Research Article

 

 

Comparison the Effect of Fungi (Beauveria bassiana) and Bacteria Bacillus thuringiensis (israelensis) on a Cumulative Mortality of Culex Quinquefasciatus (Diptera: Culicidae)

 

Aliaa Abdul Aziz Hameed1*, Enas Hatem Kareem Al-Bander2, Fatima Ali Ghanim Al-Obadi3

1College of Science; 2College of Veterinary Medicine; 3College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad, Iraq.

 

Editor | Muhammad Nauman Zahid, Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan.

Received | October 22, 2016; Accepted | January 22, 2017; Published | January 29, 2017

*Correspondence | Aliaa Abdul Aziz Hameed, College of Science, University of Baghdad, Iraq; Email: firas_rashad@yahoo.com

Citation | Hameed AAA, Al-Bander EHK, Al-Obadi FAG (2017). Comparison the effect of fungi (Beauveria bassiana) and bacteria Bacillus thuringiensis (israelensis) on a cumulative mortality of culex quinquefasciatus (Diptera: Culicidae). S. Asian J. Life Sci. 5(1): 1-4.

DOI | http://dx.doi.org/10.14737/journal.sajls/2017/5.1.1.4

ISSN | 2311–0589

Copyright © 2017 Hameed et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

Chemicals insecticide play an important role in elimination insect that cause several dangerous diseases to human and cancers through the transition to food chain or directly. Although, the chemicals insecticide represents the common approach for the control of the mosquito but the repeated use of chemicals insecticide led to increasing the resistance of insect and increasing the environmental pollution and costs (Allsop et al., 2015). Thus the alternative methods are needed to control noisome insects and limit diseases caused by them. The alternative methods include microbial insecticide (Biological control) which specialized toxic to special species of insect and have no effect on human and other mammals (Rawlins, 1989; Scholte et al., 2004).

 

Biological control included of bacteria, fungi, viruses, fermons, extract plant (botanical extracts) (McDonald and Lind, 2002) fish, predator like toxorhynchites and protozoa (Meisch et al., 1985; Collins and Blackwell, 2000). The genus culex (Diptera: Culicidae) is one of the most dangerous insect in the nature which play a role in the transmission of pathogens to human. There are two species of this genus: culex pipiens which caused west Nile virus disease and culex quinqiefasciatus which could be transmitted by the nematodes and causing filariasis and Encephalitis (Rawlins, 1989).

 

Life cycle of culex consist of egg, Larva, pupa and adult stages (Kaddou et al., 2015). Larval stage most infected with pathogens in nature (live in aquatic until adult emergence), adults consider vectors disease because its natural feeding depend on blood of mammals (Helall and Husen, 2013). Beauveria bassiana is a pathogenic and killer to larva and adult mosquito (Bezalwar et al., 2014). So this study was conducted to compare the effectiveness of Beauveria bassiana and Bacillus thuringiensis in controlling the Mosquito.

 

Materials and Methods

 

Insect Rearing

 

The adults of mosquito were collected from Lake of Political Science Collage, University of Baghdad. The diagnosis of the insect as a culex quinquefasciatus was confirmed by the Natural History Museum, Iraq. The adults reared in a box with 20x20x20 cm a rounded with organza cloth. A piece of cotton saturated with 5% sugar solution was placed inter rearing box to feed male of mosquito, while females were fed on the blood of rabbit which linking its parties tightly to prevent movement and putting above the box (Hellal and Husen, 2013). The glasses with 50 ml capacity filled with water inter rearing box to collect egg boats after adult mating then transferred to aquariums with 1 L capacity (used to rear larvae) filled with free chlorine water, fish diet was added constantly as larvae food. The water was replaced every 3 days to keep aquarium clean. Aquariums were reared continually. The experiment performed at the room temperature of 29 ±3 °C.

 

Treatment of Insect with Bacteria

 

A total of 15 larvae with 1st instars distributed with 3 replications subjected to Biopesicide ANTROL (Bti.) with concentration of 0.7g/m2 according to the manufacture company. The larvae were monitored every day until they died.

 

Treatment of Insect with Fungi

 

Beauveria bassiana SAI were brought from Biotechnology Research Center, General Authority for Plant Protection, Ministry of Agriculture, Iraq. The fungi were refreshed by putting it in the petri dish with PDA media (potato dextrose agar) and chloromingol antibiotic to prevent bacterial growth. The petri dishes incubated in 25 ±2°C and 80 ±5% humidity for 7-10 days (Olivera and Neves, 2004; Dewan, 2010).

 

Preparation of Fungal Dilution

 

After the period of fungi incubation, the spores were isolated by harvester (L-shape) and treated with distilled water 5 ml then filtered by a piece of muslin cloth to get stock fungal spores. Three dilutions of fungi were prepared (7.3 x 104, 7.3 x 106, 7.3 x 108) that used to treat 15 larvae with 1st instars distributed with 3 replications. The method of prepare dilution performed according to Kirkland et al. (2004) and Lacy (1997). The number of spores was counted by neubauer counting chamber (haemocytometer) using the following equation (Norris, 1997; Lacey, 1997).

 

 

Where;

 

N is spores collected in 5 haemocytometer chambers, 80 is number of mini boxes in 5 boxes of haemocytometer chamber, 106 is dilution correction factor and 10 is volume correction factor.

 

Statistical Analysis

 

Statistical analysis was performed using SAS (Statistical Analysis System - version 9.1). Two-way ANOVA with Interaction and Least significant differences (LSD) post hoc test was used (multiple comparisons), to assess the significant differences among means. P < 0.05 was considered statistically significant (SAS, 2010).

 

Results and Discussion

 

Table 1 shows that the differences in the cumulative mortality percentage of mosquito larvae differed significantly due to time of exposure in all treatments. The highest mortality rate was detected in the bacteria treatment as it reached to 100% mortality in the third day. In general the effect of different concentrate of fungi was found significant (P< 0.05) only in the third day. The highest mortality rate (84%) in the fungi treatment was found in the dilution of 7.3x108, while the bacteria treatment has a mortality rate of 100%.

 

Table 1: The cumulative mortality of mosquito stage treated with fungi Beauveria bassiana and bacteria Bacillus thuringiensis

 

Treatment	Dilution	Larvae			Pupa			Adult
		1	2	3	4	5	6	7
Fungi	7.3 X 104	bc20.0 ± 3.84D	b42.22± 4.44C	c66.66± 11.54B	c73.33± 10.18AB	bc84.44± 12.37A	b86.66± 13.33A	bc86.66 ± 13.33A
	7.3 X 106	C15.55± 4.44C	b42.22± 2.22B	c71.11± 8.89A	c71.11 ± 8.89A	c75.55± 11.11A	b79.99 ± 6.66A	c79.99 ± 6.66A
	7.3 X 108	C15.55± 2.55C	b42.22 ± 4.44B	b84.44± 8.01A	b86.66 ± 7.69A	ab91.11± 8.89A	ab91.11± 8.89A	ab93.33 ± 6.66A
Bacteria	0.7g/m2	a33.33± 6.66C	a64 ± 11.75B	a100.00± 0.00A	a100.00± 0.00A	a100.00± 0.00A	a100.00± 0.00A	a100.00± 0.00A
Control	-	d0.00 ± 0.00A	c2.22 ± 2.22A	d2.22 ± 2.22A	d2.22 ± 2.22A	d2.22 ± 2.22A	c2.22 ± 2.22A	d2.22 ± 2.22A

 

LSD: 13.22; Means with different small letters in the same column differ significantly (p<0.05), while means with different capital letters in same row differ significantly (p<0.05)

 

There is no significant difference among the three dilutions (7.3 x 104 , 7.3 x 106 , 7.3 x 108) in the days of pupa stage of 4, 5, 6 days until 7th day when the adult emergence. Depending on Kaddou et al. (2015) who reported that culex life cycle consist of 3 days larva, 3 days pupa and adult emergence in 7th day.

 

No adult emergence in the end period of life cycle normally and most of it die (100% mortality). The result showed that larva could developed to pupa but not developed to normal adult and died after few minutes or not complete pupa stage as compared with Bacillus thuringiensis (var. i) bacteriopesticide (ANTROL) which considered best than mycopesticide when mortality in larvae treated with ANTROL was 100% during 3 days (period of larva stage) that prohibit the complete life cycle of culex. The different between results could be attributed to the mechanism impact of micro pesticides. Beauveria bassiana effect through osculate when mycelium inter insect body through cuticle and respiratory spiracles, mycelium stimulate to germinate when contact with suitable host (Thomas and Read, 2007).

 

Fungi kill mosquito Lingeringly (George et al., 2013) so fungicide can spray on rooftops, windows and all rest area that help in reduce number of adult could transfer disease and reduce adult’s ability food (Maehara and Shimazu, 2007). This mechanism impact was seen in Anopheles, culex, Aeds (Scholte et al., 2006; Blanford et al., 2005; Mouatcho, 2010). Fungi infection mosquito larvae reduce the ability of movement and kill 3rd instars in 48 hrs (Bezalwar et al., 2014). Mohantry and Soam (2010) reported that conidia covered all larva body except the head and horns breathing this proved that mycelium inter to haemolymph through cuticle, ventral brush and papillae this consist fungi as keratinophilic. The conidia could stay in water with complete effectiveness for 7 days. The fungi effect on physiological and feeding behaviour of infection mosquito also reduce immunity of mosquito (Weiser, 1991).

 

Bacillus thuringiensis israelensis (ANTROL) effect through digestive system when spores inter larva body through feeding , so it consider a specific toxic to mosquito larva (Glare and O’Callaghan, 1998). First larva instar is more susceptible to Bti than other instars (Mulla et al., 1990) this depend on mechanism of action of Bti toxin which consist a multistage process, ingestion cry protein by larvae with feeding, solubilisation of cry in midgut which have alkaline medium, proteolytic activation in the insecticidal solubilised protein, apical microvillus membrane of epithelial midgut cell walls have a receptors which bind with toxin, changing in shape allowing toxin insertion into the membrane, toxins doing pores in the cell membrane, because of that osmotic balance disruption and cells swell then burst, thus larva stops feeding. Most larva die in few hours of ingestion (Marrone and Macintosh, 1993; Perez et al., 2005). The result of this search agree with Mario and Montserrat (2012) and Ben-Dov (2014) so it is important to choose the stronger microorganism pathogenic to kill insects (pests) then stopping vector disease with no harmful effect on environment.

 

Acknowledgments

 

We would like to thank Assistant Professor Dr. Firas R Al-Samarai for his assistance.

 

Conflict of Interest

 

The authors declare that they have no competing interest.

 

Authors’ Contribution

 

All authors contributed equally in research and writing of manuscript.

 

References

 

Allsop M, Huxdorff C, Johnston P, Santillo D, Thompson K (2015). Pesticides and our health a growing concern. Greenpeace.org. France. Pp. 56.

Annabel FV, Howard C, Koenraadt JM, Marit-Farenhorst, Bart GHK, Willem (2010). Prethroid resistance in Anopheles gambie leads to increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana. Malar. J. 9: 168. https://doi.org/10.1186/1475-2875-9-168

Blanford S, Chan BHK, Jenkins NE, Sim D, Turner RJ (2005). Fungal pathogen reduces potential for malaria transmission. Science. 308: 1638–1641. https://doi.org/10.1126/science.1108423

Bezalwar PM, Gomashe AV, Gulhane PA (2014). Laboratory-based evaluation of the potential of Beauveria bassiana crude metabolites for mosquito larvae annihilation. IOSR J. Pharm. Biol. Sci. 9: 15-20.

Ben-Dov E (2014). Bacillus thuringiensis subsp. israelensis and its dipteran-specific Toxins Open Access. Pp. 10-1243.

Collins LE, Blackwell A (2000). The biology of Toroxrhychites mosquitoes and their potential as biocontrol agents. Biocontr. News Inform. 21: 105-116.

Dewan HM (2010). Prepare biopesticide of Beauveria bassiana (Bals.) vuill and evaluate its effectiveness against Brevicoryne brassicae L. Thesis. Science Collage, Baghdad University.

George J, Jenkins NE, Blanford S, Thomas MB, Baker TC (2013). Malaria mosquitoes attracted by fatal fungus. PLoS One. 8(5): 0062632. https://doi.org/10.1371/journal.pone.0062632

Glare T.R.&O Callaghan M.(1998). Environmental and health impacts of Bacillus thuringiensis israelensis . Reported for the ministry of health . bio control &bio diversity. Grasslands division. Ag. Research. Lincoln. New Zealand.

Helall SM, Husen HM (2013). Type blood feeding of Culex pipiens L. (Diptera: Culicidae) on different vertebral family in Hella, Iraq. J. Babylon Univ. Pure Appl. Sci. 7(21): 2371.

Kaddou AK, Husen AA, Mustafa KA (2015). General insect science. Iraq. Pp. 441.

Kirkland BH, Cho EM, Keyhani NO (2004). Differential susceptibility of Amblyomma maculates and Amblyomma americanum (Acari: Ixodidae) to entomopathogenic fungi B. bassiana and Metarhizium anisopliae. Biol. Contr. 31: 414-421. https://doi.org/10.1016/j.biocontrol.2004.07.007

Lacey LA (1997). Manual of techniques in insect pathology (Biological techniques). Academy Press, Sandiego, London, Boston. Pp. 408.

Metcalf RL (1986). The ecology of insecticides and the chemical control of insects. In: Ecological theory and integrated pest management in practice (ed. M Kogan). John Wiley and Sons, New York. Pp. 251-297. https://doi.org/10.1007/BF01638999

McDonald BA, Linde C (2002). Pathogen population genetics, evolutionary potential and durable resistance. Ann. Rev. Phytopathol. 40: 349-379. https://doi.org/10.1146/annurev.phyto.40.120501.101443

Meisch MV, Gambusia Affinis, Chapman HC (eds.) (1985). Biological control of mosquitoes. Bull. Am. Mosquito Contr. Assoc. 6: 3-17.

Maehara N, He HY, Shimazu M (2007). Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes). J. Econ. Entomol. 100: 49–53. https://doi.org/10.1603/0022-0493(2007)100[49:MFATOB]2.0.CO;2

Mario RL, Montserrat RS (2012). Biological control of mosquito larvae by Bacillus thuringiensis subsp. israelensis. Insect. Pest Engin. Pp. 264.

Marrone PG, MacIntosh SC (1993). Resistance to Bacillus thuringiensis and resistance management. In: Bacillus thuringiensis, an environmental biopesticide: Theory and practice (eds. PF Entwistle, JS Cory, MJ Bailey, Higgs S). John Wiley and Sons, Chichester, UK. Pp. 221-235.

Mulla MS, Darwazeh HA, Zgomba M (1990). Effect of some environmental factors on the efficacy of Bacillus sphaericus 2362 and Bacillus thuringiensis (H-14) against mosquitoes. Bull. Soc. Vector Ecol. 15: 166–175.

Mohanty SS, Soam P (2010). Comparative efficacy and pathogenicity of keratinophilic soil fungi against Culex quinquefasciatus larvae. Indian J. Microbiol. 50: 299–302. https://doi.org/10.1007/s12088-010-0051-8

Mouatcho JC (2010). The use of entomopathogenic fungi against Anopheles funestus Giles (Diptera: Culicidae). A thesis of physiology. Pp. 162.

Norris K.P. & Powell, E.O.(1961). Improvement and determining total count of bacteria. J. Roy. Micr. Soc. 80-107.

Olivera RC, Neves PM (2004). Compatibility B. bassiana with Acaricides. J. Nectro. Pical. Entomol. 33(3): 353-358.

Perez C, Fernandez LE, Sun J, Folch JL, Gill SS, Soberon M. (2005). Bacillus thuringiensis sub sp. israelensis Cyt1Aa synergies Cry11Aa toxin by functioning as a membrane - bound receptor. Proc. Natl. Acad. Sci. 102 (51): 18303-18308.

Rawlins SC (1989). Biological control of insect pests affecting man and animals in the tropics. CRC Critic. Rev. Microbiol. 16: 235–252. https://doi.org/10.3109/10408418909105477

Scholte EJ, Knols BGJ, Takken W (2006). Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity. J. Invertebr. Pathol. 91: 43–49. https://doi.org/10.1016/j.jip.2005.10.006

Scholte EJ, Knols BGJ, Samson RA, Takken W (2004). Entomopathogenic fungi for mosquito control: A review. J. Insect Sci. 4: 19. https://doi.org/10.1673/031.004.1901

SAS (2010). SAS/STAT Users guide for personal computer. Release 9.1, SAS Institute Inc., Cary N.C., USA.

Scholte EJ, Ng‘habi K, Kihonda J, Takken W, Paaijmans K, Abdulla S, Killeen GF, Knols BGJ (2005). An entomopathogenic fungus for control of adult African malaria mosquitoes. Science. 308: 1641-1642. https://doi.org/10.1126/science.1108639

Thomas MB, Read AF (2007). Can fungal biopesticides control malaria? Nat. Rev. Microbiol. 5: 377–383. https://doi.org/10.1038/nrmicro1638

Wang C, Janine E (2002). Isolation and evaluation of Beauveria bassiana for control of Coptotermes formosanus and Reticulitermes flavipes (Isoptera: Rhinotermitida). Sociobiology. 41(1): 1-13.

Weiser J (1991). Biological control of vectors. John Wiley and Sons Ltd., England. Pp. 1–189.