Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.     

Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis

The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.     

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines (P<0.05) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score (P<0.01) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3-7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.     

Parasitization of fifth instar tasar silkworm, Antheraea mylitta, by the uzi fly, Blepharipa zebina; a host-parasitoid interaction and its effect on host's nutritional parameters and parasitoid development

The uzi fly, Blepharipa zebina, is a well-known larval endoparasitoid of the tropical tasar silkworm, Antheraea mylitta. The present study dealt with the effect of the number of maggots developing per host on host nutritional parameters, parasitoid development and reproduction. Nutritional indices for ingestion, digestion, approximate digestibility, relative consumption rate, relative growth rate, and gain in body weight declined significantly with the increase in parasitoid burden, but the efficiency of conversion of digested food recorded a significant increase. The efficiency of conversion of ingested food remained little affected. The developmental period was significantly extended in larvae parasitized with 5 and 10 maggots per larva (mpl). Cocoon shell weight decreased by 27-63.5% in parasitized groups (1, 2, and 5 mpl) while larvae parasitized with 10 mpl could not spin cocoons. The maggot development period, recovery percentage, and fecundity of the uzi fly declined significantly with the increase in number of maggots developing per host.     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

Genetic analysis of influences on survival following Toxoplasma gondii infection.

Survival of mice during the acute stage of Toxoplasma gondii infection was not influenced by the MHC Class I gene, L(d), but was influenced by the MHC Class II genes, Ia and Ie. As unexplained variability was noted in our initial studies of influence of the L(d) gene on survival, influence of the L(d) gene region on survival in the presence of a number of variables was studied. Although route of administration and dose of parasites, and age and gender of the mice markedly influenced outcome of T. gondii infection, the Class I L(d) gene did not modify survival in any of these circumstances. In separate studies, using mice with a differing genetic background, i.e. H-2(b), C57BL/10 mice, presence of Ia or Ie alone diminished survival even though presence of Ia reduced parasite burden. When neither or both the Ia and Ie genes were present together, survival was greater. In separate analyses of our studies of AxB BxA recombinant inbred mice, similar influences of MHC genes on survival and parasite burden following peroral infection were confirmed. Previously undescribed associations of novel genetic loci and survival and parasite burden also were identified. Genetic loci associated with enhanced survival included D8Mit42, D1Mit3, Iapls1-16, D8Mit14, Hoxb, Mpmv29, Pmv45, and Emv-2; genetic loci associated with reduced parasite burden included H-2, D17Mit62, D17Mit83, D17Mit21, D17Mit34, D17Mit47, D18Mit4, and Gln3-5. These studies demonstrate the importance of MHC region genes (but not L(d)) for survival, and the influence of other novel genes, and endogenous and exogenous variables on survival and parasite burden specified by host genes following T. gondii infection.     

Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture

A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.     

Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa

The methanol extract from the bark of Yucca periculosa F. Baker afforded 4,4'-dihydroxstilbene, resveratrol and 3,3',5,5'-tetrahydroxy-4-methoxystilbene and had growth regulatory activity against the Fall Army worm (Spodoptera frugiperda J.E. Smith, Lepidoptera:Noctuidae) an insect pest of corn. The most active compound was 3,3',5,5'-tetrahydroxy-4-methoxystilbene which had significant effects at 3 microg/g in diets. In addition to the inhibitory activity on bleaching of crocin induced by alkoxyl radicals, these compounds also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl in TLC autographic and spectrophotometric assays. Our results indicate that these compounds could be involved in interference of sclerotization and moulting. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results were fully comparable to known natural insect growth inhibitors such as gedunin and Cedrela extracts and have had a possible role as natural insecticidal agents.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.