LARVICIDAL EFFECT OF AMPALAYA (Momordica charantia) FRUIT JUICE ON Aedes MOSQUITO LARVAE Amtul. Noor, Asunala. Rajesh Kumar, Chevagoni. Suresh, Doddaka. Ghnapti Krishna Sayogi, Gollapally. Vikram Goud, Kosaraju. Narendra kumar, Koutharapu. Deepak Chandran, Pagadala. Devi Vara Prasad, Rama. Annapurna, Rama. Mounika, Rama. Nikhil Raj, Sah. Sitesh Kumar, Senguttuvan. Rahul Anand, Tamang. Nima, Uppugalla. Ruzu Rohith Reddy Mrs. Mary Grace G. Villaflor Abstract This study was intended to determine if Ampalaya fruit juice can be used as larvicidal agent for Aedes larvae and also compares its larvicidal property with that of a commercial larvicide (ABATE 1SG). This research was experimental in nature, using completely randomized design with commercial larvicide as positive control and pure water as negative control. Aedes mosquito eggs were collected from one of the researcher’s residence, breeding and hatching was done in the zoology laboratory of CDU. Two (2) to four (4) days old larvae were used as our research subject. Beaker, Measuring Cylinder, Plastic Containers, Spoon were the materials used in this research. After the setup of experiment, during each trial the numbers of dead larvae were counted after time interval of 20 minutes, 40 minutes and one (1) hour. The larvae that floated and did not respond to very slow stirring of the plastic spoon were considered as dead larvae. Based on the result, it can be concluded that Ampalaya fruit juice had a larvicidal effect, but it was inferior to the positive control ABATE 1SG. So, it might be effective to other genera of mosquito larvae or other parts of Ampalaya may be more effective. Key words: Aedes larvae, Ampalaya fruit juice, Commercial larvicide INTRODUCTION Background of study Mosquitoes are among the well-known group of insect vectors that transmit deleterious human diseases, which pose as the major public health challenges eroding development in the poorest countries of the world (Awad and Shimaila, 9 (4) : 637). ----------------------------------------------------Sah. Sitesh Kumar
[email protected] +069326640583
Their medical importance as vectors for the transmission of serious diseases that cause morbidity, mortality, economic loss and social disruption such as malaria, filariasis, yellow fever, dengue and other viral diseases as well (Becker et al. 325).
The species of Aedes are important vectors in the transmission of dengue, yellow fevers and chikungunya fevers, and the like. Dengue is a serious and ancient problem spread by the bite of the infected Aedes mosquitoes and its complications affects the kidney, liver, brain and blood which could be very fatal. Dengue fever is an actual frequent epidemic viral disease transmitted by the mosquito Aedes aegyptii. These are the burning issue now-a-days. These health conditions are endemic in the developing countries of Africa, Latin America and Asia. World Health Organization (88), Brown (2 : 123- 140) and Lee et al. (44: 105 - 112), reported about the development of physiological resistance to the chemicals by mosquito species. Some mosquito species developed resistance to malathion (Guneady et al. 50 (1) : 45- 54), a conventional pesticide, and to deltamethrin (Chen wenmei 33,(1) : 14 - 20) like the adult Culex pipiens. These mosquitoes have developed resistance against currently used insecticides, thereby hindering their effective control and possibility of eradicating them in areas where they are prevalent. Furthermore, the adverse effects of conventional insecticides on the environment and animals, including humans, and the limited number of available insecticides compel continued search for safer plant insecticides and larvicides that could be used in the control of these vectors. Medicinal plant extracts and their constituents have proved to be biodegradable, have low mammalian toxicity and induction of resistance while their activities were similar to those of the standard drugs, such as temephos and
methoprene. These viral diseases and their resistance to currently used insecticides were the reasons that researchers decided to study whether Ampalaya fruit extract could also be a possible mosquito larvicide. Theoretical framework According to Bayer Environmental Sciences and Reinert (16: 175- 188), there are about 3500 species of mosquitoes, grouped into 3 sub-families, Toxorhnychitinae, Anophelinae, and Culicinae. Although there are over 3500 species of mosquito through the world, all of which live in specific habitats, exhibit unique behavior and bite various animals. Despite these different behaviors, all mosquitoes share some common traits, such as four stage life cycle. After a female mosquito bites the animal and obtains the blood, it lays eggs on the surface of stagnant water, in a depression, or on the edge of a container where rain water may collect and flood the eggs. The eggs hatch in two to three days and mosquito larvae or wriggler emerges. Mosquitoes are most efficiently and economically destroyed when they are in the larval stage and are concentrated in their breeding site (Ellis 9). The larvae of Aedes mosquito were unable to survive in water of a high degree of salinity due to exosmosis. (Wigglesworth, V.B.). The larvae live in the water, feed and develop into the third stage of the life cycle which takes four to ten days and is called a pupa or tumbler. The pupa also lives in the water but do not feed. Finally, the mosquito emerges from the pupal case and it is ready to bite as a fully developed mosquito
(Evelyn, 6). Aedes bites only human beings. Twenty-four to 48 hours following the emergence of the adult mosquito from the pupa and after mating has taken place, the female seeks out the blood for egg production in which it has two peaks for the biting time, one at the dawn just after sunrise and the second at the dusk before the sunset. After female mosquito feeds on a person whose blood contains virus, it can transmit dengue either immediately or after an incubation period of 8 – 10 days, during which the virus multiplies (Evelyn, 7) According to T. K. Lim (344-348), Momordica charantia is tropical vine of the family Cucurbitaceae widely grown for edible fruit, which is among the most better of all vegetables. An English name for the fruit is bitter melon or bitter gourd. In the Philippines it is commonly known as Ampalaya, Hindi– karela; Nepali- Tite Karela; Tamil– pakal, pavakka; Marathi– karke. The fruit has a distinct warty looking exterior oblong shape. It is hollow in cross section with relatively thin layer of flesh surrounding a central seed cavity filled with large flat seeds and pith. It grows in areas where annual precipitation ranges from 480 mm to 4100 mm. All parts of the plant are equally important as fruits of Ampalaya (Momordica charantia) contain Charantin, Polypeptide-p as its constituent elements and also the leaves of the plant contains Charantin, Polypeptide-p as the main component whereas SEEDS contains Charantin , Polypeptide-p along with Vicine as its constituents. Along with these components the plant contains several biologically active
group, chiefly Momordican I, Momordican II and Cucurbitacian B. The plants contains also several bioactive glycosides (including momordin, charatine, charnantitosides, Goya glycosides, and momordicosides) and other terpenoids compounds (including momordicin 28, momordicinin, momordicilin, momordenol and momordol). It also contains cytotoxic (Ribosomeinactivity) proteins such as momorcharin and momordin. Also the aerial parts of the Bitter gourd / Ampalya possess a number of active ingredients including tannins, flavonoids, alkaloid’s, quinines and phenol. Fruit consist of amino acids such as Beta alanine, Gamma Alanine, Glutamic acid, Tryptamine and Gama amino-butyric acid among others. Seeds also contain amino acids and include Arginine, Aspartic acid, Glycine, Leucine, Lysine and Histidine (T. K. Lim,347-353). The juice extracted from the fresh fruit of Momordica charantia has been used in our research. Review of related literature Larvicides are chemical substances or group of insecticides used to stop mosquito larvae from maturing into biting adults that transmit various diseases. Dichloro-diphenyl-trichloroethane (DDT), pyrethruim, heptachlor, diedrin and lindane were the most commonly used larvicides in the past to achieve these control measures. The commonly and repeatedly used larvicides are fuel oils, kerosene and insecticide formulations (Truman et al., 207). Synthetic organic larvicides, although very efficacious to target species such as mosquitoes could be detrimental to a variety of animal life including man.
NICC reported that organophosphate temphos which is a Larvicide usually used in breeding site, though slightly toxic to target organism, may cause headache, loss of memory and irritability to man. Besides, the incessant use of chemical insecticides had often led to the disruption of natural biological control system, and outbreak of insect species as noted by (Chaithong et al., 31(1): 138- 144). Moreover, these chemicals could be carcinogenic, mutagenic and teratogenic. Many plant juices could be used to drive mosquitos away, eg. Eucalyptu citriodora, Pterigeron odorus, Santalum lanceolatum, Momordica charantia. (Zohara and Uriel, 183). Larvae can be killed by keeping it into hypertonic solution as due to the difference in pressure gradient and concentration gradient osmosis can take place and then cell loose its water shrink and dies. ( Biology, p.180). Plants that were termite or insect resistant, were used as fish poison and folkloric ally in treating malaria and fever as well as those with reported insecticidal, mosquito repellent and mosquitocidal activities had demonstrated promising larvicidal activities . However, no co-relation had been established between larvicidal activities and these properties but an earlier study with a common medicinal and vegetable plant of Momordica charantia Linn (Family: Cucurbitaceae), had shown the insecticidal activity of this plant against mustard saw fly. (T.K. Lim, 356) The larvicidal nature of fleshy fruit wall of Momordica charantica Linn (Family: Cucurbitaceae) was found first time, in the management of mosquitos done
in the year 2009 November by Batabya et al. ( P.1205-10) in view of the recently increased interest in developing plant based insecticides as an alternative to chemical insecticides. This study was undertaken to know the larvicidal potential of the various fruit juices of Momordica charantia (Cucurbitaceae) against culex species of mosquito vector. Significance of study This research could help the Rural and Community Health Workers to enhance rural based projects promoting locally grown plant for medical purpose which could serve as the cheaper alternative to expensive drugs available in the market. This research would also help in finding the active ingredient in Ampalaya fruit and potentially spearhead the manufacturing of commercial larvicides that would benefit the people. The farmers could gain profit by cultivating this plant as cash crops. Industries could probably get benefitted from this study since they could produce new larvicides from local resources. The botanists could also be benefitted as they would get new information about this plant. This would create an interest towards further research and exploration. Government would also be equally benefitted by exporting larvicides and nature would also be free and fresh since this juice is natural and biodegradable unlike some other commercial larvicides which are non-biodegradable.
Objectives General objectives The study aimed to determine the larvicidal effect of Ampalaya (Momordica charantia) fruit juice on Aedes mosquito larvae and also compare its larvicidal nature with commercial larvicide (ABATE 1SG). Specific objective This study aimed to: 1. keep a tally of number of Aedes mosquito larvae killed after 20, 40 and 60 minutes of exposure to A) Ampalaya fruit juice in tap water a.1) 25% a.2) 50% a.3) 75% B) commercial larvicide (ABATE 1SG) 2. Compare the number of Aedes mosquito larvae killed by the administration of Ampalaya fruit juice and commercial larvicide (ABATE 1SG). 3. Determine the significant difference in the proportion of dead wrigglers between Ampalaya fruit juice and commercial larvicide (ABATE 1SG). Scope and limitation This experimental study involved the determination of the larvicidal effect of various concentrations of juice extracted from the fruit of Ampalaya on the two (2) – four (4) days old larvae of Aedes. The determinations of chemical composition of the juice were not included in the study. The concentrations of Ampalaya fruit juice below 25% and above 75% were not considered in the experiment. The collected eggs were of Aedes mosquito as the eggs
were laid separately, were elongated and black in color. But, the species of the Aedes mosquito were unidentified. Hypothesis Statement of Null Hypothesis (Ho) There was no significant difference in the proportion of dead wrigglers between Ampalaya fruit juice as Aedes mosquito larvicide and a commercial larvicide. Statement of Alternative Hypothesis (Ha) There was a significant difference in the proportion of dead wrigglers between Ampalaya fruit juice as a mosquito larvicide and a commercial larvicide. MATERIALS AND METHODS Research design: The study was experimental in nature, using Single Variable completely randomized design to determine the larvicidal effect of Momordica charantia fruit juice (variable) on Aedes mosquito larvae (constant), which were selected and assigned randomly. Also it was compared with commercial larvicide (ABATE 1SG) as positive control and water as negative control. Research locale: The preparation of the different concentrations of Momordica charantia fruit juice was conducted in Cebu Doctors’ University research laboratory. The collection of mosquito eggs was conducted at one of the researcher’s residence located near CDU, Mandaue City. The breeding and hatching of the eggs were conducted at Cebu Doctors’ University, zoology laboratory. This area was suitable for conducting experiment as it is well lighted, well-furnished and well equipped with the materials needed for experiment.
Research subject: The research subjects were two (2) to four (4) days old Aedes larvae i.e. 2nd -3rd instar larvae which were randomly selected from the basin with naturally bred and hatched larvae. Collection of sample Mosquito larvae collection White plastic containers coated in black were used as breeding site of Aedes mosquito. The containers were lined with filter papers and half filled with clean water. The plastic containers with filter paper were placed in the area where Aedes mosquitoes naturally bred such as human surrounding. The containers were checked every morning for the presence of mosquito eggs. When the tiny black patches were seen on the filter papers, the filter papers were removed from the black container, air dried and were stored into a clean and dry container. The larvae were then brought to Dr. Emmanuel Pardiñan for authentication of identification. Hatching of Aedes Eggs The transparent plastic containers were filled with clean water. The filter paper with eggs were cut into pieces and then placed in the plastic containers to allow the hatching of eggs. The set ups were checked daily for the hatching of the eggs and once the larvae were seen, they were transferred into another plastic container and labeled accordingly as to how many days old. After the collection of desired amount of larvae (between one (1) to four (4) days), they were transferred to nine (9) different cups for experimental setups (three (3) for each concentration), three (3) for negative control and three (3) for positive
control. Using a plastic spoon, six larvae were randomly selected and assigned in each of the fifteen (15) cups. Procedure of extraction The selected, fresh and clean fruits of Momordica charantia were bought from the mall and brought to Mr. Vitaliano Fernandez for authentication of identification. After confirming that they were really Momordica charantia, they were then brought into the research laboratory for the extraction process. Nearly three and half (3.5) kilograms of fresh fruits of Momordica charantia were washed and chopped into small pieces. Then, they were crushed in a mixer. The juice was then taken out and filtered using a clean cloth. Then three (3) different concentrations of juice were prepared by mixing it with proper amount of water. For the 25% concentration, 25 ml of pure juice and 75 ml of water were mixed, for 50% concentration, 50 ml pure juice and 50 ml were mixed and for the 75% concentration, 75 ml pure juice and 25 ml water were mixed. The prepared different concentrations of juice were used in the experiment along with water as negative control and larvicide (ABATE 1SG) as positive control. Data collection: The experiment was conducted on the 6th of December 2013 in the zoology laboratory in CDU. The three plastic cups were taken and filled with 25 ml, 50 ml and 75 ml of pure water. The three different concentrations were obtained by adding 75 ml, 50 ml and 25 ml of pure Momordica charantia fruit juice respectively. As for the negative control,
100 ml of pure water was kept in one cup. Then six (6) naturally bred larvae were placed in each cup and the cups were kept on the table. The set ups were checked every 20 minutes until one (1) hour. A stop watch was used to note down the duration of time. Same steps were followed for three (3) trials along with positive and negative control set ups. At the end of each trial, the numbers of dead and alive larvae were noted. The larvae that floated and did not show any movement or even did not respond to very slow stirring of the plastic spoon were considered dead. The number of dead and alive larvae were counted and recorded in the tabulated data sheet. Definition of terms Aedes Mosquito- Aedes mosquitoes are typically small mosquitoes. They usually have black and white stripes marking on the body and legs. They usually bite only during day time. Larvae - larvae are the distinct juvenile forms of many insects which undergo complete metamorphosis. It is the feeding stage of the insect development. It is constant. Larvicide - An agent for killing larvae, in this experiment, it is the Ampalaya juice. Commercial larvicide - Insecticide that is specially targeted against larval stage of any insects, in this experiment, ABATE 1SG. Ampalaya fruit juice - It is the juice extracted from the fresh Ampalaya fruit with the help of grinder. After the extracted of juice, desired amount of water is added to make different concentration (25%, 50% and 75%). It is variable.
RESULTS AND DISCUSSIONS Result The tables below showed the summary of the data gathered from the research and the Mean of the different concentrations. Table 1 Percentage (%) of dead Wrigglers A) After 20 minutes Substance used No. of Trials Trial 1 Trial 2 Trial 3 MEAN
ABATE 1SG 50.00 66.67 33.33 50.00
25% of Ampal aya juice 0.00 0.00 0.00 0.00
50% of Ampal aya juice 0.00 16.67 0.00 5.56
75% of Ampala ya juice 16.67 16.67 0.00 11.11
Based on the result, it can be seen that the ABATE 1SG is 50% effective after 20 minutes whereas highest concentration of Ampalaya fruit juice is only 11.11%. B) After 20 minutes Substance used No. of Trials Trial 1 Trial 2 Trial 3 MEAN
ABATE 1SG 66.67 66.67 66.67 66.67
25% of Ampal aya juice 16.67 0.00 0.00 5.56
50% of Ampal aya juice 33.33 16.67 16.67 22.22
Based on the result, ABATE 1SG was 66.67% effective after 40 minutes whereas 25%, 50% and 75% concentrations of Ampalaya fruit juice were comparatively less effective and showed 5.56%, 22.22% and 27.78% effectiveness respectively.
75% of Ampala ya juice 33.33 33.33 16.67 27.78
C) After 1 hour Substance used
No. of Trials Trial 1 Trial 2 Trial 3 MEAN
ABATE 1SG 83.33 83.33 100.00 88.89
25% Ampalaya juice 33.33 16.67 16.67 22.22
As shown in the above table, the ABATE 1SG was more effective after 1 hour as
of 50% Ampalaya juice 50.00 33.33 33.33 38.89
of 75% Ampalaya juice 66.67 50.00 50.00 55.56
of
compared to 25%, 50% and 75% concentrations of Ampalaya fruit juice.
Table 2 Comparison of Results A) After 20 min Group
No. of Average % of Trials dead Wrigglers 3 50.00 With ABATE 1SG 0.00 With 25% of 3 Ampalaya fruit juice 5.56 With 50% of 3 Ampalaya fruit juice 11.11 With 75% of 3 Ampalaya fruit juice 3 50.00 With ABATE 1SG 11.11 With 75% of 3 Ampalaya fruit juice Based on the given results, the P value (0.038) was less than 0.05 level of significance. Hence, the null hypothesis (Ho) was rejected. It implies that the number of dead wrigglers using Ampalaya fruit juice in different concentrations and using
P value 0.038
Remarks
0.000
The Ho was rejected because the P value is < 0.05 level of significance.
Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
ABATE 1SG were significantly different. More dead wriggler were found when ABATE 1SG was used. Hence, Ampalaya fruit juice is not an effective mosquito larvicide.
B) After 40 min Group
No. of Trials
With ABATE 1SG With 25% of Ampalaya fruit juice With 50% of Ampalaya fruit juice With 75% of Ampalaya fruit juice With ABATE 1SG With 75% of Ampalaya fruit juice
3 3
Average % of P dead Wrigglers value 66.67 0.027 5.56
3
22.22
3
27.78
3 3
66.67 27.78
In this table it can be seen that the percentage (%) of dead larvae was only 27.78% for the 75% conc. of Ampalaya fruit juice. The P value at 40 minute is 0.027, which is less than 0.05 level of significance. Hence, the null hypothesis (Ho) was
0.000
Remarks Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
The Ho was rejected because the P value is < 0.05 level of significance.
rejected. The results also show that the number of dead wrigglers using Ampalaya fruit juice in different concentrations and ABATE 1SG differed significantly even after 40 minutes.
C) After one (1) hour Group
No. of Trials
With ABATE 1SG With 25% of Ampalaya fruit juice With 50% of Ampalaya fruit juice With 75% of Ampalaya fruit juice With ABATE 1SG
3 3
Average % of P dead Wrigglers value 88.89 0.020 22.22
3
38.89
3
55.56
3
88.89
With 75% of 3 Ampalaya fruit juice
55.56
Based on the above results, the percentage (%) of dead larvae was only 55.56% for the 75% conc. of Ampalaya fruit juice. Here, the P value after one (1) hour was 0.020 which is less than 0.05 level of significance. Therefore, the null hypothesis (Ho) was
0.000
Remarks Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
The Ho was rejected because the P value is < 0.05 level of significance.
rejected. More dead wrigglers were found by treating with ABATE 1SG (88.89). Hence, the number of dead wrigglers using Ampalaya fruit juice in different concentrations (even at 75% concentration) and ABATE 1SG were significantly
different after one (1) hour. Discussion Based on the experiment, it was seen that more number of larvae died in the highest concentration of Ampalaya fruit juice probably because of hyper tonicity (Biology, p.180) of the solution as the tonicity of Ampalaya fruit juice was more compared to cells of the larvae, exosmosis took place eventually resulting in their death. An experiment performed on Aedes mosquito larvae showed that the larvae of Aedes mosquito were unable to survive in water of a high degree of salinity due to exosmosis. (Wigglesworth, V.B. ) However, few larvae died in the lowest concentration of Ampalaya fruit juice. This showed the presence of certain chemicals in the Ampalaya fruit juice that had some larvicidal property. Therefore Ampalaya fruit juice had larvicidal effect on Aedes larvae even though it is minimal. Some researchers have shown that many plant juice like Eucalyptus citriodora, Pterigeron odorus, Santalum lanceolatum, Momordica charantia were used to drive mosquitoes away (Zohara and Uriel, 183) but Ampalaya fruit juice did not prove to be as effective as them. Some researches (Maurya Para et al., 2009) have also shown the larvicidal nature of fleshy fruit wall of Momordica charantia to Culex mosquito larvae. Thus, it can be said that the larvicidal property of Ampalaya fruit juice is selective to Culex mosquito larvae as proven by the work of (Batabyal L et al.
para.1) while not so effective in the case of Aedes mosquito larvae. CONCLUSION In conclusion, the Ampalaya fruit juice had a larvicidal effect, but it was inferior to the positive control ABATE 1 SG. RECOMMENDATION From this very research, it was found out that the larvicidal property of Momordica charantia fruit juice was minimal. Even the 75% concentration solution was not very effective in killing Aedes mosquito larvae. However, the other parts of Momordica charantia plant such as leaves, root, stem etc. might be useful in killing the Aedes mosquito larvae. Further research using other parts of Ampalaya was therefore recommended for further study if they have larvicidal effect. As mentioned before, the Ampalaya fruit juice has minimum larvicidal effect when it comes to Aedes mosquito larvae. But, it might be effective in killing mosquito larvae of other genera such as Anopheles, Culex. It can also be tried for different species of mosquitoes. BIBLIOGRAPHY Books Becker, et al. Mosquitoes and their Control. New York: Kluwer Academic/Plenum Publishers, 2003. Biology. New Delhi: National Council of Educational Research & Training. 2006. T.K. Lim. Edible Medicinal and Non Medicinal Plants. Springer Dordrecht Heidelberg London New York, 2012. Truman, et al. Scientific Guide to Pest
Control Operations 3rd ed. Cleveland Ohio:Harvest Publishing Company, 1976. Zohara Xaniv & Uriel Bachrach. Handbook of Medicinal Plants. New York: Food Products Press, Haworth Medical Press, 2005. Journal Awad, O.M. & Shimaila, A. “Operational Use of Neem oil as an Alternative Anopheline Larvicide” Part A: Laboratory And Field Efficacy. Eastern Mediterranean Health Journal, 9(4), 637– 645 (2003). Web. 5 February 2013.
Control. 9 (3). (2004). Web. 10 Feb. 2013.
and Field Efficacy.” (2003). Web.6 Feb. 2013.http://applications.emro.who.int/ mhj/0904/9_4_2003_637_645.pdf Batabyal L. et al. “Relative Toxicity of Neem Fruit, Gitter gourd, and Castor Seed Extracts Against the Larvae of Filaria vector, Culex Quinque fasciatus.” (2009). Web. 10 Feb. 2013. http://www.ncbi.nlm.nih.gov/pubmed/ 9562373 D. Raj Mohan and M. Ramaswamy. “Evaluation of Larvicidal Activity of The Leaf Extract of a Weed Plant, Ageratina adenophora, Against Two Important Species of Mosquitoes, Aedes aegypti and Culex quinque fasciatus.” (2007). Web.10 February 10, 2013. http://www.aseanbiotechnology.info/Ab
tract/21022274.pdf Okigbo, R.N. et al. “Larvicidal Effects of Azadirachta indica, Ocimum gratissimum and Hyptis suaveolens Against Mosquito Larvae.” Journal of Agricultural Technology, (2010). Web. 5 February 2013. http://www.ijataatsea.com/pdf/October v6_n4_10/8-111-IJAT2009_91R.pdf Michelle Evelyn. et al. “City of Surrey West Nile Virus Mosquito Response Plan.” (2010). Web. 10 February 2013.http://www.cpqam.fiocruz.br/nicc/ omunic/jc200101.html Wigglesworth, V.B. “The Adaptation of Mosquito Larvae to Salt Water.” (1932). Web. 5 February 2013. http://www.mosquitocatalog.org/files/p fs/141050-1.pdf
