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The effect of bacterial load on fatty acids in the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae)
JOURNAL
OF INVERTEBRATE
PATHOLOGY
30,
274-275 (1977)
The Effect of Bacterial Load on Fatty Acids in the Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae)l The biological changes produced by various microbes which contaminate the medium used to rear the boll weevil, Anthonomus grandis, can alter the nutritional value of the medium or the survival of the weevils thereon. For example, the exotoxins produced by some bacteria may either poison the weevils directly or may modify the intestinal mucosa so that the weevil is unable to assimilate food to insure proper growth and reproduction. Also, boll weevil eggs implanted in the diet can be adversely affected by microorganisms. Intestines of fieldcollected wild weevils normally contain few, if any, bacterial contaminants (P. P. Sikorowski, unpubl). Furthermore, field weevils are not normally exposed to the dense populations of the variety of microorganisms which contaminate the artificial diet used in rearing weevils on a large scale. Thus, the presence of nonpathogenic bacteria in the medium adversely affects egg production and egg viability and prolongs the time of immature stages. The larval stage was prolonged from 13 to 15 days by bacterial contamination of the diet medium. The purpose of the present study was to determine the effect of nonpathogenic bacteria on the assimilation of lipids and fatty acids by the male and female boll weevil. Bacteria isolated from apparently normal insectary-reared weevils were identified by G. M. Thomas, Division of Invertebrate Pathology, University of California, Berkeley, as Streptococcus sp., Micrococcus varians , and Enterobacter aerogenes. Plastic Petri dishes were prepared with either sterile meridic diet or with diet inoculated with a suspension (6.2 x 103 bacterial cell/
ml) composed of equal numbers of cells of each species of bacteria. Then, boll weevil eggs from the laboratory colony were surface sterilized in sodium hypochlorite (0.2%) and then were implanted (200-300/ dish) on sterile or inoculated synthetic diet. The plates were placed in gallon paper cartons that had been sterilized in an autoclave and incubated at 29°C. After 13 days, the plates were removed, and newly emerged weevils were removed aseptically over a period of 24 + 12 hr. On the day the adults were taken from the medium, they were frozen and, later, were assayed for fatty acid and plated for bacterial content. Contaminated and uncontaminated insects were each divided into five-weevil or one-weevil samples (20 replicates). Each weevil sample was transferred to a Potter-Elvehjem glass tube and was homogenized. The five-insect samples were homogenized in 25 ml of methanol-chloroform (2: 1, v/v), and the single-insect samples were homogenized in 5 ml of distilled water. In the latter samples a known volume was withdrawn for bacterial count. The lipid samples were reduced to dryness, and fatty acid methyl esters were prepared by transesterification with boron trifluoride-methanol (L. C. Metcalfe, A. A. Schmitz, and J. R. Peeks, Anal. Chem., 38, 514- 15, 1966). Quantitation was accomplished with a flame-ionization gas chromatograph equipped with a 1.8 m x 0.3 mm stainless steel column packed with 10% DEGA on 60/80-mesh HMDS-treated Gas Chrom-P2 by comparison of peak height of standard fatty acid methyl esters (A. C. Thompson, R. D. Hen-
1 In cooperation with the Mississippi Agricultural and Forestry Experiment Station, Mississippi State, Mississippi 39762.
* Mention of proprietary product in this paper does not constitute an endorsement of this product by the United States Department of Agriculture. 274
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OO??-201I
275
NOTES TABLE
I
EFFECTS OF BACTERIAL CONTAMINATION (STREPTOcoccus sp., MICROCOCCUS VARIANS, AND ENTEROBACTER AEROGENES) ON THE FATTYACIDSOFMALEAND FEMALE BOLL WEEVILS, ANTHONOMUS
GRANDIS
Percentage of difference
Calories” Bacteria/weevil 0- 100 (healthy) 1000-5000 5ooo+
d
4
1.517 1.799 0.3709*
6
9
1.564
-
-
0.644* 0.4115*
118.6 24.4
42.45 26.31
Q Sum of the calories calculated for each fatty acid. * Indicates significance at the 1% level of probability.
A. C.
Biochem.
1972). The concentration of each fatty acid found (16:0, 16: 1, 18:0, 18: 1, 18:2) was converted
THOMPSON
United States Department of Agriculture Agricultural Research Service Boll Weevil Research Laboratory
P. P. SIKOROWSKI J. M. WYATT
son, R. C. Gueldner, Comp.
and P. A. Hedin, Physiol., 43B, 883-90,
to calories (Table 1) for three levels of bacterial contamination. Comparison of individual fatty acids from the contaminated and uncontaminated showed a decrease up to 76% in the highly contaminated (5000+ bacteria/weevil) weevils. Caloric evaluation of the fatty acids in the boll weevil egg indicated that the healthy female must provide 25.1% of her total body fatty acids per day in producing eggs (seven eggs/day). The presence of these bacteria can, therefore, place a stress on oviposition.
Department Mississippi Mississippi Received
of Entomology State University State, Mississippi 39742 February 2, 1977
OF INVERTEBRATE
PATHOLOGY
30,
274-275 (1977)
The Effect of Bacterial Load on Fatty Acids in the Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae)l The biological changes produced by various microbes which contaminate the medium used to rear the boll weevil, Anthonomus grandis, can alter the nutritional value of the medium or the survival of the weevils thereon. For example, the exotoxins produced by some bacteria may either poison the weevils directly or may modify the intestinal mucosa so that the weevil is unable to assimilate food to insure proper growth and reproduction. Also, boll weevil eggs implanted in the diet can be adversely affected by microorganisms. Intestines of fieldcollected wild weevils normally contain few, if any, bacterial contaminants (P. P. Sikorowski, unpubl). Furthermore, field weevils are not normally exposed to the dense populations of the variety of microorganisms which contaminate the artificial diet used in rearing weevils on a large scale. Thus, the presence of nonpathogenic bacteria in the medium adversely affects egg production and egg viability and prolongs the time of immature stages. The larval stage was prolonged from 13 to 15 days by bacterial contamination of the diet medium. The purpose of the present study was to determine the effect of nonpathogenic bacteria on the assimilation of lipids and fatty acids by the male and female boll weevil. Bacteria isolated from apparently normal insectary-reared weevils were identified by G. M. Thomas, Division of Invertebrate Pathology, University of California, Berkeley, as Streptococcus sp., Micrococcus varians , and Enterobacter aerogenes. Plastic Petri dishes were prepared with either sterile meridic diet or with diet inoculated with a suspension (6.2 x 103 bacterial cell/
ml) composed of equal numbers of cells of each species of bacteria. Then, boll weevil eggs from the laboratory colony were surface sterilized in sodium hypochlorite (0.2%) and then were implanted (200-300/ dish) on sterile or inoculated synthetic diet. The plates were placed in gallon paper cartons that had been sterilized in an autoclave and incubated at 29°C. After 13 days, the plates were removed, and newly emerged weevils were removed aseptically over a period of 24 + 12 hr. On the day the adults were taken from the medium, they were frozen and, later, were assayed for fatty acid and plated for bacterial content. Contaminated and uncontaminated insects were each divided into five-weevil or one-weevil samples (20 replicates). Each weevil sample was transferred to a Potter-Elvehjem glass tube and was homogenized. The five-insect samples were homogenized in 25 ml of methanol-chloroform (2: 1, v/v), and the single-insect samples were homogenized in 5 ml of distilled water. In the latter samples a known volume was withdrawn for bacterial count. The lipid samples were reduced to dryness, and fatty acid methyl esters were prepared by transesterification with boron trifluoride-methanol (L. C. Metcalfe, A. A. Schmitz, and J. R. Peeks, Anal. Chem., 38, 514- 15, 1966). Quantitation was accomplished with a flame-ionization gas chromatograph equipped with a 1.8 m x 0.3 mm stainless steel column packed with 10% DEGA on 60/80-mesh HMDS-treated Gas Chrom-P2 by comparison of peak height of standard fatty acid methyl esters (A. C. Thompson, R. D. Hen-
1 In cooperation with the Mississippi Agricultural and Forestry Experiment Station, Mississippi State, Mississippi 39762.
* Mention of proprietary product in this paper does not constitute an endorsement of this product by the United States Department of Agriculture. 274
Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OO??-201I
275
NOTES TABLE
I
EFFECTS OF BACTERIAL CONTAMINATION (STREPTOcoccus sp., MICROCOCCUS VARIANS, AND ENTEROBACTER AEROGENES) ON THE FATTYACIDSOFMALEAND FEMALE BOLL WEEVILS, ANTHONOMUS
GRANDIS
Percentage of difference
Calories” Bacteria/weevil 0- 100 (healthy) 1000-5000 5ooo+
d
4
1.517 1.799 0.3709*
6
9
1.564
-
-
0.644* 0.4115*
118.6 24.4
42.45 26.31
Q Sum of the calories calculated for each fatty acid. * Indicates significance at the 1% level of probability.
A. C.
Biochem.
1972). The concentration of each fatty acid found (16:0, 16: 1, 18:0, 18: 1, 18:2) was converted
THOMPSON
United States Department of Agriculture Agricultural Research Service Boll Weevil Research Laboratory
P. P. SIKOROWSKI J. M. WYATT
son, R. C. Gueldner, Comp.
and P. A. Hedin, Physiol., 43B, 883-90,
to calories (Table 1) for three levels of bacterial contamination. Comparison of individual fatty acids from the contaminated and uncontaminated showed a decrease up to 76% in the highly contaminated (5000+ bacteria/weevil) weevils. Caloric evaluation of the fatty acids in the boll weevil egg indicated that the healthy female must provide 25.1% of her total body fatty acids per day in producing eggs (seven eggs/day). The presence of these bacteria can, therefore, place a stress on oviposition.
Department Mississippi Mississippi Received
of Entomology State University State, Mississippi 39742 February 2, 1977