a rapid pace. The formation of body appendages like the cirri and the segmental parapodia can thus be studied in the caudal regenerate of Platynereis within only a few days. RESULTS: The development of body appendages in Platynereis is regulated by a network of genes common to polychaetes but also shared by distant taxa. We isolated DNA sequences from P. dumerilii of five genes known to be involved in appendage formation within other groups: Meis/homothorax, Pbx1/extradenticle, Dlx/Distal-less, decapentaplegic and specific protein 1/buttonhead. Analyses of expression patterns during caudal regeneration by in situ hybridization reveal striking similarities related to expression in arthropods and vertebrates. All genes exhibit transient expression during differentiation and growth of segments. As was shown previously in other phyla Pdu-Meis/hth and Pdu-Pbx1/exd are co-expressed, although the expression is not limited to the proximal part of the parapodia. Pdu-Dll is prominent in parapodia but upregulated in the anal cirri. No direct dependence concerning Pdu-Dll and Pdu-sp/btd expression is observed in Platynereis. Pdu-dpp shows an expression pattern not comparable to its expression in other taxa. CONCLUSIONS: The expression patterns observed suggest conserved roles of these genes during appendage formation across different clades, but the underlying mechanisms utilizing this toolset might not be identical. Some genes show broad expression along the proximodistal axis indicating a possible role in proximodistal patterning of body appendages. Other genes exhibit expression patterns limited to specific parts and tissues of the growing parapodia, thus presumably being involved in formation of taxon-specific morphological differences.

Population structure and distribution patterns of the sibling mosquito species Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths.

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima's D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (FST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.

Quantitative analysis of cellular differentiation during early embryogenesis ofPlatynereis dumerilii.

As in many spiralian embryos with unequal cleavage, cleavage inPlatynereis follows an invariant pattern. Preceding each cleavage the cytoplasm is reorganized, allowing the spiral cleavage mode to produce cells with different cytoplasmic composition. The fertilized egg undergoes a dramatic ooplasmic segregation after the completion of the cortical reaction. As a consequence, a plug of clear cytoplasm becomes located at the animal pole. Once the four quadrants of the embryo have been established, the cleavage sequence of the D quadrant differs clearly from that of the other three quadrants. The results presented here suggest that differential distribution of the clear cytoplasm governs this sequence. The first quartet of micromeres, which will form the ectoderm and the cerebral ganglia of the head, is clearly bilaterally symmetrical from the onset of the third cleavage. Dorsoventral polarity and bilateral symmetry in the ectoderm of the trunk is expressed most markedly by the dorsal location of the large 2d cell, whose rapid proliferation is bilaterally symmetrical with respect to the median plane. As a result of this proliferation it comes to fill most of the posttrochal region (ectoderm, three pairs of anlagen for the setal sacs, and the ventral plate which forms the nerve cord). The other micromeres contribute only a minor portion of the ventral ectoderm and are involved in the formation of the stomodaeum. The mesentoblast, 4d, i.e. the stem cell of the primary mesoderm, forms at the sixth cleavage, also in a position on the dorsal mid-line. The daughter cells, which arise from 4d by strictly bilaterally symmetrical cleavage, form the mesodermal germ bands, which lie beneath the ectoderm. The trochoblasts are formed by asynchronously cleaving founder cells, but further cleavages in these cells are synchronous. This suggests that cell-cell interaction is involved in the development of this alleged mosaic embryo.

Competence of blastomeres for the expression of molecular tissue markers is acquired by diverse mechanisms in the embryo of Platynereis (Annelida).

This paper is devoted to the role of cell divisions for the establishment of histospecificity in the embryo of the spiralian, Platynereis dumerilii (Annelida). We have incubated successive cleavage stages in cytochalasin B (CCB) and observed whether the cells thereafter were able to acquire the competence for expressing histospecific antigens of larval gland cells (labelled by the monoclonal antibody OI64) and of neural components of the ventral nerve cord (labelled by mAb OI7 or by testing acety1cholinesterase activity), respectively. Incubation in CCB results in permanent cleavage arrest, but does not necessarily interfere with biochemical differentiation of such markers. Synthesis of the differentiation marker specific for larval gland cells does not require any cleavages but this capacity becomes restricted to the 1a and 1b cell lines if cleavages are allowed to occur. In contrast, the progenitors of neural cells need at least 6.5 h of normal development before they acquire the competence to synthesize two neural differentiation markers, which can be demonstrated after at least two more days of development. Thus, prespecifying and diversifying cleavages are a prerequisite for neurogenesis and production of the investigated neural markers. Competence for the expression of histospecific antigens may also depend on cell-cell interactions. If 20-24 h old embryos are treated with puromycin, pioneering fibres fail to grow out from a pair of posterior nerve cell progenitors as they would have done normally 24-48 h after fertilization. Concomitantly, a number of potential nerve cells which now do not come into contact with pioneering fibres do not express the neural antigen. This suggests that a local inductive stimulus from the pioneering fibres normally imprints cell fate onto ventral plate cells and turns them into neuroblasts.

Experimental change of cytoplasmic composition can convert determination of blastomeres inPlatynereis dumerilii (Annelida, Polychaeta).

Early development ofPlatynereis dumerilii is characterized by an extremely constant cleavage pattern in which the volumes and cytoplasmic contents of the blastomeres show remarkably little variability (Dorresteijn 1990). In order to test the necessity of a precise partitioning of the cytoplasm, we have stratified the ooplasm by mild centrifugation (10 min at 300 g) after completion of meiosis but before first cleavage. The cytoplasm of the zygote stratifies randomly with respect to the pre-existing animal-vegetal axis, but first cleavage follows the animal-vegetal axis dividing the plasm before it has rearranged to its normal distribution. As usual, first cleavage is unequal in the majority of centrifuged eggs. Different sorts of cytoplasm are always distributed abnormally in comparison to normal two-cell stages. Under two circumstances this leads to the formation of double trunk structures in the young worm. Such 'double monsters' either originate from zygotes whose clear cytoplasm has been distributed equally to the two daughter blastomeres at first cleavage, or from unequal two-cell embryos whose larger blastomere cleaved equally at second cleavage forming blastomeres with equal lots of clear cytoplasm. Cell-lineage could be followed in an individual embryo of the latter category and showed the existence of two adjacent D-quadrants, giving rise to a double monster with a forked trunk. Embryos of the former category give rise to two opponent D-quadrants and double monsters with a four-sided trunk. As in the normal embryo, the amount of clear cytoplasm in a blastomere is positively correlated with the speed of its cell cycle, and endows the cell with D-quadrant developmental capacities as can be judged by the cleavage pattern.

A correlative study of experimentally changed first cleavage and Janus development in the trunk of Platynereis dumerilii (Annelida, Polychaeta).

Among zygotes of Platynereis dumerilii treated with cytochalasin B (CCB) prior to first cleavage, a wide variety of developmental effects were observed. One effect is a delay in the first cleavage. Treated embryos may skip the first or even more than one cleavage cycle and become multinucleated. Once these eggs start cleaving their cleavage plane takes the same position as in synchronously fertilized controls. Accordingly, the first cleavage in embryos having skipped the first normal cleavage cycle is meridional and equal, but their second cleavage is equatorial as in the third cleavage in controls. None of the embryos that were observed to skip early cleavages showed normal organogenesis, but developed into vesicle-shaped embryos with little cytological differentiation. Another effect of CCB treatment is altered blastomere size in those embryos which begin cleaving in synchrony with controls. While the majority of treated embryos followed a normal cleavage pattern, i.e. they cleaved at the right time and inequally, some of them cleaved equally or almost equally (adequally). Most of these embryos showed cleavage defects in subsequent cleavage cycles and became abnormal vesicle-shaped embryos. However, some of these embryos cleaving on schedule and equally or adequally developed into juvenile worms showing complete duplication of urites and parapodial rows (0.3% of all treated eggs) and are described as Janus duplicitates. This means that the occurrence of duplicitates and geometrically altered first cleavage patterns are correlated phenomena. The character and origin of the duplications and the consequences for dorsoventral polarity are discussed.

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida, Polychaeta).

Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis.

Molecular specification of cell lines in the embryo of Platynereis (Annelida).

In this study we describe the site and moment of histospecific differentiation in developmental stages of the annelid Platynereis dumerilii by use of biochemical markers. The monoclonal antibody (mab) OI7 and uncloned hybridoma supernatants (pAb's) OI8, OI10, OI46 and OI69 recognize neural antigens that appear asynchronously during development. By an enzymatic test, acetylcholinesterase (AChE) was found specific for nervous tissue as well. The patterns of neural structures labelled by antibodies differ, however, from those revealed by AChE staining. Experimental inhibition of transcription (with actinomycin D) and of translation (using puromycin) demonstrate that the expression of histospecific neural markers depends on both zygotic transcription and subsequent translation. The mAb OI64 labels epidermal (and neural) gland cells. The antibody 4D9, raised against the engrailed protein of Drosophila, labels single rows of ciliated cells at the posterior border of segments.

Dye-coupling between blastomeres in early embryos ofPatella vulgata (mollusca, gastropoda): Its relevance for cell determination.

During the early development of the molluscPatella, the dorsoventral axis is established after the fifth cleavage due to direct interaction between the animal micromeres and one of the vegetal macromeres. This vegetal macromere is thereby induced to become the mesentoblast mother cell (3D). In this study we have examined intercellular communication in earlyPatella embryos by monitoring the transfer of the fluorescent dye, Lucifer Yellow, upon iontophoretic injection into blastomeres between the second and sixth cleavage. Up to the fifth cleavage dye transfer is detectable neither inin toto embryos nor in serial sections. Shortly after the fifth cleavage dye-coupling between blastomeres becomes apparent. This occurs approximately 40 min before the interaction between animal micromeres and the future mesentoblast mother cell. Inspection of serially sectioned embryos after dye-iontophoresis in either animal micromeres or in the central macromere 3D showed the absence of direct dye-transfer between these cells at the stage of interaction. The reduced rate of dye-transfer from the 3D macromere to its dorsal neighbour 2d2 suggests a bilateral symmetrical transfer pattern, the axis of which corresponds with the dorsoventral axis at the sixth cleavage. Cell deletion experiments demonstrated that the establishment of dye-coupling between the vegetal macromeres occurs independently of the interaction between animal and vegetal blastomeres.