Heat curing Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae) of a fitness-reducing microsporidium

Laboratory colonies of the predatory mite Metaseiulus occidentalis in Gainesville, FL were found to be infected with an undescribed microsporidium. Experiments were performed to quantify the effect of infection on the fitness of M. occidentalis and to determine if heat treatment can cure mites of the microsporidium. The colonies tested were derived from an isofemale line so that differences in performance could be attributed to the presence of microsporidia. A subcolony of an uninfected isofemale line was infected with the microsporidium by feeding females infected eggs from another colony of M. occidentalis. Infected mites had a shorter mean (+/-SD) female life span (7.4 +/- 2.9 vs. 10.0 +/- 2.8 days), lower mean oviposition (1.6 +/- 0.7 vs. 2.2 +/- 0.4 eggs/day), and a male-biased sex ratio (43 +/- 16% vs. 57 +/- 15% female progeny). The infection was reduced temporarily in colonies initiated from mites that were reared in a growth chamber at 33 degrees C from egg to adult, but healthy colonies only were established from the progeny of the heat-treated adults. These colonies remained free of infection for 10 weeks.     

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae)

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.     

Diapause behavior of genetically-improved strains of the spider mite predator Metaseiulus occidentalis (Acarina: Phytoseiidae)

Diapause behaviou of two strains of Metaseiulus occidentalis (Nesbitt) was compared as part of a project to evaluate this predator as a control agent for Tetranychus urticae Koch on roses in greenhouses in Berkeley, California. One strain had a normal diapause and the other had been genetically selected for non-diapause. Predators in diapause sought shelters on a simple bean plant test system. Females lacking the ability to diapause and the normal strain under long photophases tended to disperse aerially from the bean plant systems, particularly when prey were scarce. M. occidentalis females with the capacity to diapause reared near the critical photophase (11.2h) at fluctuating temperatures of 17.5 to 24.5°C within a 24 h period were influenced by the availability of prey: if prey were lacking, predators were more likely to enter diapause than if prey were abundant.Starved females left the plant system, probably through aerial dispersal, and mated females dispersed more readily than males and virgin females under long daylengths. No differences in aerial dispersal tendency were observed at short photophases. Aerial dispersal of adult females occurred at air speeds of ca. 1.5 m/s.

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Comportement lors de la diapause de souches génétiquement sélectionnées du prédateur Metaseiulus occidentalis (Acari; Phytoseiidae)

Résumé Au cours d'un programme destiné à évaluer la valeur du prédateur, Metaseiulus occidentalis Nesbitt pour lutter contre Tetranychus urticae Koch sur roses dans les serres de Berkeley (California) nous avons comparé chez deux souches le comportement lié à la diapause. Une souche a une diapause normale, l'autre a été sélectionnée génétiquement pour son absence de diapause. Les adultes avec diapause normale recherchent des abris sur un dispositif expérimental conçu à partir d'un pied de haricot. Les femelles perdant l'aptitude à la diapause ainsique celles de la souche normale soumise à une longue photophase ont tendance à se disperser par voie aérienne à partir du dispositif expérimental, surtout quand les proies sont rares.Les femelles de M. occidentalis aptes à la diapause élevées aux environs de la photophase critique (11.2h) avec des températures variant entre 17°5 et 24°5 pendant le nyctémère réagissent en fonction de la disponibilité en proies: quand les proies manquent, les prédateurs ont plus tendance à entrer en diapause que quand les proies sont abondantes.Avec une longue photopériode, les femelles à jeûn abandonnent le système expérimental, (probablement par dispersion aérienne) et les femelles fécondées se dispersent plus facilement que les mâles et les femelles vierges. Aucune différence dans la tendance à la dispersion aérienne n'a été observée aux courtes photophases. La dispersion aérienne des femelles adultes s'est produite à des vitesses de 1.5 m/sec.

     

Microbial diversity in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae) and its prey, Tetranychus urticae (Acari: Tetranychidae)

Microorganisms associated with the predatory mite Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) and its prey, the two-spotted spider mite Tetranychus urticae (Koch), were assessed using a high-fidelity polymerase chain reaction (PCR) protocol and primers designed to identify Eubacteria, Archaeabacteria, iridoviruses, Helicosporidia, Cytophaga-like microorganisms, Wolbachia and its bacteriophage WO, fungi and yeast-like organisms. Sequences from four bacterial species related to Wolbachia (α-Proteobacteria), Cardinium, Bacteroidetes, and Enterobacter (γ-Proteobacteria) were obtained from M. occidentalis, and three sequences related to Wolbachia, Rickettsia, and Caulobacter (α-Proteobacteria) were obtained from T. urticae. No nucleotide differences were detected between the 16S rRNA, wspA or wspB Wolbachia sequences obtained from M. occidentalis and T. urticae, which suggest that horizontal transfer of Wolbachia could have occurred. Southern blot analyses of genomic DNA from both M. occidentalis and T. urticae using wspA probes were negative, indicating that this Wolbachia sequence is not integrated into the nuclear genome of either species. Two of the T. urticae colonies tested contained the WO bacteriophage, but none of the six M. occidentalis populations were infected. New M. occidentalis-specific forward and reverse 16S rRNA primers based on the Wolbachia, Cardinium, Bacteroidetes, and Enterobacter sequences obtained were designed and used to amplify PCR products from each of two laboratory and four field-collected samples of M. occidentalis females and eggs, indicating that these infections are widespread. Likewise, species-specific primers for T. urticae were designed for the Wolbachia, Rickettsia, and Caulobacter sequences obtained and used to evaluate T. urticae from strawberries, wine grapes, hops, almonds, and cherries from California, Washington, and Florida; all were positive for Wolbachia and Caulobacter but two of the six were negative for Rickettsia. None of the M. occidentalis colonies tested were positive for the microsporidium Oligosporidium occidentalis, which previously had been associated with a pathogenic condition in some of our laboratory colonies. The Gainesville colonies of M. occidentalis and T. urticae were negative for iridovirus, Archaeabacteria, fungi, Helicosporidia, and yeast-like organisms. So far, Wolbachia is the only symbiont that is shared by this predator and its prey.     

Cardinium is associated with reproductive incompatibility in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae)

Cardinium, a bacterium from the Bacteroidetes group, is associated with reproductive manipulations such as cytoplasmic incompatibility, parthenogenesis, and feminization in some arthropod species. We, and others, have shown that Cardinium, but not Wolbachia, is an endosymbiont in some populations of Metaseiulus occidentalis, a phytoseiid mite that is an important predator of spider mite pests of agricultural crops. However, the precise biological effects that Cardinium may have on M. occidentalis remain unclear. In this study we show, in two sets of crosses between different colonies of Cardinium-free (C-) M. occidentalis females and Cardinium-containing (C+) males, that fecundity was reduced in parental females, F1 progeny survival rates were reduced, and fewer female progeny were produced when compared to the reciprocal and control crosses. There were no differences in these attributes in the reciprocal and two control crosses. Cardinium was transmitted maternally but there was no observed paternal transmission. Finally, Cardinium did not cause asexual (thelytoky) reproduction in M. occidentalis. Thus, Cardinium is associated with nonreciprocal reproductive incompatibility in M. occidentalis and our results support the hypothesis that Cardinium is a reproductive parasite in this agriculturally important predator. Cardinium may therefore affect the evolution and ecology of M. occidentalis and biological control efforts using this mite.     

Effects of temperature on the establishment potential of the predatory mite Amblyseius californicus McGregor (Acari: Phytoseiidae) in the UK

Amblyseius californicus was introduced into the UK in the early 1990s as a biocontrol agent against glasshouse red spider mite Tetranychus urticae. This study investigated the effects of temperature on the establishment potential of A. californicus in the UK in the light of recent reports of their successful overwintering outside of glasshouse environments. The developmental thresholds were 9.9 and 8.6 °C respectively using simple and weighted linear regression. Using the day-degree requirement per generation calculated by weighted regression (143 day-degrees) in combination with climate data, it was estimated that up to seven generations would be possible annually outdoors in the UK. Non-diapausing adult females froze at −22 °C, with 100% mortality after reaching their freezing temperature. Up to 90% of mites died before freezing after short exposures to low temperatures. Significant acclimation responses occurred; 90% of acclimated individuals survived 26 days exposure at 0 °C and 11 days at −5 °C (acclimated mites were reared at 19 °C, 6L:18D followed by 1 week at 10 °C, 12L:12D). Non-diapausing adult females survived over 3 months outdoors in winter under sheltered conditions and oviposition was observed. The experimental protocol used in this study is discussed as a pre-release screen for the establishment potential of other Amblyseius species, and similar non-native biocontrol agents.     

Heterochromatinization,chromatin elimination and haploidization in the parahaploid mite Metaseiulus occidentalis (Nesbitt) (Acarina: Phytoseiidae)

Embryogenic mitoses, mitoses in females and spermatogenesis are described in the predatory mite Metaseiulus occidentalis (Nesbitt). At 22° C, egg development lasts approximately 4 days. Six chromosomes are seen in mitotic metaphases and anaphases of 0–24 h eggs. Toward the end of this period some embryo squashes have patches of cells containing nuclei which are partially heteropycnotic. These patches of cells apparently increase in size with the age of the embryo. In approximately 1/2 of all 24–48 h-old eggs they encompass all or most cells of the embryo. In these embryos metaphases involved 6 chromosomes, anaphases 3. Either prior to, or following metaphase, a pairing of chromosomes appeared to take place to form 3 units which resembled meiotic diplotene chromosomes where there is opening out between homologues. At metaphase, two sets of 3 chromosomes were slightly differentially stained. One, designated the H set, was darker and slightly more contracted than the other, the E set. At anaphase, 3H and 3E chromosomes segregated in a reductional division retaining the differential contraction until telophase. No cytokinesis appeared. The H set appeared to remain contracted while the E set decontracted to assume the appearance of an interphase nucleus. Both of these entities, side-by-side, created the partially heteropycnotic nucleus mentioned above. The H set then appeared to be excluded from the cell. Mitotic meta and anaphases involving 6 chromosomes were noted in female deutonymphs. Spermatogenesis appeared to encompass an equational division of 3 chromosomes, with the formation of a binucleate spermatid. Two tail structures appeared juxtaposed at the edge of each spermatid and thereafter a separation into two individual sperms occurred. —While mitosis was not studied in known males, we believe that the embryos exhibiting heterochromatinization and elimination of chromosomes in most or all cells were in fact demonstrating parahaploidization.This paper is dedicated to the memory of Professor S.W. Brown and is presented to Professor H. Bauer in honor of his 75th birthday     

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.     

Nosema ceranae, a new microsporidian parasite in honeybees in Europe

Twelve samples of adult honey bees from different regions of Spain from colonies with clear signs of population depletion, positive to microsporidian spores using light microscopy (1% of total positive samples analysed), were selected for molecular diagnosis. PCR specific primers for a region of the 16S rRNA gene of Microsporidia were developed and the PCR products were sequenced and compared to GenBank entries. The sequenced products of 11 out of the 12 samples were identical to the corresponding Nosema ceranae sequence. This is the first report of N. ceranae in colonies of Apis mellifera in Europe. The suggested link of the infections to clinical disease symptoms makes imperative a study of the virulence of N. ceranae in European races of honey bees.     

The mitochondrial genome of the predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) is unexpectedly large and contains several novel features

The complete mitochondrial genome of the phytoseiid Metaseiulus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) has been sequenced. It is 24,961 bp in length and contains a 14,695-bp unique region, a 345-bp triplicated region, and a 9921-bp duplicated region, in that order. The A+T content of the unique region is 76.9% and contains 11 protein coding (COI-III; ATP6-8; CytB; ND1, 2, 4, 5 and 4L), two ribosomal RNA (srRNA and lrRNA), 22 transfer RNA (tRNA) genes, and two copies of D-loop control sequence. Two genes (ND3 and 6) appear to be missing, but there is a large intergenic spacer (390 bp) present, which could contain ND3 if a different codon usage is employed. The gene order is completely different from the pattern in all other known chelicerates, including the horseshoe crab Limulus polyphemus [Lavrov et al., Mol. Biol. Evol., 2000; 17:813-824]. All the inferred tRNA genes are missing the TPsiC arm, but this arm has fused with the variable arm to generate a TV replacement loop. The duplicated region (9921 bp) contains 18 genes in the same order as in the unique region from CytB to tRNA-His, plus one copy of D-loop control sequence (311 bp) and a partial tRNA-Leu2 sequence (34 bp). The small triplicated region (345 bp) contains a D-loop control sequence (311 bp) and a partial tRNA-Leu2 sequence (34 bp). Because of these anomalies, amplifying sequences posed technical difficulties, but were accomplished by using a primer-walking strategy and increasing the AT content to 75% in the high-fidelity PCR dNTP mix. This is the first phytoseiid mitochondrial genome to be completely sequenced and the largest (25 kb) detected from the Chelicerata.     

A new genus and species in the mite family Eupodidae (Acari,Eupodoidea) from Crimea

A new genus Pseudoeupodes Khaustov, gen. n. and new species Pseudoeupodes porosus sp. n. are described from moss in Crimea. The taxonomy of the Eupodidae and some other families and genera of Eupodoidea is reviewed. The genus Turanopenthalodes Barilo, 1988 is transferred from Penthalodidae to Penthaleidae. The family Cocceupodidae Jesionowska, 2010 and the genus Filieupodes Jesionowska, 2010 are considered as junior synonyms of Eupodidae Koch, 1842 and Cocceupodes Thor, 1934, respectively. A key to genera of the family Eupodidae is provided.     

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).     

Life-history of predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) on four spider mite species as prey, with special reference to Tetranychus evansi (Acari: Tetranychidae)

The commercially available strains of Phytoseiulus persimilis Athias-Henriot, the biological control agent of Tetranychus urticae Koch, perform poorly in the Western Mediterranean, probably because they are not well adapted to local climatic conditions. For that reason, efforts are being focused on the development of a biological control programme using native phytoseiid mites. Four species of red spider mites can be found in vegetable crops in eastern Spain: T. urticae, Tetranychus turkestani Ugarov and Nikolski, Tetranychus ludeni Zacher and the recently introduced Tetranychus evansi Baker and Pritchard. To evaluate their potential role as biological control agents, the present study evaluates the life-history of local populations of Neoseiulus californicus (McGregor) and P. persimilis when fed on T. urticae, T. turkestani, T. evansi, and T. ludeni in the laboratory. Results indicate that N. californicus and P. persimilis are able to feed and complete their development on the four tested red spider mite species. The predators may exhibit a particularly high capacity for population increase when fed on T. urticae, T. turkestani, and T. ludeni, thus may be able to provide effective control of these species in the field. When fed T. evansi, however, predator performance was poor; significant increase in development and preoviposition times, and a reduction in oviposition period and fecundity were recorded. The resultant low capacity for population growth suggests poor ability of the two tested predators to suppress T. evansi populations on commercial crops. It is unlikely therefore that P. persimilis and N. californicus, now being widely used to control T. urticae in greenhouse crops in Central Europe, will be able to halt any spread of T. evansi to greenhouse crops in temperate areas.     

Morphology, molecular characteristics and prevalence of a Cystosporogenes species (Microsporidia) isolated from Agrilus anxius (Coleoptera: Buprestidae)

A microsporidium was isolated from the bronze birch borer, Agrilus anxius Gory (Coleoptera: Buprestidae), collected near Sudbury and Sault Ste Marie, Canada. Light and electron microscopic investigations showed that gross pathology and ultrastructure of the investigated Cystosporogenes species was similar to those characterized and described for other Cystosporogenes species. Small subunit rRNA gene sequence data and comparative phylogenetic analysis confirmed that the microsporidian species from A. anxius is most closely related to the genus Cystosporogenes clade of microsporidia. Infection average in the Sudbury and Sault Ste Marie beetle populations was >80% and relatively stable in 2006-2007 but declined in 2008. Field prevalence of the A. anxius isolate, mechanisms that may potentially be involved in its horizontal (autoinfection) and vertical (transovarial) transmission, and disease dynamics are discussed. The congeneric relationship between Agrilus planipennis and A. anxius makes it imperative to study the virulence of this Cystosporogenes species in A. planipennis.     

Prey-related odor preference of the predatory mites Typhlodromalus manihoti and Typhlodromalus aripo (Acari: Phytoseiidae)

Typhlodromalus manihoti and Typhlodromalus aripo are exotic predators of the cassava green mite Mononychellus tanajoa in Africa. In an earlier paper, we showed that the two predators were attracted to odors from M. tanajoa-infested cassava leaves. In addition to the key prey species, M. tanajoa, two alternative prey mite species, Oligonychus ossypii and Tetranychus urticae also occur in the cassava agroecosystem. Here, we used a Y-tube olfactometer to determine the attraction of the predators to odors from O. gossypii- or T. urticae-infested cassava leaves and their prey-related odor preference. T. aripo but not T. manihoti was slightly attracted to odors from O. gossypii-infested leaves. Both predator species showed a stronger response to odors from cassava leaves infested by M. tanajoa over odors from cassava leaves infested by O. gossypii. Neither predator species was attracted to odors from T. urticae-infested leaves and the predators preferred the odors from M. tanajoa-infested leaves over those from T. urticae-infested leaves. When O. gossypii was present together with M. tanajoa on the same leaves or on different sets of leaves offered together as an odor source the two predators were attracted. In contrast, after mixing non-attractive odors from T. urticae-infested leaves with attractive odors from M. tanajoa-infested leaves, neither T. aripo nor T. manihoti was attracted. Ecological advantages and disadvantages of the predators’ behavior and possible implications for biological control of M. tanajoa are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.