Cluster analysis of Anopheles Stephensi fourth instar larvae behavior to explore sequential organisation of larvae movement in a microcosm

Experiments were conducted on 20 fourth instar Anopheles stephensi larvae to explore behavior organisation. Twenty fourth instar larvae were placed in a glass aquarium and filmed using a handy cam video recorder and the recordings analysed in a laptop computer. Data of transitions from one behavior to another for all observations were collated into a matrix of preceding and succeeding behaviors to study sequential organisation and relationship among behaviors. Significant testing for first-order transition was done using G test at P< 0.005 and a kinematic graph constructed from significant transitions. A time budget and transition frequency data constructed for each behavior were subjected to cluster analysis to explore relationship between the behaviors. Result of the analysis showed that fourth instar Anopheles stephensi larvae behaviors occur in clusters in specific locations in their aquatic environment. Furthermore, the sequential organisation of behaviors is influenced by behavior frequency and the amount time a larva spent doing that behavior. When food is kept constant, other factors such as gas exchange requirements, behavior variation due to day-night cycle, presence of a predator, interlarval competition for food and the size of the aquarium (depth and width) maybe determining behavior organization.

Differences in submergence time and alarm reaction rate in Anopheles stephensi, Aedes egypti and Culex pipiens fourth instar larvae in response to vibration stimulus

Studying mosquito larvae behavior can provide insights into developing new ways in controlling vector borne diseases. Feeding is an aspect of larval behavior that has been studied extensively. Surface and underwater feeding behaviors are linked by diving. The larvae use dive behavior to feed underwater or to escape predators. We elicited dive responses in fourth instar larvae of three different mosquito species using vibration stimulus and compared their response rate and submergence time. Marked differences in the response rate and submergence time were recorded for the three species of mosquitoes. These differences can be exploited to develop new larvae control methods to reduce mosquito borne diseases.

Laboratory evaluation of traditionally produced coconut oil as a surface larvacide against Anopheles Stephensi fourth instar larvae

Environmental concerns have resulted in the search for environmentally friendly natural oils for use as mosquito larvacide. Methylated coconut oil has been found to be toxic to mosquito larvae. However, the use of methylated coconut oil is limited by resource constraints in rural communities in Papua New Guinea and other Pacific Island countries where coconut oil is produced by traditional methods. This study evaluated the toxicity of traditionally produced coconut oil to fourth instar Anopheles stephensi larvae. The results showed that traditionally produced coconut oil is toxic to fourth instar Anopheles stephensi larvae. The results showed that coconut oil produced by traditional method is toxic to fourth instar Anopheles stephensi larvae. The coconut oil can be used as a larvacide for malaria vector control in community based programs utilizing community participation in the production and use of coconut oil for large scale use of coconut oil. However, a suitable surfactant needs to be identified.

Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis.

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

Superoxide-dependent and -independent pathways are involved in the transmission blocking of malaria.

Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on day 5 post-infection in X-CGD and control mice and then decreased significantly after day 5 post-infection. However, on day 7 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.

A histochemical study of the secretory gland cells of Cercaria shikokuensis and their role during development from cercaria to metacercaria.

The roles of secretory glands during the developmental process from an immature cercaria to a metacercaria in Cercaria shikokuensis were studied. Four types of secretory cells were identified in this species. On maturation of the cercaria in redia, the products of ventral gland cells and mucoid gland cells formed a thick surface coat on the mature cercaria, and the products of cephalic gland cells also formed a thin cover on the surface coat. In the process leading to the formation of a metacercaria, the surface coat constituted the outer layer of the cyst, mucoid gland cells secreted mucous substances inside the wall, and then cystogenous gland cells discharged their products to the inner wall. The cyst wall was composed of four layers, and it was thought that the outermost surface layer helped the cyst wall to adhere to the matrix and the intermediate layers helped to put together outer and inner walls.

Archaebacterial relationships of the phosphoenolpyruvate carboxykinase gene reveal mosaicism of Giardia intestinalis core metabolism.

A gene encoding a putative GTP-specific phosphoenolpyruvate carboxykinase has been cloned and sequenced from the type I amitochondriate protist Giardia intestinalis. The deduced amino acid sequence is related most closely to homologs from hyperthermophilic archaebacteria and only more distantly to homologs from Eubacteria and Metazoa. Most enzymes of Giardia core metabolism, however, are related more closely to eubacterial and metazoan homologs. An archaebacterial relationship has been noted previously for the unusual acetyl-CoA synthetase (ADP-forming) of this organism. The results suggest that phosphoenolpyruvate carboxykinase and acetyl-CoA synthetase have been acquired from different sources than most enzymes of Giardia core metabolism.

The role of saliva of Anopheles stephensi in inflammatory response: identification of a high molecular weight neutrophil chemotactic factor.

Mosquito bites can elicit dermal hypersensitivity reactions, but little is known about the chemotactic factors for host leukocytes in mosquito saliva. In this study, we determined that saliva from a malarial vector mosquito, Anopheles stephensi, possesses intense neutrophil chemotactic activity. In contrast, the midgut extract had only marginal neutrophil chemotactic activity. Eosinophil chemotactic activity was detected in the midgut but not in the saliva. According to the results of size-exclusion HPLC on a G3000SW column and Western blot analysis, the apparent molecular weight (MW) of the main neutrophil chemotactic factor (NCF) was estimated to be 200 kDa. NCF could bind with IgG from the pooled serum of Solomon islanders, whereas not with that of healthy Japanese. NCF activity was increased upon heating to 56 degrees C for 30 min or protease digestion, whereas it was affected by periodate treatment. Protease-digested NCF and naive NCF bound to lentil lectin-Sepharose, and both were eluted with a competitive sugar, methyl-alpha-D-glucoside. These results indicate that A. stephensi saliva-derived NCF is a high MW glycoprotein, and its protein moiety is important for neutrophil chemotactic activity. This NCF is thought to contribute to the inflammatory reactions through the accumulation of neutrophils at the site of the mosquito bite.

Estimation of the sporozoite rate of malaria vectors using the polymerase chain reaction and a mathematical model.

We developed a sensitive polymerase chain reaction (PCR) method for the detection of Plasmodium falciparum DNA from mosquitoes collected in the field. Plasmodium falciparum was detected from 15.2% of 1-parous mosquitoes, Anopheles farauti, in the Solomon Islands through use of the PCR method. A novel mathematical model was developed to estimate the sporozoite rate based on the malaria-positive rate of 1-parous mosquitoes. Using this model, the sporozoite rate of Anopheles farauti in the Solomon Islands was calculated to be 0.09%. This method enables estimation of the sporozoite rate based on a relatively small number (100-200) of mosquitoes compared with the number needed for the ELISA method.

Detection of Pneumocystis carinii sequences in serum by polymerase chain reaction.

The clinical significance of the detection of Pneumocystis carinii DNA was evaluated, as well as the detection of circulating P. carinii antigen from serum using previously collected samples. Fourteen serum samples from 13 patients were diagnosed positively for P. carinii DNA by polymerase chain reaction (PCR). Ten of 14 episodes (71.4%) of pulmonary complications were compatible with P. carinii pneumonia. Two patients were definitely diagnosed as having had P. carinii pneumonia at autopsy. All patients positive for circulating antigens were also positive for P. carinii DNA, suggesting that the detection of P. carinii DNA by PCR is more sensitive compared to the detection of circulating antigens by the Ouchterlony method. It is concluded that the detection of P. carinii DNA in serum by PCR provides useful information for identifying P. carinii pneumonia.

Acanthamoeba-specific human T-cell clones isolated from healthy individuals.

T-cell responses to pathogenic free-living amoebae, Acanthamoeba sp., were analyzed in healthy Japanese individuals. Of 20 healthy subjects, 10 (50%) showed significant proliferative responses of peripheral blood mononuclear cells to the soluble amoebic antigens in vitro. The antigens used were not mitogenic, and no evidence of amoebic superantigens was available. We established human T-cell clones reactive to Acanthamoeba, all of which were CD3- and CD4-positive, CD8-negative, and TCR-alpha beta-positive. We isolated two strains of Acanthamoeba from two patients, one from a patient with meningoencephalitis (CSF strain) and the other from a patient with keratitis (K strain). Of 13 clones, 11 were reactive to the K-strain as well as to the CSF-strain antigen under human leukocyte antigen (HLA)-DR restriction, whereas the other two were specific for the K-strain antigen. All but one clone tested showed TH1-equivalent functions because these cells produced interferon (IFN)-gamma in response to the amoebic antigen but produced no detectable level of interleukin 4 (IL-4). These results suggest that immunocompetent hosts might have acquired protective immunity mediated by Acanthamoeba-specific T-cells during natural sensitization.

Identification of human blood in mosquitoes (Diptera: Culicidae) using nonradioactive DNA dot blot hybridization.

A dot blot hybridization procedure was developed to detect human blood meals in engorged mosquitoes. A biotinylated DNA probe allowed the detection of 10-100 ng of human DNA, discriminated well between human and nonhuman sources of blood, and cross-reacted only with monkey DNA. Results showed that this method was a specific and sensitive technique for the identification of blood meals up to 100 h after ingestion. The nonisotopic label offers easy handling without the problems inherent in the use of radioisotopes, and it can be adapted for use in routine field tests.

Vector mosquitoes of Wuchereria bancrofti at Bicol region in the Philippines. 1. Transmission capability.

Mosquitoes were surveyed for the capability of filaria transmission at Sitio Bacolod and Barrio Salvacion, in Bicol region, Luzon, Philippines. The results of indoor collections and experimental infections showed that the infective rates and the median density of infective larvae per infected mosquito were significantly higher in Aedes poicilius than in Culex quinquefasciatus. Aedes poicilius was found to be the principal vector of bancroftian filariasis in this region.

An epidemiological study of filariasis in Sorsogon province, Republic of the Philippines, with notes on experimental mosquito infection.

An epidemiological survey was conducted on Bancroftian filariasis in Sorsogon province, the Philippines, with special regard to low density microfilaremia (LDF). Filtration technique using nuclepore membrane examining 1 ml blood revealed 31% positive rate for microfilaria out of 262 volunteers. Out of 82 positives 21 (25.6%) had LDF (less than 10 in 1 ml blood). No conspicuous grouping of LDF was noted in younger age groups or sexes. In three out of 12 clinical filariasis, microfilaria was detected but none of them were LDF. Among 22 cases treated with diethylcarbamazine, nine subsequently had LDF suggesting necessity of improvement of drug administration regimen to avoid incomplete treatment. A series of feeding experiments using two mosquito species, Aedes poicilius and Culex quinquefasciatus, on volunteers showed that both species could act as vectors in the region. No larva was detected in either species fed on LDF carriers. It was concluded that there is little possibility that LDF plays an important role in the transmission of Bancroftian filariasis in the region.

[Ultrastructural changes of the tegument on Diphyllobothrium erinacei and Hymenolepis nana treated with paromomycin sulfate in vitro].

The effects of paromomycin sulfate on Diphyllobothrium erinacei and Hymenolepis nana in vitro were examined morphologically with a scanning and a transmission electron microscope. D. erinacei was incubated for 3 and 6 hours at 37 degrees C in a culture medium, 0.85% physiological saline solution, containing 0.5% paromomycin sulfate. H. nana was incubated in the same medium for 3 hours only. The concentration of paromomycin sulfate was set basing on the results which Kitamoto (1968) reported as the concentration level in feces after administrations of this drug in a clinical survey. The effect of the drug on the surface structure in both worms appeared markedly in the neck region. Mechanisms of breakdown on the tegument were supposed as follows. First, microtriches were disconnected from the tegumental surface and many vesicles were formed in the cytoplasm of the tegument. Finally, the tegument layer was excoriated to exposed the basal lamina. In 6 hours incubation, this surface of the worm suffered more damage than that in 3 hours. The damage of the basal lamina as in the case of D. latum expelled from a man by paromomycin (Y. Tongu et al.), however, could not be observed in the present study in vitro. It suggests that the destruction of basal lamina usually observed with the expelled worms from clinically treated human might be due to the combined effect of digestive enzymes secreted from host and the mechanical impact of intestinal peristalsis. Some of the vesicles in the tegument may originate from mitochondria because the fine structure of cristae were occasionally observed remaining in the vesicles.