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Analysis of Bacillus sphaericus in controlling mosquito populations in urban catch basins.

Introduction

West Nile virus is an arbovirus that causes a number of symptoms, including encephalitis, an inflammation of the brain. Most commonly found in Africa, West Asia, and Eastern Europe, the virus was confirmed present for the first time in North America in New York City in 1999 (Pennsylvania Department of Health, 2000). By 2000, cases were found as far south as Pennsylvania.

The virus can be spread quickly by mosquitoes. Sixty species of mosquitoes inhabit Pennsylvania (Pennsylvania Department of Environmental Protection, 2001). Of these, only a few have been found to carry West Nile virus. The virus is primarily transmitted by the Culex pipiens species. Mosquitoes serve only as a vector to transmit the virus from one host to another. The primary hosts for the virus are birds, especially corvids and raptors. The virus is spread when female mosquitoes feed on infected birds, then bite humans and animals when taking their blood meal. During each blood meal, they inject some saliva into their host, and the saliva can transmit any disease the mosquito is carrying (Crans, 2000). Mosquitoes can also transmit West Nile virus to their eggs.

Female mosquitoes feed on animal and human blood to obtain the protein and other nutrients they need to lay their eggs (Crans, 2000). After taking a blood meal, female mosquitoes deposit their eggs on wet substrates or standing water. The incubation period for the eggs varies by species and environmental conditions, and may range from a few days to over a year. When the eggs hatch, the mosquito larvae develop in the water. Larval development consists of four stages, known as instars, and collectively these four stages can last from three days to several weeks. Mature larvae then metamorphose to the pupal stage. Pupae usually transform within a few days into adult mosquitoes (University of Florida, American Mosquito Association, 2001). Adult mosquitoes are able to fly and transmit disease, and are harder to target with control measures than are the larval stages.

Mosquitoes in general inhabit a wide range of moist habitats, including damp soil, containers, tires, and ponds. The Culex pipiens species breeds primarily in receptacles such as catch basins that hold stagnant water and organic material. This habitat provides mosquitoes with shade, standing water, and decomposing organic material. Even without rain, runoff from home sprinkling systems may provide enough water to facilitate mosquito breeding throughout the summer (Wade, 2001).

In order to prevent the spread of these potential vectors of West Nile virus, control measures must be taken to prevent them from completing their life cycle in catch basins and similar habitats. Three main control measures are used to eliminate mosquitoes: source reduction, larviciding, and adulticiding. Source reduction is the most effective and permanent method of mosquito control, and includes removing empty containers that collect rainwater in and around residential areas, covering swimming pools that are not in use, retrofitting catch basins to eliminate standing water, and constructing dikes in salt marshes (University of Florida, American Mosquito Control Association, 2001). All of these actions reduce mosquito breeding grounds and therefore control future generations. Source reduction is not always a plausible option, however, because elimination of some breeding grounds may be too costly or harmful to the surrounding environment.

Where source reduction is not possible, larviciding is the next best option for mosquito control. Larviciding is the application of agents to prevent mosquito larvae from maturing. There are several types of larvicide, and a specific larvicide may be more effective against one species than another. Larval surveillance and knowledge of various larvicides is important in order to apply the most effective larvicide to each mosquito breeding site.

Examples of larvicides include concentrations of certain types of bacteria found in nature, monomolecular surface films, oils, and chemical growth inhibitors (University of Florida, American Mosquito Control Association, 2001). One naturally occurring bacteria used as a larvicide is Bacillus sphaericus, which attacks mosquito larvae from the first to third instars. When ingested, B. sphaericus damages the gut of the larvae, resulting in its death. B. sphaericus is commercially produced in various forms suited to specific environments (State of New Hampshire, Department of Health and Human Services, 2000).

B. sphaericus also is packaged as a water soluble pouch (WSP) and is used to treat standing water in environments such as catch basins. The pouches quickly dissolve and release granules, which distribute evenly, horizontally and vertically, throughout the water. A single pouch can be used to treat each catch basin for approximately 30 days (State of New Hampshire, Department of Health and Human Services, 2000). Larviciding effectively eliminates the next generation of mosquitoes when specific mosquito breeding sites such as catch basins can be located and treated. It provides a more efficient solution than adulticiding because it is target specific and less disruptive to the surrounding habitat. It is also less controversial than other forms of mosquito control.

Adulticiding is a last resort for mosquito control when source reduction and larviciding are inefficient. Adulticiding is the aerial or ground application of insecticides to eliminate an adult mosquito population. Chemicals approved for this use are applied by techniques such as fogging, space sprays, and barrier treatments (University of Florida, American Mosquito Control Association, 2001). Adulticiding is the least efficient method of mosquito control because it is not target specific and may have a negative impact on other organisms. Therefore, it is primarily used when other actions fail to eliminate a mosquito population.

Method

The study reported here first evaluated whether catch basins in an urban area in southeastern Pennsylvania were used by mosquitoes as breeding sites to complete their life cycle. Second, the study set out to determine the effectiveness of B. sphaericus as a control agent and the amount of time B. sphaericus takes to go into effect.

Seventy catch basins were selected from the streets of the city during the summer months. All catch basins were located within an estimated 55-square-block area in the west and southwest sections of the city to ensure that all the catch basins would be in a similar environment. Each catch basin was examined for mosquito larvae with a catch basin dipper according to a standard dipping procedure. The dipper was submerged and moved throughout the water to fill the net. After several seconds, the dipper was lifted from the catch basin. The researchers washed the contents of the net into a white collection tray using a water bottle. The presence or absence of larvae was recorded. If no larvae were present from the first dip, the dipping process was repeated twice to ensure accuracy. Dipping results showed larvae inhabiting 60 of the 70 catch basins (Table 1).

The second part of the study was designed to determine the effectiveness of B. sphaericus in preventing mosquito larvae from developing into adult mosquitoes in urban catch basins being used as breeding sites. Only 60 catch basins were used in this part of the study because the remaining 10 did not contain larvae. The catch basins were divided randomly into two groups and marked according to whether they would be treated with B. sphaericus or left as controls.

Half of the 60 catch basins were each treated with one B. sphaericus pouch while the other half served as untreated controls. Seven to 16 days after the catch basins were treated, each catch basin was examined again for the presence of viable mosquito larvae with the same dipping procedure as before.

When the experiment had been completed, B. sphaericus product also was added to the catch basins that served as controls to eliminate a potential public health risk. The post-treatment results were compared with the initial quantities of larvae to analyze the effectiveness of B. sphaericus in eradicating mosquito larvae. A Chi-square test was performed to determine if results were statistically significant.

The study also observed the rate at which B. sphaericus eradicated mosquito larvae. To determine this rate, an aquarium with dimensions 11.5 in. X 9.5 in. X 19.5 in. was used to model the habitat of an urban catch basin. Mosquito larvae were collected from two catch basins in the area with the same standard dipping procedure previously described. The contents of the tray, including larvae and a quantity of water from the catch basin containing decomposing organic material and other organisms, were transferred to the aquarium. The entire process was repeated several times to obtain a sufficient number of larvae. The aquarium was transferred to a cool, shady room where it was treated by insertion of one pouch of B. sphaericus. The rate at which B. sphaericus dissolved and spread throughout the aquarium was observed and recorded. The amount of time necessary for B. sphaericus to take effect was analyzed by the rate at which it killed the mosquito larval instars.

Results

Of the 70 catch basins examined in the study, 87 percent had larvae during the initial dipping (Figure 1). Only 30 of the catch basins were treated in the second part of the experiment because not all of the catch basins contained larvae; B. sphaericus eliminated larvae in 97 percent of the catch basins that were treated (Figure 2). The remaining 3 percent of the basins were inaccessible during the final dipping, and the effectiveness of the product could not be ascertained from these basins. Of the 30 catch basins used as controls, 80 percent still contained larvae, while 17 percent did not and 3 percent were inaccessible (Figure 3).

Results of the study were tabulated, and a Chi-square test was performed to assess the statistical significance of the effect of the larvicide. A cutoff p-value of .05 was established. If the calculated p-value were below .05, it was concluded that the larvicide did have an effect. If the calculated p-value were above .05, the larvicide was considered not to have had an impact on the larvae in the catch basins. The Chi-square value obtained was 40.9412, and the p-value was [less than or equal to].001. Thus, it was postulated that larvae in the catch basins were eradicated by B. sphaericus and not by random chance.

In the second part of the experiment, the B. sphaericus killed larvae present in the aquarium within 24 hours, except for those in the fourth instar. Larvae in the fourth instar became pupa and then adult mosquitoes. This result was expected because the product containing B. sphaericus is designed to kill larvae only in the first through third instars. Mosquito larvae in the fourth instar stop feeding before they metamorphose into pupae, so B. sphaericus is not effective on them. B. sphaericus did not affect any other organisms, such as worms, in the aquarium.

Discussion and Conclusion

The study reported here demonstrated that B. sphaericus WSP is effective in killing mosquito larvae in urban catch basins for a period of at least two weeks, the period of time for which the study was conducted. The results from the study were significant because the p-value from the Chi-square test was <.05.

One hundred catch basins in the west and southwest sections of the city were targeted for study. Because of limitations found after the study was designed, however, only 70 catch basins could be used. Many could not be dipped because their design precluded dipping or because there was a lack of standing water.

The experiment represented the conditions of catch basins in a particular section of a large city and determined the effectiveness of B. sphaericus over a two-week period; the study was limited by time constraints. It would be desirable to conduct longer studies to gain additional knowledge on the viability of B. sphaericus for a longer period of time. Such studies should expand the target area to include a larger sample of catch basins, conduct additional dipping after three to four weeks to determine the length of time for which B. sphaericus is effective, and develop different dipping methods to include the various types of catch basins. Studies like these are important and should be comprehensive because as seen in this study, mosquitoes breed in numerous urban catch basins. To eliminate some of the potential threat of the West Nile virus, it is vital to know if the biological agents being used are working effectively so that the mosquito population can be eradicated from urban catch basins.
TABLE 1 Initial and Final Dipping Results

Initial Final
Dipping Dipping
Catch Control or Larvae Larvae
Basin # Vectolex Present Present
 (C/V) (Y/N) (Y/N)

 1 V Y N
 2 C N
 3 V Y N
 4 C Y Y
 5 V Y N
 6 C Y (A lot) Y
 7 V Y N
 8 C Y N
 9 V Y N
10 C Y N
11 V Y (A lot) N
12 C Y Y
13 V Y (A lot) N
14 C Y (A lot) Y
15 C Y Y
16 C N (Deep)
17 V Y N
18 C N
19 V Y N
20 C Y Y
21 V Y N
22 C Y Y
23 V N
24 C Y Y
25 V Y N
26 C Y Y
27 V Y Inaccessible
28 C Y Y
29 V Y N
30 C Y Y
31 V Y N
32 C Y Y
33 V Y N
34 C Y Y
35 V Y N
36 C Y Y
37 V Y N
38 C Y N (Yellow line)
39 V Y N
40 C N
41 V N
42 V Y N
43 C Y N (Moving water)
44 V Y N
45 C Y N
46 Y N
47 V Y N
48 C Y Y
49 V Y N
50 C Y Y
51 V Y N
52 C Y Y
53 V Y N
54 C Y Y
55 C Y Y
56 C N
57 V Y N
58 C Y Y
59 V Y N
60 C Y Inaccessible
61 V Y N
62 C Y Y
63 V Y N
64 C Y Y
65 C N
66 V Y N
67 V Y N
68 C Y Y
69 V Y N
70 C Y Y

FIGURE 1 Percentage of Catch Basins with Larvae During Initial Dipping

Catch basins with larvae 87%
Catch basins without larvae 13%

Note: Table made from pie chart.

FIGURE 2 Percentage of Treated Catch Basins without Larvae During Final
Dipping

Catch basins without larvae 3%
Inaccessible catch basins 97%

Note: Table made from pie chart.

FIGURE 3 Percentage of Control Catch Basins with Larvae During Final
Dipping

Catch basins with larvae 80%
Catch basins without larvae 17%
Inaccessible catch basins 3%

Note: Table made from pie chart.


Acknowledgements: The authors thank Randall B. Hirschhorn for his assistance and moral support.

REFERENCES

Crans, W. (2000). Frequently asked questions about mosquitoes. Rutgers Cooperative Extension, FS900. http://www.rce.rutgers.edu/pubs/pdfs/fs526.pdf, (21 Oct. 2004).

Pennsylvania Department of Environmental Protection. (2001). Mosquito identification manual: West Nile surveillance and control program manual. Harrisburg, PA: Author.

Pennsylvania Department of Health. (2000). What Pennsylvanians should know about West Nile virus [Brochure]. Harrisburg, PA: Pennsylvania Department of Environmental Protection.

State of New Hampshire, Department of Health and Human Services. (2000). Vectolex (Bacillus sphaericus): What is Vectolex?. http://www.dhhs.state.nh.us/commpublichealth/WestNile.nsf (23 July 2002).

University of Florida, American Mosquito Control Association. (Mar 2001). Public health pest control: Public health pest control manual--Mosquitoes. http://www.ifas.ufl.edu/~pest/vector/chapter_03.hym (27 June 2002).

Wade, B. (Feb. 2001). Mosquito control: Trench warfare and beyond. American City & County, 3-5. http://americancityandcounty.com/mag/government_mosquito_control_trench/index.html (21 Oct. 2004).

Palak Raval-Nelson, M.P.H.

Ketki Soin

Suzy Tolerud

Corresponding Author: Palak Raval-Nelson, 8201 Chelwynde Ave., Philadelphia, PA 19153. E-mail: palak.raval-nelson@phila.gov.
COPYRIGHT 2005 National Environmental Health Association
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Title Annotation:FEATURES
Author:Raval-Nelson, Palak
Publication:Journal of Environmental Health
Article Type:Cover Story
Geographic Code:1USA
Date:Mar 1, 2005
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