The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.
An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).
Morphological and host-plant relationship studies were conducted to differentiate two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice (Oryza sativa) and the other from Leersia hexandra, a weed grass. In morphometric studies based on esterase activities, an UPGMA dendrogram using 17 quantitative morphological characters, including stridulatory organs (courtship signal-producing organs) between two sympatric populations of N. lugens, one from rice and the other from L. hexandra, a weed grass revealed that both populations were separated from each other. An out-group, N. bakeri, was found to be completely different from the two sympatric populations of N. lugens. Rice plants were best suited for the establishment of the rice-infesting population, and L. hexandra was a favourable host for the Leersia-infesting population. The individuals derived from one host did not thrive on the other host, as shown by a significant reduction in survival and nymphal development, ovipositional preferences, ovipositional response, and egg hatchability. Therefore, morphological and host-plant relationship studies indicate that rice-associated population with high esterase activities and L. heaxandra-associated population with low esterase activities are two closely related sibling species.
The inhibitory effect of active fractions of Eurycoma longifolia (E. longifolia) root, namely TAF355 and TAF401, were evaluated against Toxoplasma gondii (T. gondii). In our previous study, we demonstrated that T. gondii was susceptible to TAF355 and TAF401 with IC₅₀ values of 1.125 µg/mL and 1.375 µg/mL, respectively. Transmission (TEM) and scanning electron microscopy (SEM) observations were used to study the in situ antiparasitic activity at the IC₅₀ value. Clindamycin was used as positive control. SEM examination revealed cell wall alterations with formation of invaginations followed by completely collapsed cells compared to the normal T. gondii cells in response to the fractions. The main abnormality noted via TEM study was decreased cytoplasmic volume, leaving a state of structural disorganization within the cell cytoplasm and destruction of its organelles as early as 12 h of treatment, which indicated of rapid antiparasitic activity of the E. longifolia fractions. The significant antiparasitic activity shown by the TAF355 and TAF401 active fractions of E. longifolia suggests their potential as new anti-T. gondii agent candidates.
Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
Most studies on human immunity to malaria have focused on the roles of immunoglobulin G (IgG), whereas the roles of IgM remain undefined. Analyzing multiple human cohorts to assess the dynamics of malaria-specific IgM during experimentally induced and naturally acquired malaria, we identified IgM activity against blood-stage parasites. We found that merozoite-specific IgM appears rapidly in Plasmodium falciparum infection and is prominent during malaria in children and adults with lifetime exposure, together with IgG. Unexpectedly, IgM persisted for extended periods of time; we found no difference in decay of merozoite-specific IgM over time compared to that of IgG. IgM blocked merozoite invasion of red blood cells in a complement-dependent manner. IgM was also associated with significantly reduced risk of clinical malaria in a longitudinal cohort of children. These findings suggest that merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity.
This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria-host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.
Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.
BACKGROUND: Sequestration of parasitized red blood cells from the peripheral circulation during an infection with Plasmodium falciparum is caused by an interaction between the parasite protein PfEMP1 and receptors on the surface of host endothelial cells, known as cytoadherence. Several lines of evidence point to a link between the pathology of severe malaria and cytoadherence, therefore blocking adhesion receptors involved in this process could be a good target to inhibit pRBC sequestration and prevent disease. In a malaria endemic setting this is likely to be used as an adjunct therapy by reversing existing cytoadherence. Two well-characterized parasite lines plus three recently derived patient isolates were tested for their cytoadherence to purified receptors (CD36 and ICAM-1) as well as endothelial cells. Monoclonal antibodies against human CD36 and ICAM-1 were used to inhibit and reverse infected erythrocyte binding in static and flow-based adhesion assays.
RESULTS: Anti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates.
CONCLUSIONS: The results show that, as a proof of concept, disturbing existing ligand-receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.
Platelets are understood to assist host innate immune responses against infection, although direct evidence of this function in any human disease, including malaria, is unknown. Here we characterized platelet-erythrocyte interactions by microscopy and flow cytometry in patients with malaria naturally infected with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium knowlesi Blood samples from 376 participants were collected from malaria-endemic areas of Papua, Indonesia, and Sabah, Malaysia. Platelets were observed binding directly with and killing intraerythrocytic parasites of each of the Plasmodium species studied, particularly mature stages, and was greatest in P vivax patients. Platelets preferentially bound to the infected more than to the uninfected erythrocytes in the bloodstream. Analysis of intraerythrocytic parasites indicated the frequent occurrence of platelet-associated parasite killing, characterized by the intraerythrocytic accumulation of platelet factor-4 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling of parasite nuclei (PF4+TUNEL+ parasites). These PF4+TUNEL+ parasites were not associated with measures of systemic platelet activation. Importantly, patient platelet counts, infected erythrocyte-platelet complexes, and platelet-associated parasite killing correlated inversely with patient parasite loads. These relationships, taken together with the frequency of platelet-associated parasite killing observed among the different patients and Plasmodium species, suggest that platelets may control the growth of between 5% and 60% of circulating parasites. Platelet-erythrocyte complexes made up a major proportion of the total platelet pool in patients with malaria and may therefore contribute considerably to malarial thrombocytopenia. Parasite killing was demonstrated to be platelet factor-4-mediated in P knowlesi culture. Collectively, our results indicate that platelets directly contribute to innate control of Plasmodium infection in human malaria.
Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.
Schistosomiasis is a major parasitic disease of humans, second only to malaria in its global impact. The disease is caused by digenean trematodes that infest the vasculature of their human hosts. These flukes are limited externally by a body wall composed of a syncytial epithelium, the apical surface membrane of which is a parasitism-adapted dual membrane complex. Annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the first structural and immunobiochemical characterization of an annexin from Schistosoma mansoni. The crystal structure of annexin B22 confirms the presence of the previously predicted α-helical segment in the II/III linker and reveals a covalently linked head-to-head dimer. From the calcium-bound crystal structure of this protein, canonical type II, type III and B site positions are occupied, and a novel binding site has been identified. The dimer arrangement observed in the crystal structure suggests the presence of two prominent features, a potential non-canonical membrane binding site and a potential binding groove opposite to the former. Results from transcriptional profiling during development show that annexin B22 expression is correlated with life stages of the parasite that possess the syncytial tegument layer, and ultrastructural localization by immuno-electron microscopy confirms the occurrence of annexins in the tegument of S. mansoni. Data from membrane binding and aggregation assays indicate the presence of differential molecular mechanisms and support the hypothesis of annexin B22 providing structural integrity in the tegument.
Leucocytozoon coracinae sp. nov. is described from the avian family Campephagidae and Hepatozoon apodis sp. nov. from the Apodidae. The distribution of these parasites within their respective families is discussed.
Two new and two previously described species of diplectanid monogeneans (Heteroplectanum flabelliforme n. sp., Diplectanum sumpit n. sp., D. jaculator Mizelle & Kritsky, 1969 and D. toxotes Mizelle & Kritsky, 1969) were collected from archerfish Toxotes jaculatrix off the Island of Langkawi, Kedah and off Perak, Malaysia. The reproductive systems and squamodiscs of D. jaculator and D. toxotes are described for the first time. D. sumpit n. sp. differs from D. toxotes and D. jaculator in a having a small curved copulatory tube with a distinct accessory piece, compared to the long, tubular copulatory tube of D. jaculator and the slender tube of D. toxotes. D. sumpit n. sp. also differs from D. toxotes in having a larger ventral bar and larger squamodiscs. H. flabelliforme n. sp. differs from all known Heteroplectanum species in the shape and size of the squamodiscs, the arrangement of the sclerites in the squamodiscs, the extremely large ventral bar and the short, curved, non-spinous copulatory tube.
A parasitological survey of 16 pangolins, confiscated from the Department of Wildlife and Nature Park Peninsular Malaysia (DWNP) at Kelantan and Pulau Pinang, Malaysia was conducted in 2011. Amblyomma javanense (family: Ixodidae) was the only ectoparasite found on the pangolins. The prevalence, intensity and life cycle of A. javanense were observed together with the respective pangolins' age and sex. It was found that 68.8% of the pangolins were infected, and significant difference, χ(2)(1, N=16)=4.02, p=0.05 were observed with males higher in infestation (88.9%) as compared to the females (42.9%). However, the mean intensity was higher on females (72) as compared to males (31.6). In addition, significant difference, χ(2) (2, N=16)=6.73, p=0.05 was recorded between adults and juveniles with juveniles found to be 100% infected as compared to adult (63.6%). Nevertheless, the mean intensity was slightly higher on adults (47) than juveniles (35). Adult ticks were found in higher numbers as compared to the nymph and larvae with number of male ticks higher (236) as compared to the females (53). Similarly, a high significant difference χ(2)(2, N=469)=203.47, p=0.05 was recorded in the composition of the tick's life stages with a higher number of adult ticks (61.6%) followed by nymph (30.3%) and larvae (8.1%). As such, the results of this study revealed a picture of the A. javanense life cycle which is related to the age and gender of the Malayan Pangolin.
Frogs caught from two States (Selangor and Langkawi) in Malaysia were examined for spargana of Spirometra sp. Infected frogs usually show no marks of infection but some had swelling and bleeding at the infection site. The size and weight of the infected frogs did not correlate with the infection status. The infection status in relation to human health is discussed.
Empruthotrema stenophallus n. sp. (Monogenea: Monocotylidae) is described from specimens from the nasal tissue of the blue-spotted maskray Dasyatis kuhlii (Muller and Henle, 1841) collected in shallow waters off Pulau Banggi and Pulau Mabul, Sabah, Borneo, Malaysia. This is the first monogenean species to be described from an elasmobranch collected from Sabah. E. stenophallus can be distinguished from the other 6 members of the genus by the morphology of the sclerotized male copulatory organ, which is narrow, short, and distally tapered. E. dasyatidis Whittington and Kearn, 1992, previously documented from the nasal tissue of several of elasmobranch species from Australia, is recorded from 8 host species distributed around Malaysian Borneo. These represent new host and locality records for this monocotylid. The difficulties in identifying species of Empruthotrema and the apparent lack of host specificity by some members of the genus are discussed.
Wildlife are now recognised as an important source of emerging human pathogens, including parasites. This paper discusses the linkages between wildlife, people, zoonotic parasites and the ecosystems in which they co-exist, revisits definitions for 'emerging' and 're-emerging', and lists zoonotic parasites that can be acquired from wildlife including, for some, estimates of the associated global human health burdens. The paper also introduces the concepts of 'parasite webs' and 'parasite flow', provides a context for parasites, relative to other infectious agents, as causes of emerging human disease, and discusses drivers of disease emergence and re-emergence, especially changes in biodiversity and climate. Angiostrongylus cantonensis in the Caribbean and the southern United States, Baylisascaris procyonis in California and Georgia, Plasmodium knowlesi in Sarawak, Malaysia, Human African Trypanosomiasis, Sarcoptes scabiei in carnivores, and Cryptosporidium, Giardia and Toxoplasma in marine ecosystems are presented as examples of wildlife-derived zoonotic parasites of particular recent interest. An ecological approach to disease is promoted, as is a need for an increased profile for this approach in undergraduate and graduate education in the health sciences. Synergy among scientists and disciplines is identified as critical for the study of parasites and parasitic disease in wildlife populations. Recent advances in techniques for the investigation of parasite fauna of wildlife are presented and monitoring and surveillance systems for wildlife disease are discussed. Some of the limitations inherent in predictions for the emergence and re-emergence of infection and disease associated with zoonotic parasites of wildlife are identified. The importance of public awareness and public education in the prevention and control of emerging and re-emerging zoonotic infection and disease are emphasised. Finally, some thoughts for the future are presented.
Clostridium bifermentans serovar malaysia (C.b.m.) is highly toxic to mosquito larvae. In this study, the following aquatic nontarget invertebrates were treated with high C.b.m. concentrations (up to 1,600-fold the toxic concentration for Anopheles stephensi) to study their susceptibility towards the bacterial toxin: Planorbis planorbis (Pulmonata); Asellus aquaticus (Isopoda); Daphnia pulex (Cladocera); Cloeon dipterum (Ephemeroptera); Plea leachi (Heteroptera); and Eristalis sp., Chaoborus crystallinus, Chironomus thummi, and Psychoda alternata (Diptera). In addition, bioassays were performed with mosquito larvae (Aedes aegypti, Anopheles stephensi, and Culex pipiens). Psychoda alternata larvae were very susceptible, with LC50/LC90 values comparable to those of mosquito larvae (about 10(3)-10(5) spores/ml). The tests with Chaoborus crystallinus larvae showed significant mortality rates at high concentrations, but generally not before 4 or 5 days after treatment. The remaining nontarget organisms did not show any susceptibility. The investigation confirms the specificity of C.b.m. to nematocerous Diptera.