Johannes A. J. Breeuwer

Phylogeny of cytoplasmic incompatibility microorganisms in the parasitoid wasp genus Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences

Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids, when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been identified as intracellular micro‐organisms. Microbeinduced cytoplasmic incompatibility is found in many insect taxa and may play a role in reproductive isolation between populations. Such micro‐organisms cause bidirectional incompatibility between species of the parasitoid wasp genus Nasonia. The phylogenetic relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and belong to the alpha group of the Proteobacteria.

Diseases of mites

An overview is given of studies on diseases of mites. Knowledge of diseases of mites is still fragmentary but in recent years more attention has been paid to acaropathogens, often because of the economic importance of many mite species. Most research on mite pathogens concerns studies on fungal pathogens of eriophyoids and spider mites especially. These fungi often play an important role in the regulation of natural mite populations and are sometimes able to decimate populations of phytophagous mites. Studies are being conducted to develop some of these fungi as commercial acaricides.Virus diseases are known in only a few mites, namely, the citrus red mite and the European red mite. In both cases, non-occluded viruses play an important role in the regulation of mite populations in citrus and peach orchards, respectively, but application of these viruses as biological control agents does not seem feasible. A putative iridovirus has been observed in association with Varroa mites in moribund honeybee colonies. The virus is probably also pathogenic for honeybees and may be transmitted to them through this parasitic mite.Few bacteria have been reported as pathogens of the Acari but in recent years research has been concentrated on intracellular organisms such as Wolbachia that may cause distorted sex ratios in offspring and incompatibility between populations. The role of these organisms in natural populations of spider mites is in particular discussed. The effect of Bacillus thuringiensis on mites is also treated in this review, although its mode of action in arthropods is mainly due to the presence of toxins and it is, therefore, not considered to be a pathogen in the true sense of the word.Microsporidia have been observed in several mite species especially in oribatid mites, although other groups of mites may also be affected. In recent years, Microsporidia infections in Phytoseiidae have received considerable attention, as they are often found in mass rearings of beneficial arthropods. They affect the efficacy of these predators as biological control agent of insect and mite pests. Microsporidia do not seem to have potential for biological control of mites.

Wolbachia" intracellular manipulators of mite reproduction

Cytoplasmically transmitted Wolbachia (alpha-Proteobacteria) are a group of closely related intracellular microorganisms that alter reproduction in arthropods. They are found in a few isopods and are widespread in insects. Wolbachia are implicated as the cause of parthenogenesis in parasitic wasps, feminization in isopods and reproductive (cytoplasmic) incompatibility in many insects. Here we report on the widespread occurrence of Wolbachia in spider mites and predatory mites based on a PCR assay for a 730 bp fragment of the ftsZ gene with primers that are specific for Wolbachia. An additional PCR, using two primer pairs that amplify a 259 bp region of the ftsZ gene that are diagnostic for the two Wolbachia subdivisions A and B, showed that infected mites only carried type B and not type A Wolbachia. The fact that some species tested negative for Wolbachia does not mean that the entire species is uninfected. We found that natural populations of Tetranychus urticae are polymorphic for the infection. The possible effects of Wolbachia on mite reproduction and post-zygotic reproductive isolation are discussed.

Wolbachia and cytoplasmic incompatibility in the spider mites Tetranychus urticae and T. turkestani

Wolbachia form a group of closely related intracellular Proteobacteria that manipulate reproduction of their arthropod host in various ways. They are widespread in insects, but have not been not looked for in other arthropods except a few isopods and a predatory mite. Recently, Wolbachia were demonstrated to be widespread in mites also. Here, I report that Wolbachia induce cytoplasmic incompatibility in two spider mite species, Tetranychus urticae and T. turkestani, in crosses between infected and uninfected individuals. Uninfected mites were derived from the infected lineage by antibiotic treatment. Similar to other examples of cytoplasmic incompatibility, subsequent crosses between uninfected (cured) female and infected male spider mites were different from all other cross combinations. This suggests that the dynamics and evolution in Wolbachia–mite relationships may share many features with the Wolbachia–insect systems. Interestingly, the incompatible cross showed high F1 zygotic mortality among females, but not among males. This zygotic mortality in the haplodiploid spider mites was in sharp contrast to the phenotypic effects of cytoplasmic incompatibility reported previously in haplodiploid insects. In the latter, cytoplasmic incompatibility results in all male or male-biased offspring because of haploidization of the fertilized egg with little or no zygotic mortality. Possible explanations are low bacterial densities and the different, holokinetic chromosome structure in spider mites.

Wolbachia-induced parthenogenesis in a genus of phytophagous mites.

The vertically transmitted endosymbiotic bacterium Wolbachia modifies host reproduction in several ways in order to enhance its own spread. One such modification results in the induction of parthenogenesis, where males, which are unable to transmit Wolbachia, are not produced. Interestingly, parthenogenesis-inducing Wolbachia have only been found within haplodiploid insects and it is not known whether this exclusivity is the result of functional constraints of Wolbachia. Here we find a unique pattern of Wolbachia infection that is associated with parthenogenesis in six species within the phytophagous mite genus Bryobia. Through antibiotic treatment we show that, in two species, Bryobia praetiosa and an unidentified species, the Wolbachia infection is strictly associated with parthenogenesis. Microsatellite loci show the mechanism of parthenogenesis to be functionally apomictic and not gamete duplication, with progeny identical to their infected mother. Crossing experiments within B. praetiosa showed no evidence of sexual reproduction. These results are discussed with reference to the distribution of parthenogenesis-inducing Wolbachia and the diversification of the Bryobia genus.

How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae)

ABSTRACT At least 20% of all arthropods and some nematode species are infected with intracellular bacteria of the genus Wolbachia. This highly diverse genus has been subdivided into eight “supergroups” (A to H) on the basis of nucleotide sequence data. Here, we report the discovery of a new Wolbachia supergroup recovered from the spider mite species Bryobia species V (Acari: Tetranychidae), based on the sequences of three protein-coding genes (ftsZ, gltA, and groEL) and the 16S rRNA gene. Other tetranychid mites possess supergroup B Wolbachia strains. The discovery of another Wolbachia supergroup expands the known diversity of Wolbachia and emphasizes the high variability of the genus. Our data also clarify the existing supergroup structure and highlight the use of multiple gene sequences for robust phylogenetic analysis. In addition to previous reports of recombination between the arthropod-infecting supergroups A and B, we provide evidence for recombination between the nematode-infecting supergroups C and D. Robust delineation of supergroups is essential for understanding the origin and spread of this common reproductive parasite and for unraveling mechanisms of host adaptation and manipulation across a wide range of hosts.

High temperatures eliminate Wolbachia, a cytoplasmic incompatibility inducing endosymbiont, from the two-spotted spider mite

Wolbachia can induce cytoplasmic incompatibility (CI) in the arrhenotokous two-spotted spider mite between uninfected females and infected males. Cytoplasmic incompatibility is expressed through a male-biased sex ratio and a low hatchability, and can be suppressed by removing Wolbachia from spider mites reared on a diet with antibiotics. Here we investigated whether heat-treatment can elimate Wolbachia from infected mites. Using a PCR assay with a Wolbachia-specific primer pair (ftsZ), and by standard crosses, we were able to show that 71 per cent of the mites had lost the Wolbachia infection after rearing the infected population at 32 ± 0.5 °C for four generations. The infection could be completely removed when mites were reared at 32 ± 0.5 °C for six generations. Curing through high temperatures could be one of the reasons why mixed infected/uninfected populations occur in the field. An additional consequence of rearing mites at 32 ± 0.5 °C was the shortened development time. The effect of environmental temperature on the abundance of Wolbachia and possible behavioural consequences for the spider mite are discussed.

Phylogeography of the planktonic chaetognath Sagitta setosa reveals isolation in European seas.

Abstract Numerous planktonic species have disjunct distribution patterns in the worlds oceans. However, it is unclear whether these are truly unconnected by gene flow, or whether they are composed of morphologically cryptic species. The marine planktonic chaetognath Sagitta setosa Müller has a discontinuous geographic distribution over the continental shelf in the northeastern Atlantic, Mediterranean Sea, and Black Sea. Morphological variation between these populations has been described, but overlaps and is therefore unsuitable to determine the degree of isolation between populations. To test whether disjunct populations are also genetically disjunct, we sequenced a 504‐bp fragment of mitochondrial DNA comprising the cytochrome oxidase II region of 86 individuals. Sequences were highly variable; each represented a different haplotype. Within S. setosa, sequence divergence ranged from 0.2 to 8.1% and strong phylogeographic structure was found, with four main groups corresponding to the northeastern Atlantic, Mediterranean Sea (including Ligurian Sea, Tyrrhenian Sea and Gulf of Gabès), Adriatic Sea, and Black Sea. Two of these (Atlantic and Black Sea) were resolved as monophyletic clades, thus gene flow between disjunct populations of S. setosa has been extremely limited and lineage sorting has taken place. The deepest divergence was between Atlantic and Mediterranean/Black Sea populations followed by a split between Mediterranean and Black Sea populations. The Mediterranean/Black Sea clade comprised three groups, with the Adriatic Sea as the most likely sister clade of the Black Sea. These data are consistent with a colonization of the Black Sea from the Mediterranean. Furthermore, a possible cryptic species was found in the Black Sea with 23.1% sequence divergence from S. setosa. Two possibilities for the evolutionary origin of this species are proposed, namely, that it represents a relict species from the ancient Paratethys, or that it represents another chaetognath species that colonized the Black Sea more recently. Even though the exact timing of disjunction of S. setosa populations remains unclear, on the basis of the geological and paleoclimatic history of the European basins and our estimates of net nucleotide divergence, we suggest that disjunct populations arose through vicariance resulting from the cyclical changes in temperature and sea levels during the Pleistocene. We conclude that these populations have remained disjunct, not because of limited dispersal ability, but because of the inability to maintain viable populations in suboptimal, geographically intermediate areas.

Molecular evidence of cryptic speciation in the ''cosmopolitan" excavating sponge Cliona celata (Porifera, Clionaidae)

Over the past several decades molecular tools have shown an enormous potential to aid in the clarification of species boundaries in the marine realm, particularly in morphologically simple groups. In this paper we report a case of cryptic speciation in an allegedly cosmopolitan and ecologically important species-the excavating sponge Cliona celata (Clionaidae, Hadromerida). In the Northeast Atlantic and Mediterranean C. celata displays a discontinuous distribution of its putative growth stages (boring, encrusting, and massive) leading us to investigate its specific status. Phylogenetic reconstructions of mitochondrial (COI, Atp8) and nuclear (28S) gene fragments revealed levels of genetic diversity and divergence compatible with interspecific relationships. We therefore demonstrate C. celata as constituting a species complex comprised of at least four morphologically indistinct species, each showing a far more restricted distribution: two species on the Atlantic European coasts and two on the Mediterranean and adjacent Atlantic coasts (Macaronesian islands). Our results provide further confirmation that the different morphotypes do indeed constitute either growth stages or ecologically adapted phenotypes as boring and massive forms were found in two of the four uncovered species. We additionally provide an overview of the cases of cryptic speciation which have been reported to date within the Porifera, and highlight how taxonomic crypsis may confound scientific interpretation and hamper biotechnological advancement. Our work together with previous studies suggests that overconservative systematic traditions but also morphological stasis have led to genetic complexity going undetected and that a DNA-assisted taxonomy may play a key role in uncovering the hidden diversity in this taxonomic group.

The effects of, and interactions between, Cardinium and Wolbachia in the doubly infected spider mite Bryobia sarothamni.

Many arthropods are infected with vertically transmitted, intracellular bacteria manipulating their hosts reproduction. Cytoplasmic incompatibility (CI) is commonly observed and is expressed as a reduction in the number of offspring in crosses between infected males and uninfected females (or females infected with a different bacterial strain). CI is often related to the presence of Wolbachia, but recent findings indicate that a second reproductive parasite, Cardinium, is also capable of inducing CI. Although both Wolbachia and Cardinium occur in arthropods and may infect the same host species, little is known about their interactions. We observed Wolbachia and Cardinium in the sexual spider mite Bryobia sarothamni (Acari: Tetranychidae) and investigated the effects of both bacteria on reproduction. We performed all possible crossing combinations using naturally infected strains, and show that Cardinium induces strong CI, expressed as an almost complete female mortality. B. sarothamni is the third host species in which Cardinium-induced CI is observed, and this study reveals the strongest CI effect found so far. Wolbachia, however, did not induce CI. Even so, CI was not induced by doubly infected males, and neither singly Wolbachia-infected nor doubly infected females could rescue CI induced by Cardinium-infected males. Possibly, this is related to the differences between Cardinium strains infecting singly and doubly infected individuals. We found a cost of infection in single infected individuals, but not in doubly infected individuals. We show that infection frequencies in field populations ranged from completely uninfected to a polymorphic state. In none of the populations infections were fixed.