Response of the mosquito vector , Culex pipiens to Manzamine-Aas infection barrier against Wuchereria bancrofti filaria

Ahmed I. Hasaballah 1 and Walaa A. Moselhy 2 . 1. Department of Zoology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt. 2. Department of Zoology, Faculty of Science (Girls), Al-Azhar University, Nasr City, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Culex pipiensmosquitoes thrive in temperate and tropical areas and serve as efficient vectors of Wuchereria bancrofti the causativeagent of Bancroftian lymphatic filariasis. The antifilarial effect of Manzamine-A, oneof unique alkaloid groups isolated from indo-pacific sponges was evaluated by exposing the mosquito vectors that were treated with this drug to human individual infected with W.bancrofti as well as untreated mosquitoes. After the extrinsic incubation period of the nematode parasites, random samples of the fed mosquitoes were dissected to determine the infection rate. The survival rate of mosquitoes infected with microfilaria (mf) was calculated for both treated and control group. In addition, another group of mosquitoes were examined ultrastructurally to compare between the microfilarial course in both treated and untreated mosquitoes. Analysis of results indicated that Manzamine-A reduced the infection rate and the mf in the infected once didn't penetrate the mosquito midgut to reach the thoracic muscles, the site from which the filarial larval stagesdevelops and moves to the head to be transmitted to the next host during a subsequent blood meal.

Introduction:
Wuchereria bancrofti is one of three mosquito-borne nematodes that cause lymphatic filariasis. Over 120 million people around the world are infected with W.bancrofti and another 1.2 billion are at risk (WHO, 2012). The pathogenesis of lymphatic filariasis is linked to host inflammation invoked the death of the parasite, resulting in altered lymphatic system and the abnormal enlargement of limbs and male genitalia, causing pain and severe disability (Al-Abd et al., 2013). Culex pipiens is principle vector of W.bancrafti in urban areas of Africa. This species oviposit in stagnant polluted water, and populations are increasing and expanding due to urbanization, irrigation and in the Nile Delta, creation of Aswan High Dam (Michalski et al., 2010). As infectious diseases evolve and develop resistance to existing pharmaceuticals, the marine environment represents a treasure of useful products a waiting discovery for the treatment of fungal, parasitic, bacterial and viral diseases. A small number of marine plants, animals and microbes have already yielded more than 12000 novel chemicals, with hundreds of new compounds still being discovered every year (Donia and Hamann, 2003). One of these chemicals is Manzamine-A, which belongs to a group of unique Beta-carboline alkaloids isolated from Indo-pacific sponges of genus Haliclona. These compounds exhibit a diverse range of bioactivities including cytotoxicity, insecticidal, antibacterial and antimalarial activities.

Mosquito rearing:
The colony of Culex pipienswas established from larvae and pupae obtained from breeding sites in Abu Rawash, Giza Governorate, Egypt.Twenty-fiveindividuals of 3 rd instar larvae from the second generation were placed in plastic cups (diameter 12cm x height 7cm) containing 250 ml of Manzamine-A solution. Another group of larvae with the same number were placed in 250 ml dechlorinated tap water as control. The larvae were daily provided with fish food as a diet, which proved to be the most preferable food for their development and a well female fecundity (Kasap and Demirhan, 1992). The emerged adult females were provided with 10% sugar solution on cotton pads.

The test compound:
The antiparasitic compound used in this work was Manzamine-A, polycyclic marine-derived alkaloids, isolated from the sponge Haliclonasp. It was from sigma chemical Co. with suggested used concentration: 0.5-1 g/ml. It was solubilized in dimethyl sulfoxide (DMSO) and kept in a refrigerator under sterilized conditions. The concentration used for treatment was 1 g/ml.

Parasites and Parasite exposure:
Source of mf for this study was a volunteer that was acquainted with the details of the study and the nature of her participation. Before exposure to the infected volunteer, the mosquito females emerged from Manzamine-treated and untreated larvae were starved for 24h. The starved females were exposed to volunteer hand and forearm for 20-30 minutes. The infection of the vectors was conducted between 10 PM and midnight (the peak of microfilaraemia activity), Rocha et al., 1991.After exposure the engorged mosquitoes were maintained according to standard procedures in the laboratory at 27C.

Calculation of the infection rate:
Ten engorged mosquitoes were anaesthetized and each female was dissected separately after removal of wings and legs for detection of W. bancroftimf. The mosquito's head, thorax and abdomen were teased a part in saline and were examined carefully under binocular microscope and the infection rate was calculated according to Hassan

Results:
The infection rates with Wuchereriabancrofti in females of Culex pipiens that emerged from 3 rd larval instar which treated with Manzamine-A as well as the untreated ones were shown in table 1 and figure 1. The treatment with Manzamine-A resulted in reduction of the infection rate, that it was 90% in untreated group while the treated group recorded 70%.    In contrast to the two previous micrographs, figs. 5 and 6 showed that the microfilariae did not penetrate the borders of the midgut and failed to reach the flight muscles. This means that Manzamine-A has an effect on the motility of the parasite and prevents it from reaching the flight muscles.Micrograph 6 showed the tracheoles dispersed between the fibrils of the muscles.  In a susceptible host, W.bancroftimicrofilariae migrate from the midgut and develop in the indirect flight muscles. The metamorphosis from the mf to infectious L 3 stage-a process that necessitates a physical transformation from mf to sausage-shaped first stage larvae L 1 , and two subsequent molting events. Under optimal conditions, development takes approximately 10-12 days and the parasites increase in size 4-6 times ( Bartholomay and Christensen, 2002). The ultrastructure microscopy of the normal mosquito female in the present work demonstrated this course of the parasite, that the electron micrographs showed the penetration of the mosquito's midgut by mf and the presence of larvae in the indirect flight muscles. On the other hand the electron micrographs of a female emerged from treated mosquito larva showed the failure of mf to reach the muscles. Gupta et al. 2012 found that the sponge alkaloids affect the motility of filaria as a mechanism of antifilarial activity that may explain the present work findings.
The parasite development is not benign process; W. bancrofti microfilariae inflict debilitating or even lethal damage on the mosquito hosts by disrupting normal cellular and physiological processes in the flight muscles (Bartholomay 2014). These effects supported the present work data, that the survival rate of the normal female mosquitoes was less than that of females emerged from treated larvae. In mosquitoes, midgut infection and escape barriers that have been described include inhospitable chemical environment of midgut lumen that destroys incoming parasites (Higgs 2004).The treatment of mosquito larvae with Manzamine-A might change the chemical environment of midgut resulting in incapability of the mf to escape from the midgut and transform to infectious L 3 stage.