A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

广州小斑螟发生与环境因子的关系

广州小斑螟是红树林的一种灾害性的食叶害虫.通过室内饲养和野外观察,对广州小斑螟的发生和环境因素的关系进行了详细的研究.结果表明,随着龄级的增加,取食量增大;在不同样地不同滩位的虫口密度差异性规律不同;在单株白骨壤的不同方位虫口密度差异显著,正南方向虫口密度最高,正西、正北虫口密度最低;在单株白骨壤的中上部明显高于下部;广州小斑螟大龄幼虫较耐水淹,水淹6h的死亡率为0;不同地区温度的差异可导致广州小斑螟的发育进度的不同.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at −80 °C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.     

Fate of resting cysts of Alexandrium spp. ingested by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca)

The ingestion of resting cysts of Alexandrium spp. by Perinereis nuntia (Polychaeta) and Theola fragilis (Mollusca) was experimentally examined in the laboratory. P. nuntia and T. fragilis were cultured in bottom sediment containing a high density of Alexandrium cysts under dark conditions. Moreover, to evaluate the degree and consequence of being ingested, the density of cysts in the control sediment (no macrobenthic organisms) and the germination capability of the cysts in the faecal pellets of the two species of macrobenthos were examined.Cysts in the culture sediment were found to be ingested by both P. nuntia and T. fragilis. No difference in the density of cysts between the sediments cultured with and without P. nuntia was observed. However, the density of cysts in the sediments with T. fragilis decreased by 24% compared to the density in the control sediment. It is possible that most of the cysts ingested were digested by T. fragilis. The rate of Alexandrium cyst digestion by this species is estimated 594 cysts/individual/day. It is estimated that 91% of the cysts ingested by T. fragilis were partially or totally digested and only 9% were excreted in a viable state during the experiment. Thus, T. fragilis has a stronger affect on the abundance of Alexandrium cysts compared with P. nuntia.No significant difference was observed between the germination success of the cysts from faecal pellets of P. nuntia and T. fragilis compared to the cysts in the control sediment. If, however, the necessary light for the cysts to germinate is cut off by being enclosed within the faecal pellet, the germination rate of cysts from the faecal pellets may be suppressed.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

太白虎凤蝶的生物学与生境研究

太白虎凤蝶Luehdorfia taibai主要分布于太白山南坡的局部地点,是我国特有种,种群数量稀少,并且呈下降趋势。研究了野生状态下该种蝴蝶的生活史、习性、生命表、产卵地需求,并对部分栖息地要素进行了调查分析。分析了濒危原因,提出了保护建议。太白虎凤蝶1年1代,以蛹越冬,蛹期约300d。翌年4月中下旬为成虫羽化盛期,产卵于幼虫寄主植物马蹄香Saruma henryi叶片背面。1—2龄幼虫聚集生活,3龄即扩散,白天藏入枯叶层中躲避天敌,晚上出来取食。老熟虫于枯叶或石缝中化蛹。产卵地多位于海拔1000—1400 m之间,森林郁闭度低于60%,寄主植物密度低于2株/m2,并且伴有较厚的枯叶层,较多石块的环境中。成虫访花、交尾活动和产卵地环境有所不同,多在沟底较为开阔、多蜜源植物的地点。对太白虎凤蝶的保护建议采取适度干扰原则,对栖息地生境定期实施矮林作业、割灌等经营管理措施,保持1个栖息地斑块内有几块较为开阔的林间空地和寄主植物和寄主植物即可。对于人为干扰过重的秦岭南坡地区,需进行人工管理恢复生境,辅以人工繁殖恢复种群数量。此外,应严格限制对太白虎凤蝶卵、幼虫和成虫的大量采集活动。     

Characterization of Xenorhabdus isolates from La Rioja (Northern Spain) and virulence with and without their symbiotic entomopathogenic nematodes (Nematoda: Steinernematidae)

Eighteen Xenorhabdus isolates associated with Spanish entomopathogenic nematodes of the genus Steinernema were characterized using a polyphasic approach including phenotypic and molecular methods. Two isolates were classified as Xenorhabdus nematophila and were associated with Steinernema carpocapsae. Sixteen isolates were classified as Xenorhabdus bovienii, of which fifteen were associated with Steinernema feltiae and one with Steinernema kraussei. Two X. bovienii Phase II were also isolated, one instable phase isolated from S. feltiae strain Rioja and one stable phase from S. feltiae strain BZ. Four representative bacterial isolates were chosen to study their pathogenicity against Spodoptera littoralis with and without the presence of their nematode host. The four bacterial isolates were pathogenic for S. littoralis leading to septicemia 24 h post-injection and killing around 90% of the insect larvae 36 h post-injection, except for that isolated from S. kraussei. After 48 h of injection, this latter isolate showed a lower final population in the larval hemolymph (10⁷ instead of 10⁸ CFU per larvae) and a lower larval mortality (70% instead of 95-100%). The virulence of the nematode-bacteria complexes against S. littoralis showed similar traits with a significant insect larvae mortality (80-90%) 5 days post-infection except for S. kraussei, although this strain reached similar of larval mortality at 7 days after infection.     

Effect of the snail Ilyanassa obsoleta (Say) on dynamics of the amphipod Corophium volutator (Pallas) on an intertidal mudflat

We investigated interactions between two dominant invertebrate species of intertidal soft-sediment environments of the northwest Atlantic, the mud snail Ilyanassa obsoleta and the burrowing amphipod Corophium volutator, on a mudflat of the upper Bay of Fundy, Canada. Distribution of I. obsoleta on the mudflat was highly patchy and negatively correlated with density of C. volutator. Manipulation of snail density in cages showed that I. obsoleta influences C. volutator; specifically, increasing density of snails reduced density, increased patchiness in distribution, decreased recapture rates and decreased immigration of C. volutator. Ilyanassa obsoleta seems to be affecting C. volutator through an influence on survival rate and emigration rate, although temporal variation in these effects was observed. Given that both I. obsoleta and C. volutator show a preference for tide pools, an important microhabitat on mudflats, snails might have a profound impact on C. volutator population dynamics.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

First record of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) infection in larvae and pupae of Rhizophagusgrandis Gyll. (Coleoptera, Rhizophaginae) from Turkey

The predator beetle Rhizophagus grandis Gyll. (Coleoptera, Rhizophaginae) is one of the most important biological control agents, mass-bred and used to suppress populations of an important pest: the great spruce bark beetle, Dendroctonus micans. The achlorophyllous alga Helicosporidium sp. was first discovered in the pest. Later it was also found in the predator, but only in the adults. In this study, the pathogenic alga Helicosporidium sp. was discovered in larvae and early pupae of R. grandis for the first time. The morphological characteristics of the pathogenic alga were revealed by light and electron microscopy. Infection rates of Helicosporidium sp. in the larvae and pupae of R. grandis were 23.5% and 6.25%, respectively.     

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae)

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10⁵, 10⁶, 10⁷ spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10⁶ and 10⁷ spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.