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The effects of vitamin deficiencies on the growth and mortality of Tribolium castaneum infected with Nosema whitei
IOURNAL
OF INVERTEBRATE
PATHOLOGY
31, 303-306 (1978)
The Effects of Vitamin Deficiencies on the Growth and Mortality Tribolium castaneum Infected with Nosema whitei
of
EARLENE ARMSTRONG Department
of Entomology,
University
of Maryland,
College
Park.
Maryland
20742
Received June 27, 1977 The effects of deficient diets on Tribolium castaneum parasitized with Nosema whitei were investigated. Larval mortality of the host on vitamin-deficient diets, e.g., biotin, thiamine, pantothenic acid, vitamin B,,, pyridoxine, niacin, and riboflavin, was higher than that of the beetles on the vitamin B-complete diet. Less than 80% of the larvae pupated on the biotin-, thiamine-, and pantothenic acid-deficient diets, while less than 50% pupated on the vitamin B,,-, pyridoxine-, niacin-. and riboflavin-deficient diets. When infected T. castaneum were fed the deficient diets, less than 26% of the larvae pupated. In general, the average life span of infected T. castaneum on deficient diets was shorter than that of controls on the same diet. However, the life span of the host on the vitamin B-complete diet, even when infected, was much longer than that of infected and urn-nfected hosts on deficient diets. KEY WORDS: Tribolium castaneum, larva; Nosema whitei; vitamin deficiencies.
INTRODUCTION
Vitamins are organic molecules of diverse chemistry that are required for the growth and normal functioning of cells (Giese, 1962). Of the water-soluble vitamins (B series), thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, choline, and folic acid are generally considered indispensable for most insects (Hinton et al., 1951; Dadd, 1961; House, 1965; Dadd et al., 1967). The purpose of the present study was to investigate the effects of vitamin B-complete and deficient diets on the red flour beetle Tribolium castaneum parasitized with the microsporidan Nosema whitei. MATERIALS
AND METHODS
Experimental host. Second instar larvae of T. castaneum were selected from stock cultures which had been maintained in the laboratory on a medium consisting of bleached enriched flour and brewers yeast (19: 1). Half of the larvae were permitted to 303
feed ad libitum for 48 hr on a flour-yeast mixture containing N. whitei (IO’ spores/g of medium). Control larvae were allowed to feed only on a flour-yeast mixture. The larvae in both groups were brushed free of medium and placed individually in l-dram glass vials containing fresh diets. The number of larvae on each diet varied from 34 to 40. The larvae were reared in an incubator at 30°C and a 60-70% RH. At 2day intervals, all vials were checked for the presence of dead larvae, pupae, and/or adults. Infection was determined by squashing the dead host between a microscope slide and coverglass and examining it under phase microscopy for the presence of the parasite. All insects surviving after the 125th day on the vitamin B-complete diet were killed, and the data were recorded. Experimental diets. The vitamin Bcomplete and -deficient diets were obtained commercially from Nutritional Biochemical Corp., Cleveland, Ohio. The complete diet contained the following ingredients:
0022-201 l/78/0313-0303$01.00/0 Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved
304
EARLENE
ARMSTRONG
Concentration (g/100 lbs)
Nutrient Vitamin B concentrate (200,000 units/g) Vitamin D concentrate (400,000 units/g) cY-Tocopherol Ascorbic acid Inositol Choline chloride Menadione p-Aminobenzoic acid Niacin Riboflavin Pyridoxine hydrochloride Thiamine hydrochloride Calcium pentothenate (Pantothenic acid)
Statistical evaluation. The data on the longevity of beetles were analyzed statistically using the t test.
4.5 RESULTS
0.25 5.0 45.0 5.0 75.0 2.25 5.0 4.5 1.0 1.0 1.0
Table 1 shows the growth and mortality of control and infected T. castaneum on the various diets. In general, larval mortality of the host on diets lacking vitamins was much greater than the mortality of the beetles on the vitamin B-complete diet in both the control and infected groups. For the control groups, 77% of the larvae pupated on the biotin-deficient diet, 74% on the thiamine-deficient diet, 64% on the pantothenic-deficient diet, 42% on the vitamin B,,-deficient diet, 24% on the pyridoxinedeficient diet, 18% on the niacin-deficient diet, and 13% on the riboflavin-deficient diet. In 50 days, all the beetles were dead on the deficient diets. On the vitamin Bcomplete diet, 100% of the larvae developed to the adult stage. After 125 days, 82% of the adults were still alive. The complete diet, therefore, appears to provide the essential B vitamins necessary for the growth, development, and survival of the host. In groups exposed to the microsporidan and fed the deficient diets, the additional
3.0 (mg/lOO lbs)
Biotin Folic acid Vitamin B,,
20 90 1.35
Diets deficient in thiamine, biotin, riboflavin, vitamin B12, niacin, pantothenic acid, and pyridoxine were selected for the experiments. These diets contained the same ingredients found in the complete diet except for the vitamin tested. TABLE GROWTH
AND
MORTALITY
OF CONTROL
FED
VITAMIN
AND
B-COMPLETE
AND DISCUSSION
1
NOSEMA
TRIBOLIUM
WHITEI-INFECTED
AND
DEFWENT
Mortality
(%)
Controls Diets
Larvae
Vitamin B-complete Biotin-deficient Thiamine-deficient Pantothenic-deficient Vitamin B,,-deficient Pyridoxine-deficient Niacin-deficient Riboflavin-deficient LI Figures
reflect
specimens
0
0
12 0 13 3 0 0 2
dead before
Infected
Pupae
23 26 36 58 76 82 87
125th day
CASTANEUM
DIETS
Adults
Larvae
18” 65 74 51 39 24
33 95 75 9s 90 98 98 100
18 11
when
experiment
was terminated
Pupae
Adults 20” 5 23 0 10 2 2 0
VITAMINS
AND NOSEMA-INFECTED
burden of the parasite drastically reduced the number of larvae that emerged into adults (Table 1). On all deficient diets, less than 26% of the larvae pupated and all adults that emerged were dead by the 46th day. Sixty-seven percent of the larvae in the infected group fed the complete diet pupated. Sixty-four percent became adults and 44% of the total number of insects on the complete diet lived more than 125 days. Occasionally, adults on both the infected and control diets showed external abnormalities varying from wings to abdominal deformities. The importance of certain B vitamins for
TRIBOLIUM
30.5
the optimal growth and development of insects has been well documented by several investigators. Van? Hoog (1935, 1936) and Tatum (1939, 1941) demonstrated that B vitamins, such as thiamine and riboflavin, were necessary to complete growth and development of Drosophila melanogaster. More recently, Dadd et al. (1967) reported that the aphid, Myzus persicae, when deprived of thiamine, nicotinic acid, and calcium pantothenate, showed greatly reduced first-generation Iarval growth, and the development of mature adults was prevented. Individual omission of other vitamins markedly reduced first-generation growth
so-
85-
SO-
75-
TO-
65.
EOQ m”
n
55.
8s
50-
5al
45-
IX
40353025ZO15x)5L
FIG. 1. Average life span (days) of control and Nosema complete and deficient diets.
whitei-
infected Tribolium castaneum on vitamin B-
306
EARLENE TABLE
ARMSTRONG
2
NOSEMA WHITEI INFECTIONS ( lo7 SPORES PER GRAM) ON VITAMIN B-COMPLETE AND -DEFICIENT DIETS IN TRIBOLIUMCASTANEUM No. of insects Diets
Infected
Vitamin B-complete Biotin-deficient Thiamine-deficient Pantothenic-deficient Vitamin B,,-deficient Pyridoxine-deficient Niacin-deficient Riboflavin-deficient
28 31 18 21 31 18 27 16
Noninfected 12 7 20 19 7 21 II 24
but did not prevent development to the adult stage. Little growth also occurred in the absence of any of the vitamins during the second generation. Rock (1969) showed that diets lacking folic acid, riboflavin , thiamine, calcium pantothenate, nicotinamide, and choline chloride resulted in 100% larval mortality for the red-banded leaf roller, Argyrotaenia vefutinana. When pyridoxine and biotin were omitted from the diet, 10 and 20% of the larvae developed to the adult stage, respectively. Baker (1975) indicated the larvae of Sitophifus oryzae did not require riboflavin or pantothenic acid but did need thiamine, nicotinic acid, pyridoxine, folic acid, and biotin for the development. Raulston (1975) reported a reduction or deletion of individual B vitamins from soy flour-wheat germ diets fed to tobacco budworms could affect the growth, development, and survival of the host. Data on the longevity of control and infected T. castaneum are presented in Figure 1. The life spans of the control and infected groups fed deficient diets were very shorter than those of the beetles on the complete diet. Also, the infected insects had a shorter life span than the uninfected controls in all the diet groups. This latter reduction was much more pronounced in the groups fed complete diet. Infection of T. castaneum also varied
among the groups (Table 2). Infection was as high as 82% on the biotin- and vitamin B,,-deficient diets, 71% on the niacindeficient diets, and 70% on the complete diet. The percentage of infection ranged from 40 to 53% on the pantothenic acid-, thiamine-, pyridoxine-, and riboflavin-deficient diets. The first spores were observed in the host as early as the tenth day on the thiamine-, pantothenic acid-, and riboflavin-deficient diets and as late as the 125th day on the complete diet. In fact, 54% of the adults that emerged on the complete diet were parasitized with the microsporidan while the adults emerging on the deficient diets were parasite-free. REFERENCES BAKER, J. 1975. Vitamin requirements
of larvae of 21, 1337- 1342. DADD. R. 1961. The nutritional requirement of locusts. IV. Requirements for vitamins of the B complex. J. Insect Physiol.. 6, 1-12. DADD, R., KRIEGER, D., AND MITTLER, T. 1967. Studies on the artificial feeding of the aphid Myzus persicae (Sulzer). IV. Requirements for watersoluble vitamins and ascorbic acid. J. lnsecf Sitophilus
Physiol.,
oryzae.
J. Insect
Physiol.,
13, 249-272.
GEISE. A. 1962. Cell Physiology.” 2nd ed. W. B. Saunders, Philadelphia. HINTON, T., NOYES, D., AND ELLIS, J. 19.51. Amino acids and growth factors in a chemically defined medium for Drosophila. Physiol. Zool., 24, 335352. HOUSE, H. 1965. Insect nutrition. In “The Physiology of Insecta” (M. Rockstein. ed.), Vol. 2. pp. 769813. Academic Press, New York. RAULSTON, J. 1975. B-vitamin supplements required for soy flour-wheat germ used in rearing tobacco budworms. Ann. Enfomol. Sot. Amer. 68,387-388. ROCK, G. 1969. Sterol and water-soluble vitamin requirements for larvae of the red-banded leaf roller, Argyrotaenia velutinana. Ann. Entomol. Sot. Amer. 62. 611-613. TATUM, E. 1939. Nutritional requirements of Drosophila
melanogaster.
Proc.
Nat.
Acad.
Sci.
25, 490-497. TATIJM, E. L. 1941. Vitamin B requirements sophila
melanogaster.
Proc.
Nat.
Acad.
27, 193-197. VAN’T HOOG. E. 1935. Aseptic culture vitamin research. Z. Vitaminforsch., VAN‘T HOOG, E. 1936. Aseptic culture vitamin research. Z. Vitaminforsch..
USA,
of Dro-
Sri.
USA,
of insects in 5, 300-323.
of insects in 118.
5,
OF INVERTEBRATE
PATHOLOGY
31, 303-306 (1978)
The Effects of Vitamin Deficiencies on the Growth and Mortality Tribolium castaneum Infected with Nosema whitei
of
EARLENE ARMSTRONG Department
of Entomology,
University
of Maryland,
College
Park.
Maryland
20742
Received June 27, 1977 The effects of deficient diets on Tribolium castaneum parasitized with Nosema whitei were investigated. Larval mortality of the host on vitamin-deficient diets, e.g., biotin, thiamine, pantothenic acid, vitamin B,,, pyridoxine, niacin, and riboflavin, was higher than that of the beetles on the vitamin B-complete diet. Less than 80% of the larvae pupated on the biotin-, thiamine-, and pantothenic acid-deficient diets, while less than 50% pupated on the vitamin B,,-, pyridoxine-, niacin-. and riboflavin-deficient diets. When infected T. castaneum were fed the deficient diets, less than 26% of the larvae pupated. In general, the average life span of infected T. castaneum on deficient diets was shorter than that of controls on the same diet. However, the life span of the host on the vitamin B-complete diet, even when infected, was much longer than that of infected and urn-nfected hosts on deficient diets. KEY WORDS: Tribolium castaneum, larva; Nosema whitei; vitamin deficiencies.
INTRODUCTION
Vitamins are organic molecules of diverse chemistry that are required for the growth and normal functioning of cells (Giese, 1962). Of the water-soluble vitamins (B series), thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, choline, and folic acid are generally considered indispensable for most insects (Hinton et al., 1951; Dadd, 1961; House, 1965; Dadd et al., 1967). The purpose of the present study was to investigate the effects of vitamin B-complete and deficient diets on the red flour beetle Tribolium castaneum parasitized with the microsporidan Nosema whitei. MATERIALS
AND METHODS
Experimental host. Second instar larvae of T. castaneum were selected from stock cultures which had been maintained in the laboratory on a medium consisting of bleached enriched flour and brewers yeast (19: 1). Half of the larvae were permitted to 303
feed ad libitum for 48 hr on a flour-yeast mixture containing N. whitei (IO’ spores/g of medium). Control larvae were allowed to feed only on a flour-yeast mixture. The larvae in both groups were brushed free of medium and placed individually in l-dram glass vials containing fresh diets. The number of larvae on each diet varied from 34 to 40. The larvae were reared in an incubator at 30°C and a 60-70% RH. At 2day intervals, all vials were checked for the presence of dead larvae, pupae, and/or adults. Infection was determined by squashing the dead host between a microscope slide and coverglass and examining it under phase microscopy for the presence of the parasite. All insects surviving after the 125th day on the vitamin B-complete diet were killed, and the data were recorded. Experimental diets. The vitamin Bcomplete and -deficient diets were obtained commercially from Nutritional Biochemical Corp., Cleveland, Ohio. The complete diet contained the following ingredients:
0022-201 l/78/0313-0303$01.00/0 Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved
304
EARLENE
ARMSTRONG
Concentration (g/100 lbs)
Nutrient Vitamin B concentrate (200,000 units/g) Vitamin D concentrate (400,000 units/g) cY-Tocopherol Ascorbic acid Inositol Choline chloride Menadione p-Aminobenzoic acid Niacin Riboflavin Pyridoxine hydrochloride Thiamine hydrochloride Calcium pentothenate (Pantothenic acid)
Statistical evaluation. The data on the longevity of beetles were analyzed statistically using the t test.
4.5 RESULTS
0.25 5.0 45.0 5.0 75.0 2.25 5.0 4.5 1.0 1.0 1.0
Table 1 shows the growth and mortality of control and infected T. castaneum on the various diets. In general, larval mortality of the host on diets lacking vitamins was much greater than the mortality of the beetles on the vitamin B-complete diet in both the control and infected groups. For the control groups, 77% of the larvae pupated on the biotin-deficient diet, 74% on the thiamine-deficient diet, 64% on the pantothenic-deficient diet, 42% on the vitamin B,,-deficient diet, 24% on the pyridoxinedeficient diet, 18% on the niacin-deficient diet, and 13% on the riboflavin-deficient diet. In 50 days, all the beetles were dead on the deficient diets. On the vitamin Bcomplete diet, 100% of the larvae developed to the adult stage. After 125 days, 82% of the adults were still alive. The complete diet, therefore, appears to provide the essential B vitamins necessary for the growth, development, and survival of the host. In groups exposed to the microsporidan and fed the deficient diets, the additional
3.0 (mg/lOO lbs)
Biotin Folic acid Vitamin B,,
20 90 1.35
Diets deficient in thiamine, biotin, riboflavin, vitamin B12, niacin, pantothenic acid, and pyridoxine were selected for the experiments. These diets contained the same ingredients found in the complete diet except for the vitamin tested. TABLE GROWTH
AND
MORTALITY
OF CONTROL
FED
VITAMIN
AND
B-COMPLETE
AND DISCUSSION
1
NOSEMA
TRIBOLIUM
WHITEI-INFECTED
AND
DEFWENT
Mortality
(%)
Controls Diets
Larvae
Vitamin B-complete Biotin-deficient Thiamine-deficient Pantothenic-deficient Vitamin B,,-deficient Pyridoxine-deficient Niacin-deficient Riboflavin-deficient LI Figures
reflect
specimens
0
0
12 0 13 3 0 0 2
dead before
Infected
Pupae
23 26 36 58 76 82 87
125th day
CASTANEUM
DIETS
Adults
Larvae
18” 65 74 51 39 24
33 95 75 9s 90 98 98 100
18 11
when
experiment
was terminated
Pupae
Adults 20” 5 23 0 10 2 2 0
VITAMINS
AND NOSEMA-INFECTED
burden of the parasite drastically reduced the number of larvae that emerged into adults (Table 1). On all deficient diets, less than 26% of the larvae pupated and all adults that emerged were dead by the 46th day. Sixty-seven percent of the larvae in the infected group fed the complete diet pupated. Sixty-four percent became adults and 44% of the total number of insects on the complete diet lived more than 125 days. Occasionally, adults on both the infected and control diets showed external abnormalities varying from wings to abdominal deformities. The importance of certain B vitamins for
TRIBOLIUM
30.5
the optimal growth and development of insects has been well documented by several investigators. Van? Hoog (1935, 1936) and Tatum (1939, 1941) demonstrated that B vitamins, such as thiamine and riboflavin, were necessary to complete growth and development of Drosophila melanogaster. More recently, Dadd et al. (1967) reported that the aphid, Myzus persicae, when deprived of thiamine, nicotinic acid, and calcium pantothenate, showed greatly reduced first-generation Iarval growth, and the development of mature adults was prevented. Individual omission of other vitamins markedly reduced first-generation growth
so-
85-
SO-
75-
TO-
65.
EOQ m”
n
55.
8s
50-
5al
45-
IX
40353025ZO15x)5L
FIG. 1. Average life span (days) of control and Nosema complete and deficient diets.
whitei-
infected Tribolium castaneum on vitamin B-
306
EARLENE TABLE
ARMSTRONG
2
NOSEMA WHITEI INFECTIONS ( lo7 SPORES PER GRAM) ON VITAMIN B-COMPLETE AND -DEFICIENT DIETS IN TRIBOLIUMCASTANEUM No. of insects Diets
Infected
Vitamin B-complete Biotin-deficient Thiamine-deficient Pantothenic-deficient Vitamin B,,-deficient Pyridoxine-deficient Niacin-deficient Riboflavin-deficient
28 31 18 21 31 18 27 16
Noninfected 12 7 20 19 7 21 II 24
but did not prevent development to the adult stage. Little growth also occurred in the absence of any of the vitamins during the second generation. Rock (1969) showed that diets lacking folic acid, riboflavin , thiamine, calcium pantothenate, nicotinamide, and choline chloride resulted in 100% larval mortality for the red-banded leaf roller, Argyrotaenia vefutinana. When pyridoxine and biotin were omitted from the diet, 10 and 20% of the larvae developed to the adult stage, respectively. Baker (1975) indicated the larvae of Sitophifus oryzae did not require riboflavin or pantothenic acid but did need thiamine, nicotinic acid, pyridoxine, folic acid, and biotin for the development. Raulston (1975) reported a reduction or deletion of individual B vitamins from soy flour-wheat germ diets fed to tobacco budworms could affect the growth, development, and survival of the host. Data on the longevity of control and infected T. castaneum are presented in Figure 1. The life spans of the control and infected groups fed deficient diets were very shorter than those of the beetles on the complete diet. Also, the infected insects had a shorter life span than the uninfected controls in all the diet groups. This latter reduction was much more pronounced in the groups fed complete diet. Infection of T. castaneum also varied
among the groups (Table 2). Infection was as high as 82% on the biotin- and vitamin B,,-deficient diets, 71% on the niacindeficient diets, and 70% on the complete diet. The percentage of infection ranged from 40 to 53% on the pantothenic acid-, thiamine-, pyridoxine-, and riboflavin-deficient diets. The first spores were observed in the host as early as the tenth day on the thiamine-, pantothenic acid-, and riboflavin-deficient diets and as late as the 125th day on the complete diet. In fact, 54% of the adults that emerged on the complete diet were parasitized with the microsporidan while the adults emerging on the deficient diets were parasite-free. REFERENCES BAKER, J. 1975. Vitamin requirements
of larvae of 21, 1337- 1342. DADD. R. 1961. The nutritional requirement of locusts. IV. Requirements for vitamins of the B complex. J. Insect Physiol.. 6, 1-12. DADD, R., KRIEGER, D., AND MITTLER, T. 1967. Studies on the artificial feeding of the aphid Myzus persicae (Sulzer). IV. Requirements for watersoluble vitamins and ascorbic acid. J. lnsecf Sitophilus
Physiol.,
oryzae.
J. Insect
Physiol.,
13, 249-272.
GEISE. A. 1962. Cell Physiology.” 2nd ed. W. B. Saunders, Philadelphia. HINTON, T., NOYES, D., AND ELLIS, J. 19.51. Amino acids and growth factors in a chemically defined medium for Drosophila. Physiol. Zool., 24, 335352. HOUSE, H. 1965. Insect nutrition. In “The Physiology of Insecta” (M. Rockstein. ed.), Vol. 2. pp. 769813. Academic Press, New York. RAULSTON, J. 1975. B-vitamin supplements required for soy flour-wheat germ used in rearing tobacco budworms. Ann. Enfomol. Sot. Amer. 68,387-388. ROCK, G. 1969. Sterol and water-soluble vitamin requirements for larvae of the red-banded leaf roller, Argyrotaenia velutinana. Ann. Entomol. Sot. Amer. 62. 611-613. TATUM, E. 1939. Nutritional requirements of Drosophila
melanogaster.
Proc.
Nat.
Acad.
Sci.
25, 490-497. TATIJM, E. L. 1941. Vitamin B requirements sophila
melanogaster.
Proc.
Nat.
Acad.
27, 193-197. VAN’T HOOG. E. 1935. Aseptic culture vitamin research. Z. Vitaminforsch., VAN‘T HOOG, E. 1936. Aseptic culture vitamin research. Z. Vitaminforsch..
USA,
of Dro-
Sri.
USA,
of insects in 5, 300-323.
of insects in 118.
5,