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.

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.

The moleskin gene product is essential for Caudal-mediated constitutive antifungal Drosomycin gene expression in Drosophila epithelia.

The homeobox gene, Caudal, encodes the DNA-binding nuclear transcription factor that plays a crucial role during development and innate immune response. The Drosophila homologue of importin-7 (DIM-7), encoded by moleskin, was identified as a Caudal-interacting molecule during yeast two-hybrid screening. Both mutation of the minimal region of Caudal responsible for moleskin binding and RNA interference (RNAi) of moleskin dramatically inhibited the Caudal nuclear localization. Furthermore, Caudal-mediated constitutive expression of antifungal Drosomycin gene was severely affected in the moleskin-RNAi flies, showing a local Drosomycin expression pattern indistinguishable from that of the Caudal-RNAi flies. These in vivo data suggest that DIM-7 mediates Caudal nuclear localization, which is important for the proper Caudal function necessary for regulating innate immune genes in Drosophila.

Cryofracture electron microscopy of the ookinete pellicle of Plasmodium gallinaceum reveals the existence of novel pores in the alveolar membranes.

The malaria parasite invades the midgut tissue of its mosquito host as a motile form called the ookinete. We have examined the pellicle of the ookinete of Plasmodium gallinaceum by freeze-fracture and quick-freeze, deep-etch electron microscopy. The general organization is analogous to that of invasive stages of other members of Apicomplexa. The pellicle is composed of three membranes: the plasma membrane, and the two linked intermediate and inner membranes, which in the ookinete form one flattened vacuole that is located beneath the plasma membrane. The edges of this vacuole form a longitudinal suture. Beneath the vacuole is found an array of microtubules that are connected to the inner membrane by intramembranous particles. During freeze-fracture, the membranes can split along their hydrophobic planes, thus yielding six fracture faces, each of which displays a characteristic pattern of intramembranous particles. Additionally, we find that the ookinete pellicle differs from all other apicomplexan motile stages by the presence of large pores. These pores are of unknown function, but clearly might constitute a novel pathway for the transport of molecules to and from the cortex, which is independent of the well-described route through the apical micronemal/rhoptry complex. The pores may be the route by which motor proteins or other non micronemal surface proteins are trafficked, such as P25/P28 and SOAP, some of which are implicated in transmission blocking immunity.

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.

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells.

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.

Lipopolysaccharide-activated kinase, an essential component for the induction of the antimicrobial peptide genes in Drosophila melanogaster cells.

Eukaryotic organisms use a similar Rel/NF-kappaB signaling cascade for the induction of innate immune genes. In Drosophila, lipopolysaccharide (LPS) signal-induced activation of the Rel/NF-kappaB family transcription factors is an essential step in the transcriptional activation of inducible antimicrobial peptide genes. However, the mechanism by which the LPS-induced signaling pathway proceeds remains largely unknown. Here we have cloned a novel Drosophila LPS-activated kinase (DLAK) that is structurally related to mammalian IkappaB kinases. DLAK is expressed and transiently activated in LPS-responsive Drosophila cells following LPS stimulation. Furthermore, DLAK can interact with Cactus, a Drosophila IkappaB and phosphorylate recombinant Cactus, in vitro. Overexpression of dominant-negative mutant DLAK (DLAK(K50A)) blocks LPS-induced Cactus degradation. DLAK-bound Cactus can be degraded in a LPS signal-dependent fashion, whereas the DLAK(K50A) mutant-bound Cactus is completely resistant to degradation in the presence of LPS. The DLAK(K50A) mutant also inhibits nuclear kappaB binding activity and kappaB-dependent diptericin reporter gene activity in a dose-dependent manner, but the kappaB-dependent diptericin reporter gene activity can be rescued by overexpression of wild type DLAK. Moreover, mRNA analysis of various kappaB-dependent antimicrobial peptide genes shows that LPS inducibility of these genes is greatly impaired in cells overexpressing DLAK(K50A). These results establish that DLAK is a novel LPS-activated kinase, which is an essential signaling component for the induction of antimicrobial peptide genes following LPS treatment in Drosophila cells.

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.

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae.

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5' flanking sequence (-855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5' flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Identification and characterization of a putative sevenless homologue in the malaria vector Anopheles gambiae.

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles sevenless gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.

[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.

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.

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.

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.

Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae.

A cDNA clone for prophenoloxidase was isolated from the most important human malaria vector, Anopheles gambiae. The clone encoded a polypeptide of 79341 Da that contains the two copper binding domains common to all invertebrate prophenoloxidases and haemocyanins. Expression of the prophenoloxidase gene was detected throughout all life stages from egg to imago in two strains of A. gambiae; however, the strongest expression was observed in developing embryos in eggs. The prophenoloxidase gene was mapped to the inversion rich region of the right arm of chromosome-2 in region 13B.

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.

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.

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

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.