On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

Armigeres subalbatus colonization of damaged pit latrines: a nuisance and potential health risk to residents of resettlement villages in Laos.

During the resettlement of 6500 persons living around the Nam Theun 2 hydroelectric project in Laos, more than 1200 pour-flush latrines were constructed. To assess the role of these latrines as productive larval habitats for mosquitoes, entomological investigations using Centers for Disease Control (CDC) light traps, visual inspection and emergence trapping were carried out in over 300 latrines during the rainy seasons of 2008-2010. Armigeres subalbatus (Diptera: Culicidae) were nine times more likely to be found in latrines (mean catch: 3.09) than in adjacent bedrooms (mean catch: 0.37) [odds ratio (OR) 9.08, 95% confidence interval (CI) 6.74-15.11] and mosquitoes were active in and around 59% of latrines at dusk. Armigeres subalbatus was strongly associated with latrines with damaged or improperly sealed septic tank covers (OR 5.44, 95% CI 2.02-14.67; P < 0.001). Armigeres subalbatus is a nuisance biter and a putative vector of Japanese encephalitis and dengue viruses. Dengue virus serotype 3 was identified from a single pool of non-blood-fed female A. subalbatus using reverse transcription polymerase chain reaction. Maintaining a good seal around septic tanks by covering them with a layer of soil is a simple intervention to block mosquito exit/entry and contribute to vector control in resettlement villages. The scale-up of this simple, cheap intervention would have global impact in preventing the colonization of septic tanks by nuisance biting and disease-transmitting mosquitoes.

Sex determination in malaria parasites.

A century ago, W. G. MacCallum identified distinct male and female forms in malaria parasites of both birds and humans. Since then, scientists have been puzzled by the high female-to-male ratios of parasites in Plasmodium infections and by the mechanism of sex determination. The sex ratio of malaria parasites was shown to become progressively more male as conditions that allow motility and subsequent fertilization by the male parasites become adverse. This resulted from an increased immune response against male gametes, which coincides with intense host erythropoietic activity. Natural and artificial induction of erythropoiesis in vertebrate hosts provoked a shift toward male parasite production. This change in parasite sex ratio led to reduced reproductive success in the parasite, which suggests that sex determination is adaptive and is regulated by the hematologic state of the host.

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections.

BACKGROUND: Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections. RESULTS: Whereas there was a trade-off between transmission success and virulence as defined by host mortality, contradictory clone-specific patterns occurred when defining virulence by anaemia. A negative relationship between anaemia and transmission success was found for one of the parasite clones, whereas there was no relationship for the other. Notably the two parasite clones also differed in a transmission phenotype (gametocyte sex ratio) that has previously been shown to respond adaptively to a changing blood environment. In addition, as predicted by evolutionary theory, mixed infections resulted in increased anaemia. The increased anaemia was, however, not correlated with any discernable parasite trait (e.g. parasite density) or with increased transmission. CONCLUSIONS: We found some evidence supporting the hypothesis that there is an adaptive basis correlating virulence (as defined by host mortality) and transmission success in malaria parasites. This confirms the validity of applying evolutionary virulence theory to biomedical research and adds support to the prediction that partially effective vaccines may select for increasingly virulent malaria parasite strains. By contrast, there was no consistent correlation between transmission and sub-lethal anaemia, a more common outcome of malaria infection. However, overall, the data are not inconsistent with the recent proposal that sub-lethal effects may impose an upper limit on virulence. Moreover, clone specific differences in transmission phenotypes linked to anaemia do suggest that there is considerable adaptive potential relating anaemia and transmission that may lead to uncertain consequences following intervention strategies.

Isolation and characterization of the lysozyme-encoding gene from the silkworm Bombyx mori.

We have isolated and characterized a Bombyx mori (Bm) cDNA encoding a lysozyme (Lyz). A 90-bp DNA fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid (aa) sequence of the Bm Lyz. These PCR fragments were used to screen a fat body cDNA library. A clone containing the complete lys cDNA (1294 bp) was isolated and completely sequenced. The deduced 137-aa sequence showed high homology with other chicken-type Lyz. Bm lys gene expression was constitutive in fat body, cuticular epidermal tissue and at a very low level in hemocytes. This gene expression was up-regulated in fat body, hemocytes and cuticular epidermal tissue following the injection of Gram+ bacteria.

Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning.

Following injection of bacteria into the hemocoel of the fall webworm, Hyphantria cunea, several inducible genes were identified and characterized using PCR-based differential display (DD-PCR) and subtractive cloning. Ten immune-related cDNA clones (Hdd1, Hdd2, Hdd3, Hdd11, Hdd13, Hdd15, Hdd17, Hdd23, Hs106, Hs302) were isolated and characterized. The deduced amino acid sequence of Hdd2 was shown to be a member of the copper, zinc superoxide dismutase (Cu-Zn SOD) family. The H. cunea Cu-Zn SOD is novel in that it is up-regulated following a bacterial challenge and has a putative signal peptide suggesting its secretion and involvement in the insect immune response. Hdd3 was found to encode a new member of the serpin (serine protease inhibitor) family. The putative lectin corresponding to Hdd15 is of a different kind in that it has two lectin C domains in a single molecule. These two lectin C domains show significant homology to the lectin C domain of Periplaneta lipopolysaccharide binding protein (LPS-BP). Three cloned genes, Hdd17, Hs106 and Hs302, encode a homologue to Bombyx mori Gram negative binding protein, a hemolin-like protein and a attacin-like protein, respectively. The deduced amino acid sequences from Hdd11 showed weak homology with a Locusta migratoria hemolymph protein. On the contrary, Hdd1, Hdd13 and Hdd23 did not reveal any significant homology with known proteins. All of the 10 genes were clearly inducible by E. coli and M. luteus injection. Injection of distilled water only slightly induced mRNA levels. Comparison of temporal mRNA expression following E. coli injection showed three types of expression patterns.

Isolation and characterization of the cDNA encoding the prophenoloxidase of fall webworm, hyphantria cunea.

Two kinds of cDNA clones encoding prophenoloxidases (ProPO; zymogen of phenoloxidase (monophenol, L-dopa: oxygen oxydoreductase, EC 1.14.18.1)) were isolated by polymerase chain reaction (PCR) followed by screening of cDNA library that was prepared from whole larvae of the fall webworm, Hyphantria cunea (Lepidoptera, Arctiidae). The cDNAs encode 681 and 697 amino acids with molecular masses of 78.2 and 80.2 kDa, respectively. Deduced amino acid sequence homology between the two H. cunea ProPOs are only 49% whereas the homology against other insect ProPOs ranged from about 40 to 72%. The phylogenic analysis showed that the insect ProPOs are grouped mainly into two families. A putative proteolytic cleavage site for enzyme activation was identical to other insect ProPOs. The conserved copper binding sites were 84-62% homologous to arthropod ProPOs. Two additional highly conserved regions were found in the carboxy terminal. Furthermore, like other insect prophenoloxidases, hydrophobic signal peptide sequences were absent in the deduced ProPOs from H. cunea. Southern blot analysis indicated that the H. cunea ProPO1 is present as a single copy in the genome. Northern blot analysis showed that the expression of the ProPO genes were concentrated in mid-instar larvae, but were much lower in other developmental stages.

Host-parasite intimacy: how do mosquito defense reactions affect Plasmodium sporogonic development?

Here are a few sundry reflections and questions stimulated by the talks and discussions in session 3 of the Malariology Centenary Conference 1998 on the 'intimacy' that has been established between Plasmodium and Anopheles. The degree of 'intimacy' achieved in a vectorial system seems to correlate to the difficulties incurred when one attempts to interrupt parasite development in the insect host. The main questions addressed in this essay are as follows: Are antimicrobial peptides the most efficient effector molecules to be used to block sporogonic development of malaria parasites? Are the mosquito defense reactions elicited by Plasmodium invasion deleterious, advantageous or neutral to parasite development? The purpose in asking these questions is to put in perspective the direction of research in this area and to generate new ideas.

Sex ratio adjustment in Plasmodium gallinaceum.

The sex ratio of the avian malaria parasite, Plasmodium gallinaceum, was examined during the course of infection in its natural host, the chicken. Infections can have two possible outcomes: death of the host resulting from anaemia or self-cure and survival. In lethal infections the sex ratio remained female biased throughout, whereas in self-curing infections, the sex ratio became progressively less female biased. We examined the consequences of altering sex ratio for parasite transmission success using a theoretical fertilisation model and hypothesise that an immune response specifically effective against the male gametes would provide a selective explanation for the observed sex ratio adjustment. Previous studies have demonstrated that there is an efficient anti-gamete antibody response as an infection is cleared by the host. We performed in vitro mosquito infection studies comparing mosquito infection rates with naive serum replacement and showed that decomplemented serum from curing infections is transmission blocking, whereas serum from lethal infections is not. We discuss aspects of the malaria parasite-host interaction which might provide the selective pressure for such observed sex ratio adjustment.

[Paul-Louis Simond and coccidia]. / Paul-Louis Simond et les coccidies.

In 1880, during a sojourn at Constantine, Alphonse Laveran discovered the etiological agent of human malaria. During his microscopic observations of the parasite in freshly collected blood, Laveran's attention was attracted to the movement of flagellar bodies in the preparations. For Laveran these flagellar bodies corresponded to living organisms, in fact he considered them the most characteristic stage of the parasite; perhaps the sexual form of the parasite. In 1884, back in Paris at the military hospital Val de Grâce, A. Laveran showed these flagellated bodies to Pasteur, Roux and Chamberland who all thought that is was impossible not to recognize a living body in this mass of protoplasm displacing the surrounding red blood cells with its protruding flagella. As early as 1890, Elie Metchnikoff established a link between the flagellar bodies of Laveran and the stealthy stage of parasitic Coccidia infecting the intestinal epithelium of salamanders. It was the Pasteuriens and a few others scientists like Danilewski and Pfeiffer who firmly believed that the flagellar forms were indeed a normal stage during the hematozoan life cycle. On the contrary, Grassi and the Italian school, as well as the French protozoologist Labbé were convinced that the flagellar bodies corresponded to a degenerative form of the parasite provoked by the exposure of the parasites to air during slide preparation. In early 1896, P-L Simond joined Metchnikoff's laboratory and was assigned to study salamander coccidia in order to clarify the nature of the flagellar bodies. In a very short period of time, Simond clearly demonstrated that the coccidia had two types of life cycles; one of which resulted in the formation of flagellar bodies. Simond called the flagellar bodies "chromatozoites" due to the important quantities of chromatin twisted around the flagellum. From these observations and the sperm-like movement of the chromatozoites, Simond put forth the hypothesis that the chromatozoites were the male sexual forms of the parasite. He noted that they were found in all species of coccidia, as well as in the different species of malaria causing hematozoans of man and birds. Simond went even further in his interpretation to suggest that malaria parasites undergo sexual reproduction. This was indeed true, but Simond thought erroneously that sexual reproduction in hematozoans would result in the formation of resistant spores like in the Coccidia. The sexual reproduction hypothesis of malaria parasites was also formulated independently a year later by W. G. MacCallum (1898) working in the United States on Halteridium, a hematozoan infecting crows. Initially MacCallum was not aware of Simond's work, but later gave Simond full credit for his work on Coccidia. Furthermore, MacCallum observed for the first time the chromatozoite (microgamete) enter a female element (macrogamete) to form a mobile worm-like stage of the parasite know today as the ookinete. In his writings Simond is very modest, giving most of the credit to his mentors Metchnikoff and Laveran, but Laveran in an article which appeared in 1899 recognizes P.-L. Simond as the first person having put forth the hypothesis that the flagellar bodies of hematozoans causing human and bird malaria were the male forms of the parasite destined to fertilize the female elements.

Isolation and identification of cecropin antibacterial peptides from the extracellular matrix of the insect integument.

A method for the screening and isolation of antibacterial peptides from the extracellular insect cuticle using ultrafiltration and reverse-phase high-performance liquid chromatography is presented. Using this method we were able to conveniently isolate cuticular antibacterial peptides belonging to the cecropin family in 1 day. The procedure should facilitate the rapid screening of antibacterial peptides or other bioactive peptides from extracellular matrices.

Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix.

The cuticle of the silkworm Bombyx mori was demonstrated to contain pro-phenol oxidase [zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] and its activating cascade. The activating cascade contained at least one serine proteinase zymogen (latent form of pro-phenol oxidase activating enzyme). When the extracted cascade components were incubated with Ca2+, the latent form of pro-phenol oxidase activating enzyme was itself activated and, in turn, converted through a limited proteolysis of pro-phenol oxidase to phenol oxidase. Immuno-gold localization of prophenol oxidase in the cuticle using a cross-reactive hemolymph anti-pro-phenol oxidase antibody revealed a random distribution of this enzyme in the nonlamellate endocuticle and a specific orderly arrayed pattern along the basal border of the laminae in the lamellate endocuticle of the body wall. Furthermore, prophenol oxidase was randomly distributed in the taenidial cushion of the tracheal cuticle. At the time of pro-phenol oxidase accumulation in the body wall cuticle, no pro-phenol oxidase mRNA could be detected in the epidermal tissue, whereas free-circulating hemocytes contained numerous transcripts of pro-phenol oxidase. Our results suggest that the pro-phenol oxidase is synthesized in the hemocytes and actively transported into the cuticle via the epidermis.

Erythropoiesis and molecular mechanisms for sexual determination in malaria parasites.

Malaria parasites proliferate asexually within the vertebrate host but must undergo sexual reproduction for transmission to mosquitoes and hence infection of new hosts. The developmental pathways controlling gametocytogenesis are not known, but several protein kinases and other putative signal transduction elements possibly involved in this phenomenon have been found in Plasmodium. Recently, another developmental pathway, that of Plasmodium sex determination (male or female), has been shown to be triggered by erythropoiesis in the host. Rapid progress is being made in our understanding of the molecular basis of mammalian erythropoiesis, revealing kinase pathways that are essential to cellular responses triggered by the hormone erythropoietin. Although the molecular mechanisms whereby this hormone modulates the sex ratio of malaria parasites remain to be elucidated, it probably activates, within the parasite, transduction pathways similar to those found in other eukaryotes. Indeed, enzymes belonging to protein kinase families known to be involved in the response of mammalian cells to erythropoietin (such as the mitogen-activated protein kinases) have been identified in P. falciparum gametocytes. Some of these enzymes differ markedly from their mammalian homologs; therefore, identification of the transduction pathways of the parasite that are responsible for its developmental response to erythropoietin opens the way to the development of transmission-blocking drugs based on kinase inhibitors.

Studies on prophenoloxidase activation in the mosquito Aedes aegypti L.

This study, the first of its kind in a mosquito vector species, demonstrates the feasibility of studying prophenoloxidase activation in an insect containing not more than a few microliters of hemolymph. Mosquito phenoloxidase was found to be in an inactive proenzyme form, prophenoloxidase. Mosquito prophenoloxidase required bivalent cation for its activation; Ca2+ was found to be the most efficient for activation. Concomitant amidase activity was also observed prior to phenoloxidase activity. Through Western blotting, using a cross-reactive silkworm antiprophenoloxidase antibody, our results strongly suggest that mosquito prophenoloxidase activation resulted from limited proteolysis. Protease inhibitor studies reinforced this contention showing the involvement of (a) serine protease(s) with trypsin-like activity in the activation of mosquito prophenoloxidase.

Defense reactions by larvae of Aedes aegypti during infection by the aquatic fungus Lagenidium giganteum (Oomycete).

The adherence of zoospores of Lagenidium giganteum to the cuticle of mosquito larvae is the initial step in the infection process. Subsequently, a germ tube penetrates the integument, inducing a rapid melanization of the injured cuticle and epidermis. After entering the hemocoel the developing hyphae are occasionally encapsulated locally. This process is slow (6 to 12 h postincubation) and most frequently cell-free, although it can be mediated by circulating hemocytes. Sporadic hemocyte mediation of the humoral encapsulation process in larval stages of Culicidae adds a previously unreported dimension to this unusual type of defense reaction. The defense reactions of larvae of Aedes aegypti were ineffective against observed infection by Lagenidium giganteum.

Molecular cloning and characterization of a Drosophila p38 mitogen-activated protein kinase.

A mitogen-activated protein kinase (MAPK) has been cloned and sequenced from a Drosophila neoplasmic l(2)mbn cell line. The cDNA sequence analysis showed that this Drosophila kinase is a homologue of mammalian p38 MAPK and the yeast HOG1 gene and thus was referred to as Dp38. A distinguishing feature of all MAPKs is the conserved sequence TGY in the activation domain. Dp38 was rapidly tyrosine 186-phosphorylated in response to osmotic stress, heat shock, serum starvation, and H2O2 in Drosophila l(2)mbn and Schneider cell lines. However, unlike mammalian p38 MAPK, the addition of lipopolysaccharide (LPS) did not significantly affect the phosphorylation of Dp38 in the LPS-responsive l(2)mbn cell line. Following osmotic stress, tyrosine 186-phosphorylated forms of Dp38 MAPK were detected exclusively in nuclear regions of Schneider cells. Yeast complementation studies demonstrated that the Saccharomyces cerevisiae HOG1 mutant strain JBY10 (hog1-Delta1) was functionally complemented by Dp38 cDNA in hyperosmolar medium. These findings demonstrate that similar osmotic stress-responsive signal transduction pathways are conserved in yeast, Drosophila, and mammalian cells, whereas LPS signal transduction pathways appear to be different.

Tyrosinase-type prophenoloxidase distribution in the alimentary canal of strains of Anopheles gambiae refractory and susceptible to Plasmodium infection.

Tyrosinase-type prophenoloxidase distribution was investigated in the posterior midgut and salivary glands of strains of noninfected adult Anopheles gambiae refractory (Blue strain) and susceptible (G3 strain) to infection with malaria parasites. Immunocytochemical localization showed that in the posterior midgut epithelium tyrosinase-type prophenoloxidase was almost entirely restricted to apical granules. These are known to be a mixture of secretory granules and lysosomes; it was not possible to distinguish between the two in this study. The secretory product in the lumen of the distal median lobe of the salivary gland was heavily labeled; the secretory product in the lumen of the distal lateral lobes was also labeled but to a lesser extent. Subjectively, no differences in the degree or pattern of labeling of either midgut or salivary glands between the strains of mosquito were apparent.

Use of nitrocellulose membrane to activate and measure insect prophenol oxidase.

A method for the activation and measurement of insect prophenol oxidase using nitrocellulose membrane is presented. Using this method we were able to conveniently activate both crude and purified prophenol oxidase from insects belonging to three different orders. This rapid method allows for prophenol oxidase activation, in the absence of a prophenol oxidase-activating system, and in the presence of high ionic strength, protease inhibitors, or chelator.

Mode of action of the antibacterial cecropin B2: a spectrofluorometric study.

Cecropin B2 (CecB) is a 35 amino acid residue, antibacterial peptide that was isolated from the hemolymph and cuticular matrix of the silkworm, Bombyx mori. Synthetic peptides with sequences corresponding to CecB and its truncated analogue, [3-->35]CecB, were synthesized and selectively labeled at their N-terminal amino acids with either 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or rhodamine fluorescent probes. Utilization of these probes facilitated study of the interaction of cecropin with model phospholipid membranes at a high lipid/peptide molar ratio (approximately 3000:1), permitting investigation of the initial steps involved in this process. The surface partition coefficient of CecB, derived from binding isotherms of the NBD-labeled peptide, was 10-fold higher with acidic phospholipids than with zwitterionic ones, which correlates with the high efficiency of CecB and its analogues in permeating acidic phospholipid vesicles. Furthermore, a direct correlation was found between the antibacterial activity of CecB or its truncated analogues and the ability of their Rho-labeled analogues to interact with bacteria and human red blood cells. We propose that CecB binds phospholipid membranes preferentially as monomers lying on the surface, rather than cooperatively as bundles that form transmembranal pores via a "barrel stave" mechanism. This is based on the following: (i) the linearity of CecB's binding isotherms; (ii) the low energy transfer between membrane-embedded donor and acceptor-labeled CecB, even in the presence of a transmembrane potential; (iii) the surface localization of CecB's N-terminus; (iv) the need for more than 100 peptide molecules per phospholipid vesicle to induce initial ion leakage; and (v) the fact that CecB is a highly positively charged amphipathic alpha-helix, and therefore it is not expected to transverse the membrane as a monomer. We speculate that the non-cooperative binding of the peptides on the outer surface of the bacteria (i.e., no aggregation of CecB monomers) may help them to diffuse efficiently into the inner membrane, which is thought to be the target of antibacterial peptides.

Variation of larval susceptibility to Lagenidium giganteum in three mosquito species.

A significantly greater number of Lagenidium giganteum zoospores were found encysting on the dorsal thoracic surface of Anopheles gambiae larvae than on the larvae of Aedes aegypti and Culex pipiens. However, germ tube penetration in the cuticle of A. gambiae provoked an intense and diffuse melanization which encapsulated the fungus and protected 56% from death. Although a small number of zoospores attach to and penetrate the cuticular surface of A. aegypti and C. pipiens approximately 99% of both species succumb to fungal infection. Melanization in A. aegypti is slower, weaker, more localized, and generally ineffective against L. giganteum infection compared to A. gambiae. Upward migration of L. giganteum zoospores to the water surface favored encounters with mosquito larvae and was speculated to be due to negative geotaxis rather than positive aerotaxis and phototaxis. Otherwise, initial contact between larva and zoospore was random.