Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees.

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyngeal gland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees.

Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph.

The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.

A prokaryotic-eukaryotic relation in the fat body of Bombus terrestris.

The interaction between the insect host and its microbiota plays a central role in insect health and is mostly studied in relation to the digestive system. Nonetheless, there are numerous microorganisms occupying multiple habitats in and on insects. We studied microbial communities in the gut and fat body of bumblebees (Bombus terrestris) using the V4 region of the 16S rRNA gene on the Illumina MiSeq platform. In one of the two study locations, the fat body microbial composition was marked by the dominant presence of Arsenophonus sp. and Phyllobacterium sp. Bumblebees infected with Apicystis bombi, a eukaryotic parasite multiplying in the fat body, had a significant higher relative abundance of Arsenophonus sp. compared with the non-infected individuals. In general, the infection of A. bombi correlated with a more interlinked microbial association network, as we observed an increase of significant associations between the relative abundance of bacteria present in the gut and fat body of infected bumblebees. The causality within this potential prokaryotic-eukaryotic relation is important when assessing the health impact on bees.

Impact of Trypanosoma cruzi on antimicrobial peptide gene expression and activity in the fat body and midgut of Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In natural habitats, these insects are in contact with a variety of bacteria, fungi, virus and parasites that they acquire from both their environments and the blood of their hosts. Microorganism ingestion may trigger the synthesis of humoral immune factors, including antimicrobial peptides (AMPs). The objective of this study was to compare the expression levels of AMPs (defensins and prolixicin) in the different midgut compartments and the fat body of R. prolixus infected with different T. cruzi strains. The T. cruzi Dm 28c clone (TcI) successfully develops whereas Y strain (TcII) does not complete its life- cycle in R. prolixus. The relative AMP gene expressions were evaluated in the insect midgut and fat body infected on different days with the T. cruzi Dm 28c clone and the Y strain. The influence of the antibacterial activity on the intestinal microbiota was taken into account. METHODS: The presence of T. cruzi in the midgut of R. prolixus was analysed by optical microscope. The relative expression of the antimicrobial peptides encoding genes defensin (defA, defB, defC) and prolixicin (prol) was quantified by RT-qPCR. The antimicrobial activity of the AMPs against Staphylococcus aureus, Escherichia coli and Serratia marcescens were evaluated in vitro using turbidimetric tests with haemolymph, anterior and posterior midgut samples. Midgut bacteria were quantified using colony forming unit (CFU) assays and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Our results showed that the infection of R. prolixus by the two different T. cruzi strains exhibited different temporal AMP induction profiles in the anterior and posterior midgut. Insects infected with T. cruzi Dm 28c exhibited an increase in defC and prol transcripts and a simultaneous reduction in the midgut cultivable bacteria population, Serratia marcescens and Rhodococcus rhodnii. In contrast, the T. cruzi Y strain neither induced AMP gene expression in the gut nor reduced the number of colony formation units in the anterior midgut. Beside the induction of a local immune response in the midgut after feeding R. prolixus with T. cruzi, a simultaneous systemic response was also detected in the fat body. CONCLUSIONS: R. prolixus AMP gene expressions and the cultivable midgut bacterial microbiota were modulated in distinct patterns, which depend on the T. cruzi genotype used for infection.

[Peculiarities of the expression, structure, and localization of the subtilisin-like protease in the microsporidium Paranosema locustae].

Peculiarities of the expression, localization, and structure of the subtilisin-like protease from the microsporidium Paranosema locustae, a parasite of the migratory locust and other orthopteran species, are analyzed. Heterologous expression of the microsporidian ferment in the bacterium Escherichia coli allowed obtaining antibodies to the recombinant protein and to start its examination. In spite of the presence of the N-tail signal peptide in the ferment, potentially able to secret it into the cytoplasm of the infected cell, immunoblotting with obtained antibodies had demonstrated specific accumulation of the protease in the insoluble fraction of spore homogenate. At the same time, the ferment was absent in intracellular stages.of the parasite and also in the cytoplasm of infested host cells. Accumulation of mRNA, coding the studied protein in microsporidian spores was confirmed with the use of RT-PCR method. Heterologous expression of the protease in the methylotrophic yeast Pichiapastoris demonstrated the same result. The ferment of P. locustae was not secreted into a culture medium and was absent in the cytoplasm of yeast cells, accumulating in a dissoluble (membrane) fraction of the homogenate. On the whole, the obtained data testify to the fact that the subtilisin-like protease of P. locustae plays an important role in the physiology of spores rather than participates in host-parasite relations during intra-cellular development.

Melanotic pathology and vertical transmission of the gut commensal Elizabethkingia meningoseptica in the major malaria vector Anopheles gambiae.

BACKGROUND: The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission. RESULTS: Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingiameningoseptica. Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E. meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E. meningoseptica can be vertically transmitted from eggs to larvae and that E. meningoseptica-mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E. meningoseptica and Plasmodium on mosquito survival. CONCLUSION: The high pathogenicity and permanent association of E. meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.

Description and phylogeny of Zelenkaia trichopterae gen. et sp. nov. (Microsporidia), an aquatic microsporidian parasite of caddisflies (Trichoptera) forming spore doublets.

Two novel microsporidia infecting the fat body tissues in larvae of two hosts, Halesus digitatus and Micropterna sequax (Trichoptera, Limnephilidae), were investigated using light and electron microscopy and rDNA sequence analyses. The molecular and morphological characters of these isolates warrant creation of a new microsporidian genus, Zelenkaia gen. n., with two species, one named herein. Developmental stages of Zelenkaia spp. have single nuclei. In sporogony, a plasmodium with four nuclei gives rise by rosette-like budding to two pairs of uninucleate sporoblasts, each within a thin-walled, subpersistent sporophorous vesicle. Sporoblasts and mature spores adhere temporary together, forming doublets oriented in parallel, within the sporophorous vesicle. Spores are long-oval and uninucleate, and those of the type species Z. trichopterae measure 10.3×3.5µm and have 24-25 polar filament coils. Phylogenetic analysis based on rDNA places Zelenkaia spp. within the aquatic clade of microsporidia and, more specifically, in the clade containing some microporidia from amphipod hosts.

Reduced activity of juvenile hormone esterase in microsporidia-infected Lymantria dispar larvae.

Infection of the fat body of Lymantria dispar (Lep.: Lymantriinae) larvae with the microsporidium Vairimorpha disparis has severe effects on juvenile hormone (JH) metabolism of the host. Beginning 8 days postinfection, activity of the JH degrading enzyme JH-esterase was significantly lower in the hemolymph of infected than uninfected larvae. Activity remained low as microsporidiosis progressed. JH titers were slightly elevated in infected larvae; the difference was not significant in most cases. This disturbance of JH metabolism may be due to generally impaired fat body functions and high demand for resources by the developing pathogen.

Engineered anopheles immunity to Plasmodium infection.

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquito's innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control.

Cuticular encystment of Autographa nigrisigna eggs by epidermal cell migration.

It was previously established that Autographa nigrisigna loopers form cuticular cysts at the dorsal site of the 9th (penultimate) abdominal segment after parasitization by the solitary endoparasitoid Campoletis chlorideae and get rid of the parasitoid egg with the old cuticle at ecdysis. The cuticular cyst consists of a space between the old cuticle and new cuticle formed by the epidermis to enclose the parasitoid egg. The fact that A. nigrisigna loopers exclude the oviposited egg from the hemocoel using a cuticular cyst raises the question how the parasitoid egg passes through the epidermis. To exclude the endoparasitoid eggs from the hemocoel, the epidermis is required to move the location of the parasitoid egg. In the current study, we investigated the morphological process of cuticular cyst formation. First, the oviposited egg drifted to the 9th abdominal segment located at the open end of the dorsal vessel as a result of force generated by the hemolymph current from the oviposition site, and formed contacts with the integument containing the fat body (FB). The epidermis, in contact with the egg, then began to move along with the basement membrane formed on the surface of the FB, and settled under the egg, thus altering its location. This inversion was duplicated in vitro using integument from the 9th abdominal segment when parasitoid eggs were inserted between the epidermis and FB. When the integument, without the FB, was incubated on an agar plate, the epidermal cells migrated on the plate. Integument without eggs showed no signs of migration from their original sites. When the actin polymerization inhibitor latrunculin B was added to the cultures, the epidermal cells remained in their original location.

[The artificial infection of the cladoceran Moina macrocopa (Crustacea: Phyllopoda) with the microsporidia Gurleya sp. (Microsporidia: Gurleyidae)].

Microcrustaceans Moina macrocopa (Straus, 1820) have been collected in a small vernal pond and cultured under laboratory conditions. The infection of this culture with the microsporidia Gurleya sp. was detected and high virulence of this microsporidian species allowed us to carry out the transmission experiments. Horizontal transmission, the high host specificity and maximal spore production in the hypodermal and fat body cells through 5-6 days after per oral experimental infection have been revealed. Histological investigations demonstrated that Gurleya sp. reduces drastically host reproductive success without a considerable influence on the microcrustaceans' mortality.

[Six new species of microsporidia of the genus Amblyospora (Microspora: Amblyosporidae) from blood sucking mosquitoes (Diptera: Culicidae) from the west Siberia].

Microsporidia of the genus Amblyospora parasiting the adipose body of mosquito larvae of the genus Aedes and Culex has been studied with both light and electron microscopy. Six new species of microsporidia are described based on ultrastructural characteristics of spores and sporogony stages. Amblyospora flavescens sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 165 nm. Exospore is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, large vesicular, lamellar. The anisofilar polar filament with 10--11 coils (3 1/2 + 2 1/2 + 4-5). Fixed spores are 6.3 +/- 0.1 x 4.24 +/- 0.1 microm. Amblyospora kolarovi sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 265-315 nm. Exospore shapes tucks on the surface of spore. It is two-membranous. Subexospore is quagge, structural. Endospore is electron-translucent. Polaroplast consists of two parts: lamellar and large vesicular. The anisofilar polar filament with 11-13 coils (3 + 8-10). Fixed spores are 5.4-5.6 x 3.5-4.2 microm. Amblyospora orbiculata sp. n. Mature spores are widely egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 155 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, vesicular, lamellar. Polar filament is anisofilar, with 11 1/2 coils (4 1/2 + 1 + 6). Fixed spores are 6.3 +/- 0.1 x x 4.0 +/- 0.1 microm. Amblyospora rugosa sp. n. Mature spores are egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 225 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 17 1/2 coils (3 1/2 + 1 + 13). Fixed spores are 5.3 +/- 0.1 x 3.7 +/- 0.1 microm. Amblyospora undata sp. n. Mature spores are egg-shaped. The spore wall is three-layered, about 220 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. The anisofilar polar filament with 8 coils (3 + 5). Fixed spores are 5.0 +/- 0.1 x 3.0 +/- 0.1 microm. Amblyospora urski sp. n. Mature spores have widely oval form. The back pole is concave. The spore wall with three layers, about 280 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 6 coils (2 + 4). Fixed spores are 4.4 +/- 0.1 x 2.9 +/- 0.1 microm.

[Microsporidia of the genus Parahtelohania (Microspora: Amblyosporidae) from blood-sucking mosquitoes of the genus Anopeles (Diptera: Culicidae) from the South of the West Siberia]. / Mikrosporidii roda Parathelohania (Microspora: Amblyosporidae) iz komarov roda Anopheles (Diptera: Culicidae) iuga Zapadnoi Sibiri.

Five new microsporidian species of the genus Parathelohania have been found in the fat body of blood-sucking mosquitoes collected in various water basins in the South of West Siberia: Parathelohania divulgata sp. n., P. formosa sp. n., P. sibirika sp. n., P. teguldeti sp. n., and P. tomski sp. n. Processes of merogony, sporogony, and ultrastructure of spores was were investigated by means light and electronic microscopy.

[Microsporidiosis in the wax moth Galleria mellonella (Lepidoptera: Pyralidae) caused by Vairimorpha ephestiae (Microsporidia: Burenellidae)]. / Mikrosporidioz pchelinoi ognevki Galleria mellonella (Lepidoptera: Pyralidae), vyzyvaemyi Vairimorpha ephistiae (Microsporidia: Burenellidae).

An experimental microsporidiosis of the wax moth caterpillars from laboratory population had been caused by oral infecting of early stages larvae and by intracavity injections of the spores of the microsporidian species Vairimorpha ephestiae. Peculiarities of microsporidiosis proceeding, manifestations of host defence reactions, and also an effect of the temperature of caterpillars cultivation and conditions of spores keeping on liability of the insects to the infection were studied. The effect of the microsporidia on the host organism was the early death or the delay of larvae development, but in several cases external manifestations of the effect of the parasite on the host were absent. The development of the parasites from the moment of infecting to the appearing of the mature spores congestions in the host organism proceeded 6 days. Microsporidia invaded insect fat body and caused its hypertrophy and disappearance of lipid granules. In the intestine and salivary glands microsporidia were not observed in the period from 6 to 16 day of the development. On the final stage of microsporidiosis the all contents of fatty tissue cells were replaced by spores of microsporidia. Under microscope only diplocaryotic spores of the Nozema type had been found in infected and died specimens, but not octospores. The spores threw out polar tubes under the change of pH in incubating solution from neutral to alkaline. The effects of microsporidiosis on the wax moth haemolymph were the increased rate of prohaemocytes, appearing of multinuclear free-circulating cells at 6 day after infection, and suppression of the reaction of haemolymph melanization with the mass sporogenesis of the parasite. The characteristic symptom of the wax moth microsporidiosis had been revealed, accumulation of black points and small spots of irregular form under cuticle ("reaction of attretization"). Increase of the temperature of insect cultivation up to 32 degrees C during 3 days after infection contributed to the full deliverance of the insects from the infection in first and second generations. It can be considered as a method of treatment of wax moth laboratory colonies from microsporidiosis. Oral infection of III and IV stage caterpillars by the spores being kept during 3-6 months under 4 degrees C in form of water suspension caused the death of 63.0-61.5 and 91% of caterpillars being cultivated under 25 and 21 degrees C respectively. Under the temperature of cultivation equal 30 degrees C the mortality did not differ from the control sample (8-10%). The spores extracted from dried bodies of caterpillars lost their vitality. It was demonstrated by the test on infectious ability in vivo and by acridine orange staining. This host-parasite system appears to be perspective in investigations of resistance mechanisms in insects and immunosuppressive features of entomopathogen microsporidia.

[Trehalose catabolism in microsporidia Nosema grylli spores]. / Osobennosti katabolizma tregalozy v sporakh mikrosporidii Nosema grylli.

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.

[Crepidula beklemishevi gen. et sp. n. and Dimeiospora palustris gen. et sp. n. (Microspora: Amblyosporidae)--new microsporidian genera and species from blood-sucking mosquitoes (Diptera: Culicidae) from the south of the western Siberia]. / Crepidula beklemishevi gen. et sp. n. i Dimeiospora palustris gen et sp. n. (Microspora: Amblyosporidae)--novye mikrosporidii iz krovososushchikh komarov (Diptera: Culicidae) iuga zapadnoi Sibiri.

Microsporidia parasitizing the adipose body of mosquito larvae of Anopheles beklemishevi and Aedes punctor has been studied. Two new genera of microsporidia are described based on lightmicroscopic and ultrastructural characteristics of spores and sporogony stages. The spore wall of Crepidula beklemishevi gen. n. et sp. n. is formed by two-membrane exospore, thick exospore, bilayer endospore and thin plasmolemma. Spores with single nucleus, polar filament anisofilar, with 6-7 coils (2+ 4-5), polaroplast consisting of three parts: macrochelicoidal, microhelicoidal and lamellar. Fixed spores 4.2 +/- 0.22 x 2 +/- 0.01 microns. The sporogony of Dimeiospora palustris gen. et. n. results in spore formation of two different types. Spores of the first type are oviform, with thick wall, single-nuclear, 6.1 x 4.9 microns. Spore wall with three layers, about 370 nm. Exospore electron-dense, subexospore moderately electrondense. Exospore and subexospore irregularly pleated on the almost spore surface and slightly thinner on anterior end only. Endospore electron-translucent. Polar filament anisofilar, with 9 coils (3 + 6). Polaroplas consists of three parts: lamellar, fine bubbled, and coarse bubbled. Spores of the second type broad-ovate, with apical pole narrower, distal pole concave, 4.6 x 3.7 microns. Spore wall with three layer, 355 nm. Exospore on the apical end irregularly pleated, consists of thin electrondense exospore, subexospore of variable electron density, endospore electron-translucent. Polar filament anisofilar, with 13 coils (3 + 10). Polaroplast has two parts: lamellar and vesicular.

[Accumulation of heat-shock proteins 70 in insect fat bodies as evidence of stress in Microsporidia-infected insects]. / Nakoplenie belka teplovogo shoka 70 v zhirovom tele nasekomykh, zarazhennykh mikrosporidiiami, kak svidetel'stvo stressa nasekomykh.

At present a concept prevails that pathological alterations in insect hosts infected with microsporidia, and those associated with hormone imbalance may be explained by the production of juvenile hormone-like (JH) substances by microsporidia. According to another view point, this pathology is a consequence of the host response. We suggested that the microsporidian infection can provoke a stress reaction in insects, which may cause JH secretion by these insects. To confirm this hypothesis, we have analysed major stress protein Hsp70 levels in the infected insects. Using affinity chromatography on ATP-agarose and immunoblotting, we have shown that Hsp70 was accumulated in infected crickets, and that it was the host protein. The consequence of events accompanying the infection in the insects is discussed in relation to the response of hormonal system of the host organism.

Haemolymph and fat body metallo-protease associated with Enterobacter cloacae infection in the bloodsucking insect, Rhodnius prolixus

Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.

Life cycle of a new species of Duboscqia (Microsporida: Thelohaniidae) infecting the mosquito Anopheles hilli and an intermediate copepod host, Apocyclops dengizicus.

A new species of Microsporida, Duboscqia dengihilli, was found infecting larvae of the mosquito Anopheles hilli in northern Queensland, Australia. Laboratory experiments showed that binucleate spores formed within infected female mosquitoes were responsible for transovarial transmission to the next generation. Sporogony within the larval fat body was initiated by two diplokarya, one at each end of the cell, which undergo meiosis within a single sporophorous vesicle to form 16 meiospores. These spores are responsible for horizontal transmission to the copepod Apocyclops dengizicus. The microsporidium is transmitted back to the mosquito host via uninucleate pyriform spores formed within the copepod host which are infectious to larvae of A. hilli. The meronts within larvae infected by horizontal transmission ultimately develop into the binucleate spores within adult females to complete the life cycle. Thus, the development of this microsporidium involves vertical transmission between successive mosquito generations and horizontal transmission between mosquitoes and copepods similar to the life cycles of Amblyospora and Parathelohania.