Binding sites of mosquitocidal toxins of Pseudomonas fluorescens and Bacillus subtilis on pupae and larvae of Culex quinquefasciatus.

Two of the potential bacterial isolates, viz., Pseudomonas fluorescens (VCRC B-426) and Bacillus subtilis (VCRC B-471) whose toxins kill the mosquito pupae/larvae have been identified at our center. As the mode of action of these bacteria are not known, an attempt was made to find out the binding sites of the toxic proteins through immunological methods. Antibodies were raised in BALB/c mice and egg yolk system of chicken layers against the mosquitocidal proteins. The antibodies showed specific binding on to the cephalic and thoracic cuticle of the pupae as well as the paddles of the larvae, indicating the binding of the mosquitocidal proteins.

Efficacy of a mermithid nematode Romanomermis iyengari (Welch) (Nematoda: Mermithidae) in controlling tree hole-breeding mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) in a rubber plantation area of Kerala, India.

In rubber plantations, tree holes are one of the major types of breeding habitats of Aedes mosquitoes which transmit dengue and chikungunya. A mermithid nematode, Romanomermis iyengari, was evaluated in tree holes for its efficacy in controlling Aedes albopictus. Infection of mosquito larvae by the nematode was determined through microscopic examination on the next day of application, and evaluation of immature density of mosquito was done on the seventh day. After application of the infective stage of the nematode in a host-parasite ratio of 1:3 or 1:4, the infection rates on the different larval instars of mosquito were similar, 85.7-95.8 % in first to third instars and 79.3 % in fourth instar larvae or 100 and 92.9 %, respectively. Parasite burden varied from 1.1 to 2.4, respectively, among first and third instar larvae applied at 1:3. At 1:4, the parasite burden was between 1.6 (fourth instar) and 4 (second instar). The increase in parasite burden due to parasite density was significant in all the larval instars (P < 0.05). High parasite burden is detrimental to parasite recycling as it can cause premature mortality of the host. Hence, the dosage of 1:3 could be considered as suitable for rubber tree hole habitats. In the nematode-applied tree holes, there was a significant level (P < 0.05) of reduction in the immature density of A. albopictus, especially late instars and pupae, confirming the efficacy of R. iyengari in infecting the mosquito and controlling pupal emergence.

Bacillus sphaericus in the adults of Culex quinquefasciatus mosquitoes emerged from treated larvae and its effect on development of the filarial parasite, Wuchereria bancrofti.

Bacillus sphaericus is a bio-control agent effective against Culex quinquefasciatus, the vector of bancroftian filariasis. Apart from its larvicidal effect, there are reports of reduced infection of filarial parasites in mosquitoes exposed to it. In the present study, adults of Cx. quinquefasciatus emerged from B. sphaericus treated larvae were fed on blood samples positive for microfilariae of Wuchereria bancrofti and examined at various time intervals to assess the infection level. The rate of infection was reduced from 95% on day 1 post-feeding to 75% on day 13, when fed with blood sample containing 41 mf/20 µl. The mean parasite burden was also reduced from 4.9 per mosquito on day 1 to 2.15 on day 13. When fed with another sample (30 mf/20 µl), the infection was reduced from 100% on day 1 to 80% on day 13. Reduction in parasite burden was 4.0 to 1.75. Abnormally developed second-stage larvae of the parasite were seen in treated mosquitoes. Thus, the results indicated adverse effect of B. sphaericus treatment on infection and development of the filarial parasite in mosquitoes. The possible reason for the parasite regulation was studied through the assessment of the carryover of the bacterium as well as its toxins to the surviving mosquitoes. The presence of B. sphaericus was determined through plating of homogenate of survived mosquitoes on NYSM agar. Toxic protein was detected through immunoblotting. The bacterium as well as its 41.9-kDa toxic protein was found to be transmitted from larvae to adults and affected the parasite development, directly by the toxin or indirectly by eliciting humoral immune response of the mosquito.

Up-regulation of lipophorin (Lp) and lipophorin receptor (LpR) gene in the mosquito, Culex quinquefasciatus (Diptera: Culicidae), infected with the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae).

In mosquitoes, including Culex quinquefasciatus, immune molecules are known to be upregulated or produced de novo upon exposure to parasites or pathogens. These molecules are regulatory in nature acting against parasite or pathogen infection and development. Similarly, there are molecules that are upregulated to facilitate parasite development in the vector mosquitoes. Lipophorin, a major lipid transporting lipoprotein in the hemolymph of insects, is implicated as a helper molecule in the clotting mechanism and facilitator of parasite and pathogen development in mosquitoes. In the present study, upregulation of a 240 kDa protein was detected in C. quinquefasciatus infected with the human lymphatic filarial parasite, Wuchereria bancrofti. It was identified as a lipophorin through nano-Lc-MS/MS analysis. Transcription of the lipophorin receptor gene also was identified through RACE-PCR. C. quinquefasciatus is the vector of W. bancrofti, and it allows successful development of the parasite. The role of upregulated lipophorin and transcription of its receptor gene in this mosquito could be implicated as a facilitator for the parasite development.

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain.

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC(50) dose of 1 microg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13-C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

Identification of immune-responsive genes in the mosquito Culex quinquefasciatus infected with the filarial parasite Wuchereria bancrofti.

Several antimicrobial/parasitic peptides are known to be upregulated in mosquitoes upon infection with parasites. The aim of this study was to identify immune-responsive genes in the vector mosquito, Culex quinquefasciatus Say (Diptera: Culicidae) against the human lymphatic filarial parasite, Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae). Suppression subtractive hybridization was performed using RNA from filarial infected and non-infected mosquitoes to obtain differentially expressed transcripts, and their identities were confirmed through reverse transcriptase polymerase chain reaction (RT-PCR). Out of 23 clones selected from the suppression subtractive library, three corresponded to antimicrobial peptide genes, defensins, and four corresponded to regulatory serpin peptide genes. RT-PCR using defensin-specific primers and sequencing of the product showed a 284-bp defensin cDNA. Sequence alignment with defensins of the mosquitoes Anopheles gambiae s.s. Giles and Aedes aegypti (L.) showed maximum homology with the former. Similarly, that of serpin-specific primers showed a 406-bp cDNA encoding serpins. Sequence alignment showed maximum homology with that of An. gambiae, as in the case of defensins. Hence, this investigation revealed upregulation of defensins and serpins in Cx quinquefasciatus infected with W. bancrofti. Antimicrobial peptide genes such as defensins may have limited or no specific role in regulating parasite development. Serpins may prove to be facilitating molecules, by regulating melanization of the parasite. However, the exact functions of these molecules in the immune system of the vector mosquito are yet to be investigated.

Domestic dogs as reservoir hosts for Leishmania donovani in the southernmost Western Ghats in India.

The peripheral blood samples from domestic dogs (n=47) and wild rats (n=25) in the Kani Tribe settlements, located southernmost part of the Western Ghats, Thiruvananthapuram district, Kerala, India were examined for Leishmania infection. This area is known for cases of leishmaniasis with cutaneous manifestations and sandfly abundance. The tribes domesticate dogs to protect them from untoward activities of wild animals. Leishmania donovani parasite DNA was detected only from 6.4% (n=3) of the blood samples collected from the domestic dogs by amplification of the diagnostic kinetoplast mini-circle DNA and PCR-RFLP analysis of the UTR region of heat shock protein 70 (hsp70) gene. None of the blood samples collected from rats was positive. Through sequencing, L. donovani infection among dogs was confirmed. The DNA sequences generated for hsp70 were deposited with the GenBank. The GenBank accession numbers of these samples are KR905363, KR905364 and KR905365 for hsp70 genes. The results indicated that the DNA isolates from dog blood samples matched precisely with that of our earlier isolates from skin lesions of Kani tribes and also from P. argentipes vector. Thus, the role of dogs as reservoirs for L. donovani parasite in the Kani tribe settlements is confirmed.

Development of lymphatic filarial parasite Wuchereria bancrofti (Spirurida: Onchocercidae) in mosquito species (Diptera: Culicidae) fed artificially on microfilaremic blood.

The efficiency of laboratory colonies of mosquitoes such as Anopheles stephensi Liston, Aedes aegypti (L.) Liverpool strain, Ae. aegypti wild type, Aedes albopictus (Skuse), Culex tritaeniorhynchus Giles, Culex sitiens Wiedemann, and Armigeres subalbatus Coquillett in supporting the development of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) microfilariae to infective larvae was investigated. The mosquitoes were fed on heparinized microfilaremic human blood by using a membrane-feeding unit with Parafilm as membrane. The rate of infection, parasite development, and parasite burden were compared with that in the known vector mosquito Culex quinquefasciatus Say. Cx. quinquefasciatus showed the highest percentage of infection, followed by Ae. aegypti Liverpool strain and An. stephensi. The rate of development of the parasite was more or less similar in all the three species, and infective larvae were found on day 13. When the larvae were harvested on day 17, Cx. quinquefasciatus yielded the highest numbers, followed by Ae. aegypti Liverpool strain and An. stephensi. The percentage of infection was low, and the development was slow in Cx. tritaeniorhynchus compared with the other susceptible species. The parasite developed to second-stage larvae only by day 22 and to infective larvae by day 28. When 2-wk-old Cx. tritaeniorhynchus were fed on microfilaremic blood, they could develop the parasite to infective larvae by day 13 postfeeding. All other species of mosquitoes tested were found to be refractory to parasite development. It is shown that Cx. quinquefasciatus is the most suitable mosquito host for the production of infective larvae. However, Ae. aegypti Liverpool strain, which is commonly used for Brugia malayi filarial parasite, also can be used for generation of W. bancrofti infective larvae to circumvent the problem of maintaining two mosquito species.

Toxicity of a mosquitocidal metabolite of Pseudomonas fluorescens on larvae & pupae of the house fly, Musca domestica.

BACKGROUND & OBJECTIVES: Biological control through the use of parasitoids and pathogens is one of the alternatives to the use of chemical pesticides for control of insects of public health importance. At the Vector Control Research Centre, a liquid formulation developed using the metabolite of a Pseudomonas fluorescens strain was found to be lethal to larvae as well as pupae of vector mosquitoes. The lethal fraction of the metabolite is a protein with a molecular mass of 44 kDa and toxicity studies showed that it is safe to mammals. In the present study, this formulation was evaluated against immatures of the common house fly, Musca domestica, to find out whether it could be developed into a potential biocontrol tool. METHODS: Early second instar larvae of house fly were introduced into rearing medium incorporated with the formulation at concentrations of 1, 5, 10, 15, 20 and 25 per cent, which were equivalent to respectively 1.13, 5.63, 11.25, 16.88, 22.50 and 28.13 microg of the toxic protein/ g of rearing medium. Mortality was monitored until the emergence of adult house fly. Net mortality of larvae and pupae were calculated and the LC50 and LC90 values were determined through probit regression analysis. RESULTS: Larval mortality was obtained from day 3 to 6 post-treatment. Net mortality of larvae was higher at the concentration of 20 than at 25 per cent. However, it was higher at 25 per cent on day 5 and continued to day 6 when there was no larval mortality at other concentrations. The net mortality of pupae was higher than that of larvae at all the concentrations except at 20 per cent. The LC50 and LC90 values calculated from the net mortality of larvae and pupae together, from day 1 to 12 post-treatment, were respectively, 8.25 and 51.79 microg protein/g of the fly rearing medium. INTERPRETATION & CONCLUSION: The formulation prepared from the exotoxin of P. fluorescens was toxic to the house fly. Pupae were more susceptible than larvae and the activity of the toxin might have been through cuticular absorption. The results are indicative of the possibility of development of the mosquitocidal metabolite for house fly control through appropriate field evaluations.

A report on the demonstration of microfilariae of Brugia malayi in the brain of an experimental animal host, Mastomys natalensis.

The brain tissues of microfilaraemic animals, Mastomys natalensis, which were earlier inoculated (s.c.) with Brugia malayi infective larvae (100 each) were examined for the occurrence of Mf. This was done by staining squash preparations of the brain tissues which were cleared off from the vascular piamater. Animals with blood Mf count of 50 >/per 20 cu. mm were found to harbour Mf in the brain tissues. The Mf count in the brain varied from 5-86/81 cu. mm (sum of Mf detected in 3 tissue pieces, each of 27 cu. mm collected from 3 parts of the brain, viz., the cerebral hemispheres, cerebellum and medulla oblongata). Teh presence of Mf in the brain was confirmed by its detection in 20-micrometers-thick cryosections of the tissue. Also, fine needle aspirates of cerebral hemispheres of an animal showed live Mf.

An improved method of mass culturing of Romanomermis iyengari, a mermithid nematode parasite of mosquito larvae.

An attempt was made to develop an alternative method of mass culturing for R. iyengari instead of the usual sand culture method. Fifty pairs each of post-parasitic juveniles were seeded in moist sandbed and beakers containing tap water and distilled water and examined for exsheathing, egg-laying and egg-hatching. All post-parasitic juveniles in the moist sandbed had moulted by 7th day whereas in tap water and distilled water it lasted up to 11th day. Maximum numbers of eggs were observed on day 14 in sandbed (15/ml), day 16 in tap water (24/ml) and day 29 in distilled water (28/ml). The preparasites obtained from moist sandbed, tap water and distilled water did not exhibit any difference in their infectivity to mosquito larvae. The eggs of the nematode obtained from a culture in distilled water maintained at 30 +/- 2 degrees C for 60 days, when treated with CO2 (18 to 556 ppm) showed enhanced rate of egg hatching (73-98% compared with 11.5% in the untreated ones). CO2 treatment did not affect the infectivity of the preparasites that hatched from the CO2-treated eggs.

Enhanced recovery of fourth stage larvae of Wuchereria bancrofti from mongolian gerbil, Meriones unguiculatus & their in vitro maintenance.

Earlier attempts to produce different stages of W. bancrofti, such as fourth stage larvae (L4), in small animal models have yielded very low recovery rates. In order to enhance the recovery of L4, two routes of inoculating a small animal, M. unguiculatus, with infective larvae (L3) viz., intraperitoneal and intrathoracic routes, were compared. On day 17 post-inoculation, higher percentage (23-25%) of L4 were recovered from animals inoculated intrathoracically compared to that from animals inoculated intraperitoneally (2-8%). Also, comparatively higher proportion of worms (75-92%) remained within the intrathoracic region, unlike in the intraperitoneal region (50-80%). A few worms (1-4%) could be recovered even on 31 days post-inoculation from animals inoculated intrathoracically. When the L4 produced in animals were cultured in modified Frank's medium, all of them survived for 15 days and 50 per cent survived till the 25th day. The higher yield and ease of recovery from the thoracic cavity makes this route of inoculation a suitable method for production of L4. In vitro maintenance of L4 for prolonged period is significant with respect to excretory/secretory products or for drug screening.

Isolation of a Pseudomonas fluorescens metabolite/exotoxin active against both larvae and pupae of vector mosquitoes.

A formulation was developed from the metabolite(s) of a novel Pseudomonas fluorescens Migula strain (VCRC B426) and tested against 4th-instar larvae and pupae of three species of vector mosquitoes, Anopheles stephensi Liston, Culex quinquefasciatus Say and Aedes aegypti (L). The larvae and pupae of An. stephensi were the most susceptible to the formulation, followed by those of C. quinquefasciatus and Ae. aegypti, in that order, and the dosage requirement for pupal mortality was less than that required for larval mortality. The LC50 dosage requirements for larvae of these mosquito species were, respectively, 70.4, 511.5 and 757.3 microg protein ml(-1), whereas for pupae they were, respectively, 2.0, 9.4 and 19.2 microg protein ml(-1). The lethal fraction was purified from the culture broth and its molecular mass, as determined by high performance liquid chromatography, was 44kDa. This is the first report of a microbial formulation acting upon mosquito pupae, a non-feeding stage. Its mode of action and efficacy to control mosquitoes under field conditions need to be studied further.

In vitro cultivation of third stage larvae of Wuchereria bancrofti to fourth stage: influence of some physico-chemical factors.

It has been reported that third stage larvae (L3) of Wuchereria bancrofti strain from Jakarta, molted to the fourth stage (L4) in vitro, in a simple culture medium supplemented with 10% human serum. In the present study, this culture medium has been used to examine the effects of some physico-chemical parameters on larval growth, development and molting of Wuchereria bancrofti from India. Lymph at 10% concentration enhanced the in vitro survival time of larvae. Molting of larvae from L3 to L4 stage has been obtained using human fetal lung cells in cellular co-culture and as a source of conditioned medium. Given these improvements in the medium supplementation, it has been observed that the age of L3s (duration of L3s maintenance within the mosquitos) is one of the most important parameters for the development of L3s in vitro. No molting was observed when one day L3s were used whereas, molting occurred with one or two weeks old L3s. On the contrary, when more than 3 weeks old L3s were used molting failed to occur even though duration of survival of L3s was improved and in this case, most of the larvae were degenerated.

Effect of temperature and host-parasite ratio on sex differentiation of Romanomermis iyengari (Welch), a mermithid parasite of mosquitoes.

The effect of temperature and host-parasite ratio on the percentage infection and sex differentiation of R. iyengari was studied. Significant differences were observed in the percentage infection due to different host-parasite ratios and temperatures. At 25 degrees and 30 degrees C, the host parasite ratio of 1:3 resulted in 86-92% infection of Culex quinquefasciatus larvae. At 20 degrees and 35 degrees C, a higher host-parasite ratio was required to get this level of infection. More number of post-parasites per mosquito larva emerged at 20 degrees (1.5-5.8) and 25 degrees C (1.9-6.3) than at 30 degrees (1.5-3.9) and 35 degrees C (1.6-3.6). More than 50% of the post-parasites were females at 20 degrees and 25 degrees, 30 degrees and 35 degrees C at 1:1-1:10, 1:1-1:4 and 1:1-1:3 host-parasite ratios, respectively.

Infectivity of a mermithid nematode Romanomermis iyengari (Welch) in different conductivity levels under laboratory and field conditions.

Infectivity of R. iyengari was examined by exposing mosquito (Culex quinquefasciatus) larvae to the preparasite at different conductivity levels. The preparasite infected 63.5, 30, 11, 1.5 and 0.5% of the mosquito larvae respectively at 2000, 2500, 3000, 3300 and 3600 mu ho/cm. Although, 62-69% of the preparasite survived at 4000-5400 mu ho/cm, it did not infect. Application of preparasite to tree-holes resulted in 53-63% infection of Aedes albopictus larvae initially. On 6th day the infection level was 40% which decreased further to 7% by 15th day. The infection reappeared on 38th day indicating that R. iyengari has not only infected mosquito larvae as soon as they were applied to tree-holes in which the conductivity was 600-2800 mu ho/cm but also got established there.

Observations on population density of Culex quinquefasciatus and transmission indices of Bancroftian filariasis during and after Integrated Vector Management strategy.

An Integrated Vector Management strategy, implemented as an alternative to the conventional control operations that include mainly chemical control in Pondicherry, South India, reduced very substantially the population density of Culex quinquefasciatus. This resulted in drastic decrease in the intensity of transmission of bancroftian filariasis transmitted by Culex quinquefasciatus and consequently the incidence of new infections in children of 0-5 age group was minimized. When the IVM strategy was withdrawn after five years of implementation and conventional control measures were re-adopted, resilience of Culex quinquefasciatus population was observed and human exposure to the risk of infection increased. The results suggest that maintenance of vector density at reduced levels for prolonged periods, is necessary to control infectious diseases like filariasis, which is difficult in the present day urban situations in developing countries. Hence the emphasis should be on chemotherapy to achieve control of lymphatic filariasis.