Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation.

BACKGROUND: Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. METHODS: This study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA. RESULTS: Malpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed. CONCLUSIONS: Our data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity.

Obligate development of Blastocrithidia papi (Trypanosomatidae) in the Malpighian tubules of Pyrrhocoris apterus (Hemiptera) and coordination of host-parasite life cycles.

Blastocrithidia papi is a unique trypanosomatid in that its life cycle is synchronized with that of its host, and includes an obligate stage of development in Malpighian tubules (MTs). This occurs in firebugs, which exited the winter diapause. In the short period, preceding the mating of overwintered insects, the flagellates penetrate MTs of the host, multiply attached to the epithelial surface with their flagella, and start forming cyst-like amastigotes (CLAs) in large agglomerates. By the moment of oviposition, a large number of CLAs are already available in the rectum. They are discharged on the eggs' surface with feces, used for transmission of bugs' symbiotic bacteria, which are compulsorily engulfed by the newly hatched nymphs along with the CLAs. The obligate development of B. papi in MTs is definitely linked to the life cycle synchronization. The absence of peristalsis allow the trypanosomatids to accumulate and form dense CLA-forming subpopulations, whereas the lack of peritrophic structures facilitates the extensive discharge of CLAs directly into the hindgut lumen. The massive release of CLAs associated with oviposition is indispensable for maximization of the infection efficiency at the most favorable time point.

Ophryocystis anatoliensis sp. nov., a new neogregarine pathogen of the chrysomelid beetle Chrysomela populi.

Chrysomela populi (Coleoptera: Chrysomelidae) is the most abundant and most important pest species that causes damage to poplar trees. Members of the family Chrysomelidae are frequently infected by protist pathogens but no neogregarine has been reported to date at the species level. In the present study we identify a new neogregarine pathogen from the chrysomelid C. populi. The infection was observed in the Malpighian tubules of adult beetles. A reddening of the Malpighian tubules was the most distinctive symptom of the infection. Single fusiform oocysts (9.8×4.7µm) were formed within a gamontocyst. The polar plugs were very thin, varying from 380 to 525nm in thickness. The oocyst wall was smooth and also quite thin (90-120nm). Morphological and ultrastructural characteristics of the pathogen indicate that the described neogregarine in C. populi is clearly different from known Ophryocystis species which infect coleopterans. Therefore, the neogregarine pathogen was determined to be a newly discovered species and named Ophryocystis anatoliensis sp. nov.

Prevalence and intensity of infection, metacyclogenesis and nuclear phenotypes in Panstrongylus megistus (Burmeister, 1835) after ingestion of Trypanosoma cruzi (Chagas, 1909) II and subjection to heat shock.

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.

Prevalence and intensity of infection, metacyclogenesis and nuclear phenotypes in Panstrongylus megistus (Burmeister, 1835) after ingestion of Trypanosoma cruzi (Chagas, 1909) II and subjection to heat shock / Prevalência e intensidade de infecção, metaciclogênese e fenótipos nucleares em Panstrongylus megistus (Burmeister, 1835) após ingestão de Trypanosoma cruzi (Chagas, 1909) II e choque de temperatura

This study aimed to contribute to our knowledge of the parasite-vector interaction associated with Trypanosoma cruzi (Chagas, 1909) infection in Panstrongylus megistus (Burmeister, 1835), an important vector of Chagas' disease in Brazil. The prevalence and intensity of T. cruzi infection, the incidence of metacyclogenesis and the frequency of nuclear phenotypes in Malpighian tubules were investigated in nymphs of P. megistus, reared at 28 °C and subjected to heat shock (40 °C, 1 hour) two days after infection with T. cruzi II (Y strain). Following the 45-day post-infection period, the frequency of epimastigotes was much higher than that of trypomastigotes in both heat-shocked and non-shocked insects, and the prevalence of infection was not altered by heat shock. Fewer epimastigotes and trypomastigotes were found in the infected insects subjected to the heat shock, indicating that the multiplication and metacyclogenesis of the parasites were affected by the stress. In infected specimens heat shock promoted an increased frequency of cell nuclei with heterochromatin decondensation, a cell survival response to stress, and did not affect insect survival. The effects of infection and heat shock, especially on the multiplication and metacyclogenesis of T. cruzi, and the observed resistance to heat shock developed by P. megistus nymphs are suggestive that they should be considered when adequate conditions for rearing these infected insects in the laboratory are pursued.

O presente estudo teve como objetivo contribuir para o conhecimento sobre a interação parasita-vetor associada à infecção por Trypanosoma cruzi (Chagas, 1909) em Panstrongylus megistus (Burmeister, 1835), importante vetor da doença de Chagas no Brasil. A prevalência e a intensidade da infecção por T. cruzi, a incidência de metaciclogênese e a frequência de fenótipos nucleares presentes em túbulos de Malpighi foram investigadas em ninfas de P. megistus criadas à temperatura de 28 °C e submetidas a choque térmico por 1 hora a 40 °C, dois dias após alimentação em camundongos infectados com T. cruzi II (linhagem Y). A análise realizada 45 dias pós-infecção revelou que tanto nos insetos submetidos ao choque térmico como nos respectivos controles a frequência de epimastigotos se apresentava muito maior do que a dos tripomastigotos; a prevalência da infecção não foi alterada pelo choque térmico. Menos epimastigotos e tripomastigotos foram encontrados nos insetos submetidos ao choque térmico, indicando que a multiplicação e a metaciclogênese dos parasitas possam ser afetadas com o estresse. Nos espécimes infectados, o choque térmico induziu aumento na frequência de núcleos com descompactação da heterocromatina, uma resposta de sobrevivência celular ao estresse, e não afetou a sobrevivência propriamente dita do inseto. Os efeitos da infecção e do choque térmico, especialmente sobre a multiplicação e a metaciclogênese de T. cruzi, e a resistência ao choque térmico desenvolvidos pelas ninfas de P. megistus são indicativos de que devam ser considerados quando se buscam condições adequadas de criação em laboratório de insetos infectados.

A single fluorescence-based LAMP reaction for identifying multiple parasites in mosquitoes.

Vector-borne diseases, such as malaria and lymphatic filariasis, are co-endemic in large parts of the world. To develop a multiplex amplification method for the simultaneous detection of multiple insect-borne infectious diseases, we used LAMP with fluorescently labeled primers to identify the SPECT2 gene of Plasmodium berghei and the cytochrome oxidase subunit I gene of Dirofilaria immitis in mosquitoes. This technique could detect as few as 100 P. berghei-infected red blood cell-equivalents or one D. immitis microfilaria. Moreover, individual species of parasites in mosquitoes could be identified when a mixture of fluorescently labeled primer sets was used. These findings suggest that the multiplex LAMP assay is sensitive and specific enough to identify parasite-bearing mosquitoes in areas where several diseases occur simultaneously. This procedure could increase the efficiency and effectiveness of arthropod-borne disease elimination programs.

Unikaryon phyllotretae sp. n. (Protista, Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae).

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74+/-0.17 x 1.93+/-0.17 microm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39+/-0.18 x 1.61+/-0.20 microm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.

Susceptibility of geographically distinct Aedes aegypti L. from Florida to Dirofilaria immitis (Leidy) infection.

In the present study, five geographically distinct populations of Aedes aegypti L. from various regions of Florida were compared for their susceptibilities to Dirofilaria immitis (Leidy) infection. The populations were from Gainesville (North Central), Jacksonville (North Eastern), Naples (South Western), Sarasota (West Central), and Vero Beach (East Central). Mosquitoes of all the populations had fewer parasites in their Malpighian tubules 6 and 13 days after blood feeding compared to the number of microfilariae initially ingested. There was no significant difference in the mean number of the total parasites per female 6 and 13 days after taking an infectious blood meal. In comparison, significantly larger numbers of the microfilariae completed development in the laboratory-selected Vero Beach susceptible strain. Females excreted microfilariae within 72 hr. of engorgement on an infected dog and the Gainesville, Jacksonville, Naples, and Sarasota populations had 20-23% females that lacked ovarian development six days after blood feeding. Out of these, in 16-22% of the females from the Jacksonville, Naples, and Sarasota populations, parasite development was arrested mostly as moribund prelarvae and only few developing larvae were observed. All females of the Gainesville population that did not develop eggs (20%) also lacked parasites. In the Jacksonville population, 23% of the females that matured eggs had no infection. Eight percent of the females from the Sarasota population exhibited a similar response. Low survivorship was observed at 6 and 13 days after imbibing an infectious blood meal. Females showed melanization of microfilariae in the Malpighian tubules, on the midgut wall, and in the hemocoel. Cellular melanization response was observed in the hemocoel, whereas, a humoral melanization response was observed in the Malpighian tubules. The Vero Beach susceptible strain showed maximum vector efficiency and lowest survival at 13 days post-blood meal compared to the Jacksonville and the Gainesville populations.

Ultrastructural investigation of the spore-forming protist Nephridiophaga blattellae in the Malpighian tubules of the German cockroach Blattella germanica.

Multinuclear plasmodia of the sporogenic protist Nephridiophaga blattellae are found intracellularly and in the lumen of the Malpighian tubules of the German cockroach Blattella germanica. Spore formation occurs only in the lumen. During sporogony, about 10-35 spores measuring 5.5x3.2 microm are endogenously formed within a plasmodium. Sporoblasts arise by the fusion of cisternae of the endoplasmic reticulum into a double membranous wall, which encloses a generative nucleus plus a portion of cytoplasm. Several somatic nuclei remain in the residual cytoplasm. Sporoblast and residual cytoplasm include mitochondria of the tubular type, endoplasmic reticulum, and many free ribosomes. During spore maturation, electron-dense wall material is deposited between the spore membranes, and the spores gain their typical oval, biconcave form. Freeze-etched spores reveal a small, central, cap-like structure, which may be the site where an infectious sporoplasm could emerge. Mature spores always have one nucleus, whereas early sporoblast stages with two small nuclei were found by transmission electron microscopy. Many nuclei of different developmental stages contain granules within the nuclear envelope. The systematic position of N. blattellae is unresolved. In certain respects it is reminiscent of Haplosporidia. However, the organisms of the two groups have different spore-forming processes and haplosporosomes are missing in the nephridiophagids. Therefore a new phylum might have to be erected for members of the family Nephridiophagidae.

Ultrastructure of infection, development and gametocyst formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in its mosquito host, Aedes albopictus (Diptera: Culicidae).

The life history of the protozoan parasite Ascogregarina taiwanensis in mosquito larvae (Aedes albopictus, collected in southern Taiwan) was shown to consist of two consecutive stages--intracellular and extracellular. Light microscopy showed that most trophozoites moved into the Malpighian tubules and developed into giant trophozoites during the first day pupa. The locomotion may be associated with bristle-like ridges of the trophozoite. The stage for sexual reproduction, i.e., the gamete, was then formed by segmentation of the giant trophozoite and twisting off the anucleate extremities of the body. Sexual reproduction occurred via fertilization by fusion of two resulting gametes, presumably two opposed sexes. The fused gametes finally generate the formation of the gametocyst, within which oocysts develop by budding from the cytoplasmic mass. This type of sexual reproduction has not been reported previously in any gregarine protozoa. We here proposed it as a new hypothesis for further elucidation of the protozoan reproduction.

Dirofilaria (Filarioidea: Onchocercidae) infection in Aedes albopictus (Diptera: Culicidae) collected in Louisiana.

First-stage Dirofilaria larvae were found infecting the Malpighian tubules in 3 of 163 Aedes albopictus collected from New Orleans, LA, during June-July 1994. Further studies will be necessary to provide definitive identification of the parasite species, and to better describe the vectorial status of Ae. albopictus.

The development of species of Leishmania Ross, 1903 in Lutzomyia longipalpis (Lutz & Neiva, 1912)

The development of four isolates of Leishmania from foci of American cutaneous leishmaniasis was studied in Lutzomyia longipalpis. The suggestion that the differences in the development of the Leishmania in the invertebrate host are of great taxonomic significance was confirmed. The pattern of development of three strains was typical of parasites of the subgenus Leishmania, the other was similar to Leishmania of the subgenus Viannia. The identification of the strains using other criteria is in agreement with biological characterization. The results show that the morphological and morphometric study of promastigotes do not clearly define the taxonomic position of the parasites but other studies are needed to confirm this.

The development of species of Leishmania Ross, 1903 in Lutzomyia longipalpis (Lutz & Neiva, 1912).

The development of four isolates of Leishmania from foci of American cutaneous leishmaniasis was studied in Lutzomyia longipalpis. The suggestion that the differences in the development of the Leishmania in the invertebrate host are of great taxonomic significance was confirmed. The pattern of development of three strains was typical of parasites of the subgenus Leishmania, the other was similar to Leishmania of the subgenus Viannia. The identification of the strains using other criteria is in agreement with biological characterization. The results show that the morphological and morphometric study of promastigotes do not clearly define the taxonomic position of the parasites but other studies are needed to confirm this.

An ultrastructural study on the early cellular response to Dirofilaria immitis (Nematoda) in the Malpighian tubules of Aedes aegypti (refractory strains).

Dirofilaria immitis living in Aedes aegypti refractory strains were studied in relation to ultrastructural events in primary cells of Malpighian tubules and to defense mechanisms activated by host-cells. When the microfilaria reaches the Malpighian cells, its intracellular development is blocked by defense mechanisms activated by the host, resulting in lysis of the outermost cuticle of the parasite without melanin involvement. Ultrastructural evidence suggests that lysis is brought about by Malpighian cell products.

The susceptibility of the mosquitoes Aedes notoscriptus and Culex annulirostris to infection with dog heartworm Dirofilaria immitis and their vector efficiency.

The mosquitoes Aedes notoscriptus (Skuse) and Culex annulirostris Skuse were fed on a dog infected with Dirofilaria immitis (Leidy) and a blood parasite count of approximately 5000 microfilaria per ml. Cx annulirostris ingested almost 4 times as much blood and almost 4 times as many microfilariae as Ae.notoscriptus (mean 26.0 compared to 6.6). Attrition of the filarial numbers occurred primarily within the midgut during the first 24 h following ingestion and was greater in Cx annulirostris than Ae.notoscriptus. Aedes notoscriptus sustained development of almost 8 times as many third-stage infective larvae as Cx annulirostris (mean of 3.8 compared to 0.5), and thus had a vector efficiency index of 57.6 compared to 1.9 for Cx annulirostris. In a series of investigations Ae.notoscriptus has now been shown to be an important vector for dog heartworm in southern Australia and may be a significant factor in the apparent burgeoning of the disease.

Membrane dynamics in insect malpighian tubules.

Urine formation in insects occurs in the Malpighian tubules by means of active ion transport and osmotically coupled water flow. The rates of urine formation can vary with time and can be modulated by diuretic hormones, developmental events, and intracellular parasitism. This paper reviews a number of recent studies in which it has been demonstrated that variations in transport rate are associated with substantial changes in tubule ultrastructure in the form of membrane insertion into and deletion from the apical microvilli. The principal driving force for fluid movement in Malpighian tubules is thought to be a common cation pump located in the apical membranes. It is proposed that modulation of the apical microvillar membrane may reflect regulation by the cells of the number of common cation pump units involved in fluid secretion.

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy).

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.

Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans.

Population density and percentage of the different stages of an established infection of Trypanosoma cruzi were determined for two parts of the excretory system and for the rectum of fifth instars of Triatoma infestans unfed and 4 hr after feeding. These data were also evaluated for feces and urine of the fed bugs. In the first unfed group only small populations of the flagellate occurred in the Malpighian tubules and ampullae and not in all bugs. The three rectal populations (rectal lumen and anterior and posterior rectal wall) consisted of approximately equal numbers. About 10% were spheromastigotes and about 10% were stages intermediate to epimastigotes. Significantly fewer epimastigotes and more trypomastigotes were present on the rectal wall than in the lumen. Two intermediate forms leading to the trypomastigote stage occurred in similar numbers. In nearly all bugs the initial excretion (feces) contained the highest number of flagellates as compared to the following drops of urine. More flagellates were excreted through the urine than were contained in the excretory system of unfed bugs. The population in the feces reflected the percentage of forms present in the rectal lumen of unfed bugs, but in the urine the percentage of trypomastigotes increased up to 100%. Four hours after blood uptake, dissection of bugs still showed parasites in the Malpighian tubules and ampullae; the total number of parasites in the rectum was reduced by more than 50%. This reduction was more pronounced in the rectal lumen and on the posterior rectal wall. In stained smears from all three rectal populations there were rarely spheromastigotes but high percentages of epimastigotes. The intermediate stages leading to trypomastigotes mainly originated from short epimastigotes. Comparison of the T. cruzi populations before and after feeding demonstrates that the trypomastigotes in the urine should originate from the rectal wall, especially from the posterior part.

An ultrastructural study of Dirofilaria immitis infection in the Malpighian tubules of Anopheles quadrimaculatus.

An ultrastructural study was conducted of the Malpighian tubules of Anopheles quadrimaculatus, both uninfected and following infection with Dirofilaria immitis. The Malpighian tubules in Anopheles are composed of primary and stellate cells. The primary cells are the predominant cell type and are characterized by the presence of membrane-bound, intracellular, mineralized concretions and large apical microvilli containing mitochondria. Following the infective blood meal, the microfilariae enter the primary cells of the Malpighian tubules and reside in the cytoplasm in a clear zone without a delimiting membrane. Cells in infected tubules differ from those in uninfected tubules in that the membranes of the vacuoles surrounding the concretions are disrupted in many specimens. The apical and basal cell membranes and the mitochondria associated with these are not disrupted during the first 6-8 days of infection. These observations differ sharply from those previously described in Aedes taeniorhynchus infected with D. immitis. The observations are consistent with the hypothesis that the extended transport capacity observed in previous physiological studies of An. quadrimaculatus infected with D. immitis are dependent on the prolonged normal ultrastructure of the apical microvilli, mitochondria, and basal membranes.