Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

Stress-related proteins compared in diapause and in activated, anoxic encysted embryos of the animal extremophile, Artemia franciscana

Previous work indicated similarities between diapause and anoxic quiescence in encysted embryos (cysts) of the brine shrimp Artemia franciscana. That possibility was examined further in the present study through an immunochemical study of the following stress-related proteins in low speed supernatants and pellets: hsc70, artemin, p26, hsp21, LEA Group 1 protein and p8. Changes in the amounts and locations of these proteins occurred during the initial period after release of diapause cysts from females, and after activated (diapause-terminated) cysts were made anoxic. However, with the passage of incubation time the patterns seen in both kinds of cysts were more similar than different, lending further support to the possibility that activated anoxic embryos retain many of the mechanisms operative in the previous diapause condition.     

Temperature effects on embryonic development of Paratlanticus ussuriensis (Orthoptera: Tettigoniidae) in relation to its prolonged diapause

Paratlanticus ussuriensis eggs overwinter by entering diapause, which can be prolonged to more than 1 year depending on environmental conditions. To determine temperature effects on diapause duration of P. ussuriensis eggs, the rates of embryonic development and hatching were compared at various temperatures conditions by measuring embryonic stages and egg weights. Most eggs stayed in a very young stage (blastoderm formation, stage 4) when reared at 15 and 20 °C, 10–30% eggs developed into middle or late stages when reared at 25 °C, and most embryos developed fully (stage 23/24) when reared at 30 °C. Egg weight at 30 °C was 1.5 times higher than those reared at 20 °C. Chilling induced hatching in embryos at stage 23/24. Chilling caused stage 4 embryos to develop into stage 24, but they failed to hatch in response to a second warm period. Thus, P. ussuriensis eggs can overwinter either as young embryos (initial diapause) or as fully-developed embryos (final diapause). Eggs that experience an initial diapause overwinter again the second year in a final stage diapause. The post-diapause period was shorter when embryos overwintered in a final stage diapause. The hatching rate was highest in a temperature range of 7.5–15 °C. Our results suggest that temperature is an important environmental factor for the control of prolonged diapause in P. ussuriensis and initial diapause plays an important role in the control of its life cycle.     

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410 bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.     

Enhancing bio-suppression of Parthenium hysterophorus L.: Diapause in Zygogramma bicolorata Pallister and its manipulation through insulin-like peptides (ILPs)

In-depth investigations on diapause behaviour of Z. bicolorata revealed that the adults entered diapause at any time from August to December and that the number peaked (42.00%) during the last half of November. The percentage of adults entering diapause increased with a decrease in day length. Weight of diapausing adults was significantly higher than weight of non-diapausing adults. The percentage of adults undergoing diapause at 30 °C was significantly lower than those undergoing diapause at 15 and 25 °C. The percentage of adults burrowing increased with increasing moisture. In silty soil and soil with high organic matter, 46.7% and 49.2% of adults entered diapause, respectively, whereas in sandy soil, only 23.5% of adults entered diapause. When newly emerged beetles were exposed to 5 μg of human insulin 30/70, a significantly lower percentage of treated adults underwent diapause compared to untreated adults under both feeding and no feeding conditions. Insulin treatment also influenced the emergence period from diapause (93.92 ± 1.73 days), percent emergence (81 ± 1.54%) and fecundity/month (512.7 ± 25.38 eggs) of Z. bicolorata in treated adults as compared to untreated adults (109.05 ± 2.2, 74.00 ± 1.82 and 438.3 ± 19.33 eggs, respectively). However, there was no significant impact of insulin on adult longevity. These findings are of great utility in the biological suppression of Parthenium as it will enhance the effectiveness of this beetle through manipulation of diapause.     

Performance of diapausing parasitoid wasps,Habrobracon hebetor,after cold storage

The ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is an important potential biological control agent for lepidopterous pests of stored products. We investigated the effects of long-term cold storage of diapausing and nondiapausing H. hebetor on their performance after cold storage. Mortality during storage increased with increasing storage duration, and the mortality of diapausing females was lower than that of nondiapausing females after 8, 12, and 16 weeks of storage. Longevity, egg laying, number of progeny produced, and time to 50% egg laying were all reduced, as compared with the culture females when parasitoids were reared at conditions that do not induce diapause. But, for females reared at 20 °C at conditions that induce diapause, all of these quality parameters did not differ from those of culture insects when the storage duration was 8 weeks or less. The percentage of female F1 offspring was always lower for cold stored insects than for the culture insects. Presence of a male after cold storage did not impact any of the quality parameters measured. Thus, rearing parasitoids at 20 °C and 10L:14D and then storing them for up to 8 weeks at 5 °C would produce parasitoids that are similar to culture parasitoids, except that the percentage of females is lower than that in the cultures (36% vs. 52%).     

Embryonic diapause in the Australian plague locust relative to parental experience of cumulative photophase decline

The Australian plague locust Chortoicetes terminifera (Walker) exhibits facultative embryonic diapause during autumn. To approximate natural photoperiod changes during late summer and autumn, locust nymphs were reared under different total declines in laboratory photophase (−0.5, −0.75, −1.0, −1.25, −1.5, −1.75, −2 h each lowered in 15 min steps) in a 24 h photoperiod to quantify any effect on the subsequent production of diapause eggs. Induction of diapause eggs was significantly affected by accumulated photoperiod decline experienced by the parental generation throughout all development stages from mid-instar nymph to fledgling adult. The incidence of embryonic diapause ranged from nil at −0.5 h to 86.6% diapause at −2 h. Continued declines in photoperiod for post-teneral locusts (transitioned from −1 h until fledging to −1.75 h) produced a further increase in the proportion of diapause eggs. The results were unaffected by time spent at any given photoperiod, despite a previously indicated maximal inductive photoperiod of 13.5 h being used as the mid-point of all treatments. Implications for the seasonal timing processes of photoperiodism in C. terminifera, which has a high migratory capacity and a latitudinal cline in the timing of diapause egg production across a broad geographic range, are discussed.     

Heat coma temperature,relative contents of saturated/unsaturated fatty acids and reproductive maturation in the oceanic sea skaters Halobates micans

This study was performed to clarify how the relative volume of saturated/unsaturated lipid and reproductive maturation relate to resistance to high temperature in the oceanic sea skaters, Halobates micans. Heat coma temperature (HCT) was measured in H. micans adults collected from a fixed sampling location (12°00′N, 135°00′E) in the western tropical Pacific Ocean. After measuring HCT, the specimen were dissected to measure the testes size and to determine the presence and number of oocytes in females. Bodies of the specimen were assessed by lipid analysis to evaluate saturated and unsaturated lipid content. A negative trend was seen between heat coma temperature and percentage of a saturated fatty acid, myristic acid (ratio of carbon number to number of double bonds = 14:0) (Pearson's correlation test: r = − 0.520, p = 0.101). In contrast, a positive trend was detected between heat coma temperature and percentage of an unsaturated fatty acid, palmitoleic acid (16:1) (r = 478, p = 0.137). Young males with small testes showed lower heat coma temperatures, whereas females that showed relatively high heat coma temperatures of 36–40 °C tended to have fewer mature oocytes in their ovaries than those that showed low heat coma temperatures of 30–34 °C. As Halobates appears to exhibit embryonic diapause rather than adult diapause, males of H. micans may develop both testes and resistance to high temperature in the parallel as they grow. In females, a trade-off may occur between heat tolerance function and oogenesis in the oceanic sea skaters.     

Multigenerational maternal inhibition of prepupal diapause in two Trichogramma species (Hymenoptera: Trichogrammatidae)

It is known that in some insect species the incidence of diapause among the progeny of females that had undergone diapause is relatively low or zero even under strong diapause-inducing conditions. Moreover, the maternal inhibition, preventing the induction of a maladaptive diapause in spring, can persist over several generations. This multigenerational effect based on hypothetical ‘interval timer’ was thoroughly studied in Aphididae. We first described a similar phenomenon in Hymenoptera: laboratory experiments demonstrated that the proportion of diapausing progeny of Trichogramma females that had undergone diapause was practically zero independently of photoperiodic and temperature conditions used (day lengths of 12 and 18 h and temperatures of 12–15 °C). Then the ability to enter diapause recovered gradually and returned to the normal level over two (in Trichogramma telengai) or even five (in Trichogramma principium) generations. We conclude that the observed effect may be based on an interval timer similar to that in aphids.     

Multi-chaperone-peptide-rich mixture from colo-carcinoma cells elicits potent anticancer immunity

Background: Chaperones play an important role in inducing anti-cancer immunity. To explore the probability of using chaperone-peptide-rich complexes extracted from colo-carcinoma cells as anti-cancer vaccine, we extracted and prepared chaperone-peptide-rich complexes from CT26 cells, which were subsequently investigated on anti-cancer efficacy. Methods: The crude extracts of the CT26 cells treated with heat and Trichosanthin were precipitated with salt and dialyzed to remove proteins below 50 kDa and above 300 kDa in molecular weight; the proteins with the molecular weights in 70 kDa, 90 kDa, 95 kDa, 110 kDa and 170 kDa were collected through gel filtration and SDS-PAGE. After confirmation, the purified proteins were used to determine their effects on lymphocyte proliferation, the activities of NK and CTL, tumor suppression and the tumor-bearing mouse survival. Results: The majority of the chaperone-peptides of anti-cancer immunity in CT26 cells, including HSP70-antigen peptide, HSP90-antigen peptide, gp96-antigen peptide, HSP-110 antigen peptide, HSP170-antigen peptide, was satisfactorily extracted that the multi-chaperone-peptide-rich mixtures were obtained. All the mixtures prepared could elicit lymphocyte proliferation, enhance the activities of CTL and NK, reinforce the tumor suppression and prolong the mouse survival. Conclusions: The multi-chaperone-peptide-rich mixtures could be prepared via dialysis and gel filtration combining with SDS-PAGE. Both the heat stress and Trichosanthin could induce and increase the mixtures, of which that treated by 42 °C heat and Trichosanthin was found to possess the strongest anti-cancer efficacy.     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from ∼ 3–50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of ∼ 1.6 × 10⁷ CFU ml^− 1 (∼ 1.6 × 10⁴ cells spot^− 1), and bound to proteins of ∼ 50 and ∼ 39 kDa. Other MAbs, binding to proteins ranging from ∼ 3–14 and ∼ 40 kDa, detected VVB (but not VVC) with high sensitivity at ∼ 1.6 × 10⁵ and 4 × 10⁶ CFU ml^− 1 (∼ 1.6 × 10² and 4 × 10³ cells spot^− 1), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

Sarcocystis tupaia,sp. nov., a new parasite species employing treeshrews (Tupaiidae,Tupaia belangeri chinensis) as natural intermediate hosts

The range of vertebrates that serve as intermediate hosts for parasites in the genus Sarcocystis remains incompletely defined. Here, we provide the first report of infections in treeshrews, describe the morphology of encysted parasites using light and transmission electron microscopy, and place this agent within a phylogenetic context by sequencing and comparing its 18 S ribosomal DNA to that of related parasites. Muscle infections were diagnosed in four of 45 wild treeshrews captured in the vicinity of Kunming, Yunnan Province, Mainland China. Thread-like cysts (10.773 ± 2.411 mm in length, 0.106 ± 0.009 mm in width) had walls (0.538–0.746 µm thick) that lacked perpendicular protrusions. The interior of the cyst was packed full with cyst merozoites, the shape of which was typical of Sarcocystis. The primary cyst wall consisted of a thin membrane supported by osmiophilic material, 31–60 nm in thickness. The ground substance was about 105–526 nm thickness. Cysts conformed to typical of ‘type 1’ sarcocysts. Freshly examined and frozen specimens did not differ in their cyst wall structure, however, the appearance of bradyzoites did differ: the conoid, rhoptries and micronemes were all visible in fresh bradyzoites; in stored bradyzoites, by contrast, the rhoptries appeared smaller, and although the conoid was visible, the micronemes were not. 18 S rRNA gene was distinct from any previously reported sequence in GenBank. Their genetic and morphological uniformity suggest that these parasites, derived from treeshrews, represent a single biological species, Sarcocystis tupaia, sp. nov.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Effects of constant temperature,exposure period,and age on diapause induction in Trichogramma dendrolimi

Trichogramma dendrolimi can be successfully reproduced in fresh eggs dissected from ovaries of the Chinese tussah silkworm (Antheraea pernyi) and is widely used in biological control of lepidopteran agricultural and forest pests in China. Diapause induction of T. dendrolimi in A. pernyi eggs was investigated through exposing the parasitoid to six constant temperatures (16, 13, 10, 7, 4, and 1 °C) for 19 exposure periods between 10 and 46 days. The sensitive age of T. dendrolimi for diapause induction was explored through a separate experiment to examine the parasitoids that had developed for 2, 3, 4, 5, 6, 7, and 8 days at 26 °C after parasitization, under the six constant temperatures, respectively. Diapause was induced at 10 or 7 °C, and the induction period was 4–6 weeks. The sensitive age of T. dendrolimi to react at the induction temperature was 2–3 days (at 26 °C). At 7 and 10 °C, the diapause rate increased with increasing exposure period and decreased with increased T. dendrolimi age at exposure. The optimum method to induce diapause in T. dendrolimi consisted of exposing hosts for parasitization at 26 °C for 8 h, and then keeping them at 26 °C for 40 h, finally, moving them into 10 °C for 4 weeks.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

Rapid phosphorylation of MAP kinase-like proteins in two species of Arctic kelps in response to temperature and UV radiation stress

Mitogen-activated protein kinases (MAPKs) are a group of cytoplasmic phosphoproteins that constitute the central core of the signalling network to respond to stress in most organisms. Their role in stress responses has been extensively studied in organisms from yeast to humans, and recently, their presence has also been described in higher plants as well as in micro- and macroalgae. In this study, we demonstrate via short experiments (1 h in duration), the rapid activation of two MAPKs similar to p38 and JNK of mammalian cells, in the Arctic kelps Laminaria solidungula and Saccharina latissima exposed to temperature and UV stress. The molecular mass of p38 is 40 kDa in L. solidungula and 42 kDa in S. latissima, while two JNKs were detected in both species, of 36 and 42 kDa in L. solidungula, and 36 and 40 kDa in S. latissima. These MAPKs are highly phosphorylated in response to temperature and UV light. In S. latissima, both p38 and the JNK showed higher phosphorylation at 2 °C than at 7 °C, while the reverse response was shown for L. solidungula. In addition, a significant increase in phosphorylation of both kinases was found following exposure to UV radiation (UVR). Exposure to PAR + UVA + UVB induced higher phosphorylation than PAR + UVA in L. solidungula, especially at 7 °C. In S. latissima, this response occurred only with JNK, and no differences in p38 phosphorylation between PAR + UVA and PAR + UVA + UVB at any temperature were observed. These results indicate the possible participation of MAPK-like proteins in response to stress in Arctic kelps, and that their activation is species-specific.     

Abundance and distribution of toxic Alexandrium tamarense resting cysts in the sediments of the Chukchi Sea and the eastern Bering Sea

Abundance and distribution of the toxic dinoflagellate Alexandrium tamarense species complex resting cyst were investigated in the eastern Bering Sea and the Chukchi Sea for the first time. Sediment samples (top 0–3 cm depth) were collected from the continental shelf of the eastern Bering Sea (17 stations) and the Chukchi Sea (13 stations) together with a long core sample (top 0–21 cm depth) from one station in the Chukchi Sea during 2009–2012. The cysts were enumerated using the primuline staining method. Species identification of the cysts was carried out with multiplex PCR assay and the plate morphology of vegetative cells germinated from cysts in the both areas. Alexandrium cysts were widely detected in the both areas, ranging from not detected (<1 cysts cm⁻³) to 835 cysts cm⁻³ wet sediment in the eastern Bering Sea and from not detected (<1 cysts cm⁻³) to 10,600 cysts cm⁻³ in the Chukchi Sea, and all isolated cysts were genetically and morphologically identified as the North American clade A. tamarense. Their cysts were mainly distributed in the shallow continental shelf where the water depth was less than 100 m in both areas. The cysts were detected from the deep layer (18–21 cm depth of sediment core) of the long core sample. The present study confirmed the abundant existence of A. tamarense with wide range of distribution in these areas. This fact suggests that A. tamarense vegetative cells have appeared in the water column in the both areas. Furthermore, these abundant cyst depositions indicate that this species originally distributed in the Arctic and subarctic regions and well adapted to the environments in the marginal ice zone.     

The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae

Heterodera avenae is a devastating plant pathogen that causes significant yield losses in many crops, but there is a lack of scientific information whether this pathogen can be controlled effectively using biocontrol agents. Here we determined the parasitic and lethal effects of Trichoderma longibrachiatum against H. avenae and the possible mechanism involved in this action. Both in vitro and greenhouse experiments were conducted. In vitro, T. longibrachiatum at the concentrations of 1.5 × 10⁴ to 1.5 × 10⁸ spores per ml had a strong parasitic and lethal effect on the cysts of H. avenae, with the concentration of 1.5 × 10⁸ spores per ml having >90% parasitism 18 days after treatments. In greenhouse, T. longibrachiatum inoculation decreased H. avenae infection in wheat (Triticum aestivum) significantly. Observations with microscopes revealed that after mutual recognition with cysts, the spore of T. longibrachiatum germinated with a large number of hyphae, and reproduced rapidly on the surface of cysts. Meanwhile, the cysts surface became uneven, with some cysts producing vacuoles, and the others splitting. Finally the cysts were dissolved by the metabolite of T. longibrachiatum. Chitinase activity increased in the culture filtrates of T. longibrachiatum and reached the maximum 4 days after inoculation in the medium supplemented with colloidal chitin (1.02 U/min per ml) and nematode cysts (0.78 U/min per ml). The parasitism and inhibition of cysts through the increased extracellular chitinase activity serves as the main mechanism with which T. longibrachiatum against H. avenae. In conclusion, T. longibrachiatum has a great potential to be used as a biocontrol agent against H. avenae.     

Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): First discovery of giant hexagonal-bilayer hemoglobin in the former “Pogonophora” group

Siboglinids are symbiotic polychete annelids having hemoglobins as essential oxygen- and sulfide-carriers for their endosymbiotic bacteria. We analyzed the structure of the hemoglobins from two species of siboglinids: the monilifera Sclerolinum contortum and the frenulata Oligobrachia webbi (i.e. haakonmosbiensis) from Norwegian cold seeps. Measured by Multi-Angle Laser Light Scattering (MALLS), Sclerolinum shows a 3190 ± 50 kDa hexagonal bilayer hemoglobin (HBL-Hb) and a 461 ± 46 kDa ring-Hb, just as vestimentifera, whereas Oligobrachia has a 409 ± 3.7 kDa ring-Hb only. Electrospray Ionization-Mass Spectrometry (ESI-MS) showed Sclerolinum HBL-Hb composed of seven monomeric globins (15–16 kDa), three disulfide-bonded globin heterodimers and three linkers. The heterodimers always contain globin-b (15814.4 ± 1.5 Da). Sclerolinum ring-Hb is composed of globins and dimers with identical masses as its HBL-Hb, but lacks linkers. Oligobrachia ring-Hb has three globin monomers (14–15 kDa) only, with no disulfide-bonded dimers. Comparison of Sclerolinum hemoglobins between Storegga and Haakon Mosby Mud Volcano, using the normalized height of deconvoluted ESI-MS peaks, shows differences in globin monomers abundances that could reflect genetic differences or differential gene expression between distinct seep populations. The discovery of HBL-Hb in Sclerolinum is a new element supporting the hypothesis of monilifera being phylogenetically more closely related to vestimentifera, than to frenulata.