Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

GPR39 up-regulation after selective antidepressants

Recent studies indicated that zinc activates neural transmission via the GPR39 Zn²⁺-sensing receptor. Preclinical and clinical studies demonstrated the antidepressant properties of zinc. To investigate whether the GPR39 receptor is involved in the mechanism of antidepressant action, we measured the expression of the GPR39 receptor (Western Blot) in the frontal cortex of mice treated intraperitoneally with imipramine (30 mg/kg), escitalopram (4 mg/kg), reboxetine (10 mg/kg) or bupropion (15 mg/kg) for 14 days. The present study shows the up-regulation of the GPR39 receptor protein level after escitalopram (by 290%), reboxetine (by 816%) and bupropion (by 272%), but not imipramine treatment. This is the first report to indicate the involvement of the GPR39 Zn²⁺-sensing receptor in the antidepressant effect of selective monoamine reuptake inhibitors.     

Cellular and molecular responses of haemocytes from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8 h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1 h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4 h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1 h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters

The study was conducted to examine the occurrence and interaction between Oestrus ovis and Haemonchus contortus in experimentally infected Ethiopian Afar breed of goats. Twenty goats were divided into four groups (O, OH, H, and C) of five animals each. Each animal of groups O and OH received weekly infections for 5 weeks with 66 first instar larvae (L₁) of O. ovis. Then animals of groups OH and H were infected with a single dose of 5000 third stage larvae (L₃) of H. contortus. Goats of group C were kept free of any infection as non-infected control. Faecal egg count (FEC), blood cell count, total serum protein level and body weight were recorded weekly throughout the study period. At necropsy worm burden, female worm length, fecundity and larval burden of O. ovis in the nasal-sinus cavities of infected animals were assessed. The results showed that the presence of H. contortus in the abomasum of goats of group OH had no influence on the development of O. ovis. On the contrary, a significant reduction (P < 0.05) in FEC, worm burden, fecundity and female worm length was revealed in group OH animals compared to the mono-infected animals (group H). This was associated with eosinophilia and reduced packed cell volume.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Cloning,molecular characterization,and spatial and developmental expression analysis of GPR41 and GPR43 genes in New Zealand rabbits

Short-chain fatty acids (SCFAs) play a regulatory role in various physiological processes in mammals and act as endogenous ligands for the G protein-coupled receptors (GPR) 41 and 43. The role of GPR41 and GPR43 in mediating SCFA signaling in the rabbit remains unclear. The present study was to investigate the sequence of the GPR41 and GPR43 messenger RNA (mRNA) and their expression pattern in different tissues and developmental stages in New Zealand rabbit. Comparison of genomic sequences in GenBank using the Basic Local Alignment Search Tool program suggested that the New Zealand rabbit GPR41 mRNA has high similarities with the human (84%), bovine (84%) and Capra hircus (84%) genes. Similarly, GPR43 mRNA has high similarity with the rat (84%) and mouse (84%) genes. Real-time PCR results indicated that GPR41 and GPR43 mRNA were expressed throughout rabbit’s whole development and were expressed in several tissues. G protein-coupled receptor 41 and GPR43 mRNA were most highly expressed in pancreas (P<0.05) and s.c. adipose tissue (P<0.05), respectively. The expression levels of GPR41 mRNA was down-regulated in duodenum, cecum (P<0.05) and pancreas and up-regulated in jejunum, ileum, adipose tissue and spleen during growth. G protein-coupled receptor 43GPR43 mRNA was highly expressed in the duodenum, jejunum, ileum, colon, cecum and lung at 15^th day (P<0.05), whereas the expression levels in the pancreas and spleen increased later after birth, with the highest expression at 60^th day (P<0.05).     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Nutritional value of the cryptophyte Rhodomonas lens for Artemia sp.

Juvenile or adult Artemia sp. are often used as live prey for the rearing of early life stages of some crustacean, fish and cephalopod species. The improvements of both Artemia growth and its biochemical composition are key issues for the suitable use of Artemia biomass in these rearing processes. In this study we evaluated the growth and survival rates of Artemia fed with the cryptophyte Rhodomonas lens in comparison with different microalgal species commonly used in aquaculture: the prasinophyte Tetraselmis suecica, the prymnesiophyte Isochrysis galbana Parke, and the eustigmatophyte Nannochloropsis gaditana. Microalgae were cultured semi-continuously in nutrient saturated conditions and with a daily renewal rate of 30% of the volume of cultures, to obtain biomass of controlled and optimized composition. Considerable differences in Artemia growth were observed, as well as in the survival rate. At day 8 of rearing, Artemia fed R. lens had the highest length (4.9 ±0.6 mm, P < 0.001), followed by individuals fed T. suecica (4.2 ± 0.7 mm), I. galbana (3.6 ± 0.7 mm) and finally those fed N. gaditana (1.5 ± 0.2 mm). The survival rate of Artemia fed N. gaditana (18 ± 3%) was much lower (P < 0.001) than values found for the remaining groups (69 to 88%). The growth rate of Artemia obtained with R. lens was in general much higher than with other microalgal diets previously reported in the literature. The higher protein content of R. lens could explain the higher growth obtained with this species, but differences of Artemia growth with the different diets could not be explained solely on the basis of the gross composition of microalgae. Factors such as cell size and digestibility all seem to contribute to the results observed. Another trial was carried out to investigate differences in Artemia growth and on its biochemical composition when fed the best two diets: R. lens or T. suecica. The fatty acid (FA) and total amino acid (AA) composition of both microalgal species and the composition of Artemia were assessed as well. As found in the first experiment individuals fed R. lens (group ARHO) grew faster than those fed T. suecica (group ATET), attaining 3.6 ± 0.3 mm and 3.2 ± 0.4 mm (P < 0.001), respectively, after 5 days of rearing. The much higher AA content obtained in R. lens may be on the basis of the higher growth obtained with this species. Protein and carbohydrate levels in Artemia juveniles were very similar in both groups (64-68% of dry weight, and 8-10%, respectively). Lipid was slightly lower in ARHO (12%) than in ATET (15%, P < 0.01). Regarding the FA composition, juveniles from group ARHO contained higher levels of eicosapentaenoic acid (EPA, 6.2%) than juveniles from ATET (4.1%, P < 0.01), whereas docosahexaenoic acid (DHA) was only found in juveniles from ARHO (1.1%). Taking into account that the daily productivity of R. lens culture was higher than, or at least equal, the remaining microalgal species this cryptophyte is confirmed as an excellent diet to optimize the growth of Artemia, as well as to improve its biochemical composition.     

Effects of Bayluscide on Bithynia siamensis goniomphalos, the first intermediate host of the human liver fluke, Opisthorchis viverrini, in laboratory and field trials

The molluscicidal effects of Bayluscide (niclosamide) were investigated on Bithynia siamensis goniomphalos, the first intermediate host of human liver fluke, Opisthorchis viverrini. Lethal concentrations of 50% (LC₅₀) and 95% (LC₉₅) against young and adult males were 0.38 and 0.80, 0.42 and 0.86 ppm, respectively. The LC₅₀ and LC₉₅ against young and adult females were 0.42 and 0.86, 0.46 and 0.97 ppm, respectively. No significant differences in mortality rate between sexes or snail size (p > 0.05) was detected. Bayluscide-related tissue damage in B. siamensis goniomphalos included detachment of cilia of the epithelial layer of the digestive tract and decreased number of calcium cells. In tests of lethal concentrations of Bayluscide on non-target animals, no lethal effect was observed on Filopaludina martensi martensi (Viviparous snail) but high mortality rates were recorded in Puntius gonionotus fingerling, Ricefish (Oryzias mekongensis) and shrimp (Macrobrachium lanchesteri), but lower in guppy fish (Poecilia reticulata) after 24 h exposure. For field trials, sufficient Bayluscide was sprayed in 3 roadside ditches to result in final concentrations of 5, 10 or 20 ppm, with mortality rates on B. siamensis goniomphalos of 10.94, 20.00 and 31.25%, respectively. Non-target snails died in small numbers but no effect was observed in other aquatic vertebrate animals. Field trials of Bayluscide on B. siamensis goniomphalos revealed low mortality rates, suggesting the need for application methods of higher efficacy or that Bayluscide is not suitable for application to operculate snails or snails which are able to escape by burying in mud.     

Toxoplasma gondii: A morphometric analysis of the wall and epithelial cells of pigs intestine

The aim of this study was to perform a morphometric analysis of the different layers of the jejunal wall and epithelial cells of pigs with toxoplasmosis. Experiments were conducted using 10, 88-day-old crossbred (Pietran × Wessex) pigs divided into two groups: control (n = 5) and experimental (n = 5). The experimental group consisted of animals inoculated orally with 5000 sporulated oocysts of a genotype III strain of Toxoplasma gondii. At 30 and 60 days following inoculation, the animals were anaesthetised for jejunal biopsy. The intestinal segments were processed routinely for histology. Transverse cuts (4 μm thick) were stained with haematoxylin and eosin (HE), Periodic Acid Schiff (PAS), Alcian Blue (AB), pH 2.5, and Alcian Blue (AB), pH 1.0. We observed hypertrophy of the jejunal wall, increased enterocyte height, and a decreased number of intraepithelial lymphocytes in the infected animals. There were no changes in the number of goblet cells.     

Larvae of the ectoparasitic wasp,Eulophus pennicornis,release factors which adversely affect haemocytes of their host,Lacanobia oleracea

When larvae of the ectoparasitic wasp Eulophus pennicornis were incubated for 4 h on balls of cotton wool soaked in tissue culture medium (TC-100), they released a variety of factors. Subsequent incubation of these larval wasp secretions with monolayers of haemocytes from their host, Lacanobia oleracea, demonstrated that they adversely affect haemocyte morphology, behaviour and viability. For instance, when monolayers of haemocytes were incubated for 18 h in TC-100, approximately 73% of the cells present, attached firmly to and spread over the tissue culture surface by extending pseudopods. By contrast, when incubated in TC-100 containing larval wasp secretions, only about 27% of the haemocytes present remained attached to the tissue culture surface after washing. The majority of these had a rounded configuration and neither spread nor extended pseudopods. Furthermore, viability assays indicated that approximately 36% of the attached haemocytes were dead, as opposed to 11-12% in the controls. The E. pennicornis secretions also significantly reduced the ability of L. oleracea haemocytes to move across the surface of the slide and form clumps (p≤0.0005) and to phagocytose FITC-labelled Escherichia coli in vitro (p≤0.0005). These results indicate that secretions from E. pennicornis larvae contain an anti-haemocyte factor(s) that can kill and/or alter the behaviour of host haemocytes. As a result, the ability of the haemocytes to execute important immune responses is compromised. Preliminary data suggest that the active molecules are proteins, and that their mechanism of action may involve inhibition of polymerization and/or disorganization of the haemocyte cytoskeleton.     

Effect of Acanthocephala infection on the reproductive potential of crustacean intermediate hosts

The effect of a naturally acquired infection by three acanthocephalan parasites Dentitruncus truttae, Echinorhynchus truttae, and Polymorphus minutus on the reproductive potential of their intermediate host, Echinogammarus tibaldii (Amphipoda) from Lake Piediluco (Centre of Italy) was assessed. During May 2007, 1135 amphipods were collected from two different samplings and examined for larval helminths. Forty-five amphipods were infected and of those, 16 were infected with D. truttae (intensity = 1-3 larvae), 15 with E. truttae (intensity = 1-2 larvae), and 14 with P. minutus (intensity = 1 larva). The sex ratio was nearly 1:1 in all examined amphipods. One female infected with D. truttae contained six eggs in the brood pouch and another female infected with E. truttae contained five eggs. However, none of the eight female amphipods harbouring P. minutus larva contained eggs in their brood pouch. Uninfected females of the same size and body length as that of the infected females contained between 20 and 32 eggs. No acanthocephalan species were found to co-occur.     

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5-11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.     

Cold stenothermal cave-dwelling beetles do have an HSP70 heat shock response

The response to high temperatures in adults of two cold stenothermal cave-dwelling leptodirins, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae) was evaluated by determinating levels of gene expression of two members of the family of heat shock proteins 70 kDa by qPCR. In both species, hsc70 mRNA level was constant with increasing temperature, whereas a significant increase in the inducible member (hsp70) mRNA was observed, higher in N. pasai. This difference could be due to their in-cave distribution: N. pasai colonizes the cave entrance where the temperature is more variable than the internal part where N. mancinii is confined. These results demonstrated for the first time the occurrence of a heat shock response in troglobite insects and suggest the correlation between the intensity of this response and the adaptation to the cave environment.     

Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.     

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.