​Biotin Inhibitors, Avidin and Streptavidin: Their Influence on Lethality, Growth and Survival of Callasobruchus maculatus (Chrysomelidae: Coleoptera)

Green revolution has increased food grain production in India to about 230 million tons of food grains. But Post-harvest losses in India amounts to 12 to 16 million metric tons of food grains each year, an amount that the World Bank estimates could feed one-third of India’s poor. The monetary value of these losses amounts to more than Rs. 50,000 crores per year (Singh, 2010). Among these food grains, pulses are referred as “the poor man’s meat”, as they serve as a low-cost protein to meet the needs of large section of the people are the second most important group of crops worldwide. Pulses are excellent sources of proteins (20-40%), carbohydrates (50-60%) and are fairly good sources of thiamine, niacin, calcium and iron. One of the major constraints in the production of pulses are the insect pests and  pulse beetle, C. maculatus is regarded as the most important and cosmopolitan pest, severely attacking several stored pulses leading to weight loss, market value, as well as the germination potential of the infested grains (Booker, 1967).

Biotin is an essential nutrients for vertebrates and insects which is also known as vitamin H or coenzyme R, is a water-soluble B-vitamin (vitamin B7). It is a co-factor of major carboxylases involved in lipogenesis, gluco neogenesis, fatty acid and amino acid catabolism, required for normal cellular metabolism and growth (Alban et al., 2000), but its synthesis occurs only in plants, bacteria and certain fungi. Biotin is composed of a ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring. Biotin binding proteins like avidin and streptavidin bind to dietary biotin, making it unavailable to the insects, which then die from a deficiency of this vitamin. Avidin is a water soluble tetrameric glycoprotein found in chicken egg white and streptavidin is a nonglycosylated protein present in culture supernatant of Streptomyces avidinii (Stapley) (Bayer et al., 1990) and these biotin binding proteins bind tightly to vitamins. They act as anti-nutritional proteins by inhibiting insect growth and causing biotin deficiency. The use of various synthetic insecticides, which are considered as the source of serious health and ecological problems, such as development of pest strains resistant to pesticides, toxicity to mammals and detection of residues in human food can no longer reliable. Hence this experiment was conducted to know the effect of biotin inhibitors, avidin and streptavidin on lethality, growth and survival of C. maculatus which may be thought of as an alternative tool.

Rearing of the test insect
 
The population Callosobruchus maculatus F. were maintained in the laboratory of Department of Entomology, Agricultural College, Bapatla. Black gram grains were kept in an oven for one hour at 50oC for disinfestation. Black gram grains (250 g) were taken separately in jars (each of volume 500 ml) and 10 pairs of adults of C. macculatus were released into each jar and after allowing mating and oviposition for one week, these adults were removed. These containers were covered with muslin cloth and kept at room temperature (32±1^oC) and 75±2% relative humidity throughout the period of study. The adults which have emerged from this culture were utilized for conducting experiments as well as for subculturing. Sub culturing of these insects was done at 20 days intervals to meet the continuous supply of insects for experiments.
 
Chemicals
 
Biotin binding proteins-avidin and streptavidin SRL chemicals and Himedia, Vijayawada and tested against the test insect by pellet feeding method.
 
Bioassay
 
The test insects were subjected to suitable bioassay method to work out the LC25, LC50 and LC90 values for C. maculatus against avidin and streptavidin. Different concentrations of avidin and streptavidin were prepared viz., 5, 15, 30, 75 and 100 ppm of avidin and 5, 20, 30, 75 and 100 ppm of streptavidin against C. maculatus; These concentrations were fixed based on a preliminary study using 10, 50, 100, 150, 200 and 300 ppm where zero adult emergence of C. maculatus was recorded from 150 ppm of avidin/streptavidin onwards, Hence, based on the mortality of larvae in the broad range concentrations, a narrow range was fixed between 0 to 100 ppm for C. maculatus. Different concentrations of avidin and streptavidin as mentioned above were prepared by serial dilution technique from the stock solution using double sterile distilled water.
 
Pellet feeding method
 
In this method, milled bengalgram flour was taken and sterilized by keeping in hot air oven at 60oC for one hour. One gram of flour was mixed with one ml of required concentration of avidin or streptavidin to form a paste. This prepared paste was then dried completely for 24 hours and the dried paste was ground into powder by using pestle and mortar. This ground powder was hydrated to 10% moisture using double sterile water and this flour blend was made into four small pellets. These pellets were coated by dipping in 8% aqueous gelatin solution and air dried overnight (Plate 1) (Shade et al., 1986 and Murdock and Shade (2008). Untreated control was maintained by preparing flour pellets without mixing avidin/streptavidin. Four pellets were taken in glass vial in five replications and two mating pairs of freshly emerged adults of C. maculatus were released for oviposition. The adults were removed after three days ensuring oviposition. The treated pellets were dissected after three weeks to record the number of dead and live grubs.


 
Statistical analysis
 
The mortality data of the test insects were subjected to Abbott’s correction (Abbott, 1925) and then to probit analysis (Finney, 1971) using SPSS (Statistical Package for Social Sciences) to calculate (Median Lethal Concentration) LC25, LC50 and LC90, heterogeneity (c2), intercept (a), slope of the regression line (b), regression equation and fiducial limits.
 
Effect of BBP’s on grain damage, growth and development of C. maculatus
 
Biotin inhibitors viz., avidin and streptavidin were tested at different concentrations approximately at their respective LC25, LC50, LC75, LC80 and LC90 values on growth and development of C. maculatus. Avidin 5, 15, 30, 75 and 100 ppm; streptavidin 5, 20, 30, 75 and 100 ppm, spinosad 70 and 250 ppm were tested along with untreated control in three replications to study their effect on growth and development of C. maculatus. The LC25 and LC50 values of spinosad were determined by conducting bioassay with spinosad as these values were not available from literature. The above treatments were tested by bengal gram pellet feeding method as outlined obove. Two mating pairs of freshly emerged adults of C. maculatus were released into each replication having four pellets prepared from one gram bengal gram flour. The adults were removed after one week. The treatments were kept at ambient temperature (32±1oC) and relative humidity (75±2%) for further development of the pest. The number of eggs laid by C. maculatus in each test treatment sample was counted at ten days after release by observing under the illuminated magnifying lens. Later these treatments were kept for observation under laboratory conditions till the emergence of adults and the day of first adult emergence was noted. From then onwards, the number of adults that emerged were counted and separated every day till the emergence of adults was ceased, after which the total number of adults emerged was pooled and the per cent adult emergence, development period, growth index, per cent weight loss  was calculated by the following formulae:
 

 

  

 

  

 

Where,
W1 = Initial weight of sample.
W2 = Final weight of sample.
 
Stistical analysis
 
The parameters viz., number of eggs laid, growth index, larval weight and weight loss were subjected to square root transformations, day of first adult emergence and development period were subjected to logarithm transformations, while per cent adult emergence was transformed into arc sine transformations and then subjected to analysis of variance (ANOVA) in completely randomized block design (CRBD) with three replications for the test of significance and LSD (Snedecor and Cochran, 1967).

Lethal concentrations of avidin against C. maculatus
 
Avidin incorporated pellets, when fed to C. maculatus with varying concentrations of 5, 15, 30, 75 and 100 ppm, the mortality observed in the grubs is 20, 47.6, 72.58, 89.65 and 92.68 per cent respectively. At concentrations of greater than 77 ppm of avidin, more than 90 per cent mortality is observed. The LC25, LC50 and LC90 values of avidin against C. maculatus was 6.211, 14.733 and 76.031 ppm respectively (Table 1). The slope (b) of log concentration probit (lcp) line of avidin against C. maculatus was determined as 1.79 (Fig 1). The chi-square test indicated that the C. maculatus population used in this study was homogeneous.


 


Lethal concentrations of streptavidin against C. maculatus
 
It is seen from Table 2 that the mortality of C. maculatus larvae seen is 33.33, 58.06, 78.12, 86.95 and 92.1 per cent when fed with streptavidin incorporated pellets with concentrations of 5, 20, 30, 75 and 100 ppm respectively. The probit analysis of the mortality data revealed that the respective LC25, LC50 and LC90 values of streptavidin against C. maculatus were 3.824, 11.192 and 86.134 ppm. The population of the C. maculatus was found homogeneous as evident from the chi-square test. The slope of the lcp line of streptavidin against C. maculatus was determined as 1.44 (Fig 1).



It is clearly evident from bio-assay studies that avidin is more effective than streptavidin in causing mortality of C. maculatus with its lcp line more flat than that of streptavidin (Fig 1). Avidin could cause more than 90 per cent of C. maculatus at concentrations less than 77 ppm while streptavidin at 100 ppm. Similarly Murdock and Shade (2008) indicated that avidin at concentration of 15 ppm caused 50 per cent mortality in C. maculatus larvae which was in accordance and supported the present study. He also stated that at concentrations of 20 ppm and above, nearly complete mortality is seen with only few larvae reaching the grub stage before dying and in another experiment he reported that more than 83 per cent mortality at avidin concentration of 25 ppm and above. The results from Murdock and Shade (2008) revealed that streptavidin at a concentration of 10 ppm in the diet caused about 75 per cent mortality in C. maculatus and stated that streptavidin had toxic effects similar to that of the avidin. These results were in conformity with the findings of Murdock and Shade (2008) on C. maculatus where two and three adults emerged in avidin 25 ppm and streptavidin 10 ppm, respectively against 11 adults in untreated control.
 
Bioassay experiments proved that there is a slow increase in mortality of the three test insects and increased up to third week. But at higher concentration, larval mortality was observed immediately within one week. Similar results were obtained with the red flour beetle T. confusum and flat grain beetle C. pusillus (Kramer, 2000). It is also observed that the dead larvae turned to black, as the larvae appeared to stop feeding during ecdysis, died while still attached to a partially shed larval skin. Similar results were also observed in the case of H. armigera and S. litura larvae, fed on transgenic tobacco leaves expressing BBPs (Burgess et al., 2002).

Effect of avidin and streptavidin on oviposition by C. maculatus
 
The number of eggs laid by C. maculatus on treated pellets differed significantly. Maximum number of, 26 eggs were laid on four pellets made with one gram of untreated bengal gram flour (Plate 2). The least number of eggs (6) were recorded on pellets treated with spinosad 250 ppm and streptavidin 100 ppm which were on par with 7.67 eggs recorded at streptavidin 75 ppm (Table 3). The next better treatments were 100 and 75 ppm of avidin and 30 ppm streptavidin with 9.33 and 9.67 and 10 eggs respectively which were similar without any significant difference between them. Pellets treated with 20 ppm streptavidin, 15 and 30 ppm avidin and 70 ppm spinosad recorded 11.33, 11.67, 10.67 and 11 eggs, respectively which were similar with each other. Lower concentrations of streptavidin 5 ppm and avidin 5 ppm recorded fairly higher number of eggs (15.33 and 19.67) but lesser than the untreated control and differed significantly with other treatments (Fig 2).






 
Effect of avidin and streptavidin on day of first adult emergence of C. maculatus  
 
Significant difference was observed regarding the first adult emergence of C. maculatus on treated pellets compared to untreated one. First adult emergence of C. maculatus was observed on 36^th day which was earliest/minimum in untreated control. There was no adult emergence, on pellets treated with spinosad at 70 and 250 ppm. There was only one adult emergence on 76^th day in streptavidin 100 ppm. Streptavidin 75 ppm and avidin 100 ppm have recoded first adult emergence on 66^th and 81^st day which were on par with streptavidin 100 ppm and differed significantly from other treatments. Streptavidin @ 30, 20 and 5 ppm recorded first adult emergence on 41^st, 41^st and 42^nd day and showed no significant difference with lower concentration of avidin @ 5 and 15 ppm by recording first adult on 43^rd and 37^th day respectively and were found to be on par with 30 ppm avidin which recorded 43^rd day of first adult emergence. Avidin at 75 ppm showed first adult emergence on 53^rd day which differed significantly from other treatments (Fig 2) (Table 3).

 
Effect of avidin and streptavidin on development period of C. maculatus
 
The results revealed a significant difference among all the treatments regarding the development period of C. maculatus (Table 3). Spinosad treated pellets showed no development of C. maculatus when treated with concentrations of 70 and 250 ppm. Streptavidin 30 ppm showed minimum development period of 42.33 days, because on an average only three adults emerged out of 10 eggs laid within this development period in this treatment. The short development period with better growth was observed in untreated pellets with 55.33 days  which was on par with 15 and 30 ppm avidin and 5 and 20 ppm streptavidin with development period of 57.33, 54.33, 56 and 49 days respectively (Plate 3; Fig 2).



The longest development period with delayed growth was observed in avidin 100 ppm and streptavidin 75 ppm with 81.33 and 82.67 days respectively, showed no significant difference between them and were on par with streptavidin 100 ppm with 76 days respectively, followed by avidin 75 ppm with 68.33 days.

Among avidin treated pellets, the development period at 15 ppm was 57.33 days, which is on par with avidin 5 and 30 ppm with 63 and 54.33 days respectively and differed significantly from the maximum development period of 81.33 and 68.33 days with avidin 100 and 75 ppm respectively.

Among streptavidin treated pellets, development period was delayed maximum up to 82.67 days with 75 ppm which was on par with 100 ppm with a development period of 76 days respectively and differed significantly with shorter development period of 56, 49 and 42.33 days at lower concentrations of 5, 20 and 30 ppm, respectively.

When avidin treated pellets were compared with streptavidin treated pellets, no significant difference was seen regarding the development period at avidin 15 ppm with streptavidin 5 ppm and at avidin 100 ppm with streptavidin 75 ppm. Where, at 100 ppm of streptavidin no further development was seen after first adult emergence at 76^th day.
 
Effect of avidin and streptavidin on adult emergence of C. maculatus
 
Adult emergence of C. maculatus differed significantly among all the treated pellets. The highest adult emergence of 62.83 per cent was noticed in untreated pellets and differed significantly from all other treatments. Zero adult emergence was observed with spinosad 70 and 250 ppm. The least number of adults emerged were 3.33 per cent in 100 ppm streptavidin, followed by 5.56 per cent in 100 ppm avidin which has no significant difference with each other and differed significantly from other treatments (Fig 2). These were followed by 75 ppm of avidin and streptavidin with adult emergence of 13.47 and 13.10 per cent respectively with no significant difference between them.

Among avidin treated pellets, highest number of adult emergence was found at lower concentrations of avidin 5 ppm with 40.58 per cent which differed significantly with 15 and 30 ppm avidin with similar adult emergence of 33.93 and 31.11 per cent respectively. It is followed by higher concentrations of 75and 100 ppm with 13.47 and 5.56 per cent adults respectively which differed significantly with one other.

Among streptavidin treated pellets, highest number of adults was observed at 5 ppm with 41.35 per cent followed by 20, 30, 75and 100 ppm with 34.94, 24.17, 13.1 and 3.33 per cent adults respectively which differed significantly with each other.

When adult emergence on avidin treated pellets were compared with streptavidin treated pellets, no significant difference was found at 5, 75 and 100 ppm of avidin when compared to 5, 75 and 100 ppm of streptavidin and also between 15 and 30 ppm of avidin compared with 20 ppm of streptavidin, which further differed significantly with 30 ppm of streptavidin and the untreated control.

The results conclude that the per cent adult emergence at lower concentration (30 to 5 ppm) of avidin and streptavidin were from 31 to 40 per cent and 24 to 41 per cent respectively and the per cent adult emergence at higher concentrations (100 and 75 ppm) of avidin and streptavidin were 5 to 13 per cent and 3 to 13 per cent respectively against the untreated control with 62 per cent (Table 3).
 
Effect of avidin and streptavidin on growth index of C. maculatus
 
The growth index of C. maculatus differed significantly among the treatments. The highest growth index of 1.13 was observed in the untreated pellets. It was followed by 5 and 20 ppm of streptavidin with growth index of 0.74 and 0.72 respectively, which were found to be similar and at par with 5 ppm avidin with 0.65 growth index and differed significantly with other treatments.

Zero growth index was recorded in spinosad treatments as there was no adult emergence. The lowest growth index of 0.11 is noticed at higher concentrations of 100 ppm avidin which is on par with growth index of 0.13 and 0.16 at streptavidin 100 and 75 ppm, respectively (Table 3).

Among avidin treated pellets, highest growth index of 0.65 was observed in lower concentrations at 5 ppm, which was on par with similar growth index of 0.59 and 0.58 at 15 and 30 ppm respectively and differed significantly from 75 ppm and 100 ppm avidin with growth index of 0.2 and 0.11 respectively.

Among streptavidin treated pellets, no significant difference in growth index was observed between lower concentrations of 5 and 20 ppm with higher growth index of 0.74 and 0.72 respectively and also between higher concentrations of 75 and 100 ppm with lower growth index of 0.16 and 0.13 respectively and differed significantly with 0.57 growth index at 30 ppm streptavidin.

When avidin and streptavidin treated pellets were compared with each other regarding the growth index, the lower concentrations of streptavidin at 5 and 20 were found on par with avidin at 5 and differed significantly with higher concentrations of avidin at 75 and 100 ppm respectively. Even the higher concentrations viz., Streptavidin 75 and 100 ppm were on par with avidin 75 and 100 ppm and differed significantly with lower concentrations viz., avidin 5 and 15 ppm respectively. There was no significant difference between 15 and 30 ppm avidin and streptavidin 30 ppm respectively.

The growth index recorded in 5 to 100 ppm avidin and streptavidin is in the range of 0.65 to 0.11 and 0.74 to 0.13 respectively against the control (1.13).
 
Effect of avidin and streptavidin on weight loss of Bengal gram flour pellet due to C. maculatus
 
Untreated Bengal gram pellets recorded the highest weight loss of 21.55 per cent which was on par with 5 and 15 ppm of avidin (19.54 and 17.95%) and 5 ppm of streptavidin (17.82%).

Weight loss was minimum (9.84%) in streptavidin 100 ppm and on par with 30 ppm and 75 ppm streptavidin with 12.29 and 12.25 per cent. No weight loss was recorded in pellets treated with spinosad 70 and 250 ppm, with no grub development (Fig 2).

Among avidin treated pellets, the highest weight loss was noticed at lower concentration of avidin 5 ppm with 19.54 per cent, followed by further higher concentrations of avidin 15, 30, 75 and 100 ppm with 17.95, 16.43, 16.11 and 12.53 per cent weight loss respectively, where weight loss at 30 ppm was at par with 5, 15 and 75 ppm respectively and differed significantly from 100 ppm of avidin with 12.53 per cent weight loss.

Among streptavidin treated pellets, streptavidin 20 ppm with 15.24 per cent was found on par with higher weight loss of 17.82 per cent at 5 ppm and with 12.29 and 12.25 per cent at 30 and 75 ppm of streptavidin, respectively. No significant difference regarding weight loss was found between 30 and 75 ppm streptavidin which was at par with 9.84 per cent at 100 ppm streptavidin.

When per cent weight loss in avidin and streptavidin treated pellets were compared with each other, streptavidin 5 ppm and avidin 15 ppm showed no significant difference. Minimum weight loss recorded at 100 ppm avidin, on par with 20, 30, 75 and 100 ppm of streptavidin.

From the present investigation, it is clearly evident that biotin inhibitors viz., avidin at 75 and 100 ppm, streptavidin at 30, 75 and 100 ppm against C. maculatus; have inhibited the growth and development by recording delayed first adult emergence, longer development periods, zero or least adult emergence, reduced larval weights and weight loss. There was no adult emergence at >100 ppm of BBP’s and delayed adult emergence beyond 60 days at 75 ppm of Avidin and streptavidin. These results obtained were supported by the findings of Murdock and Shade (2008), where the first adult emergence of C. maculatus was delayed up to 35th day at concentration of 15 ppm and above when compared to 27^th day of first adult emergence recorded in the control treatment.

There was zero adult emergence at 70, 250 ppm of spinosad and about 3.33 and 5.56 at 100 ppm of streptavidin and avidin with growth index 0.13 and 0.11. The findings of Morgan et al., (1993) were also in support to the results of the present study, where R. dominica when fed on 10 ppm avidin treated pellets caused 60 per cent reduction in emergence and 100 ppm avidin resulted in 100 per cent larval mortality with zero emergence.  The above results of delayed development period obtained at higher concentrations of avidin and streptavidin were in conformity of the Bruins et al., (1991), who demonstrated that avidin increased the development time of Drosophila melanogaster and also by the findings of Murdock and Shade (2008) who reported that C. maculatus larval survival at 15 ppm dose of avidin with slight delay in their development.The results obtained in this experiment were supported by the findings of Christeller et al., (2006) where the mean growth rates of the light brown apple moth, Ephiphyas postvittana larvae placed on diets at 14, 21 and 28 days later with avidin 5.52 mg were 0.61, 1.2 and 1.29 against the untreated control with 2.22, 4.17 and 4.74 growth rates respectively.

During development, the larvae have showed reduced feeding, inactivity, failure to moult and pupation. It is also evident that insects affected by BBPs generally die during the moulting period (Markwick et al., 2001, Burgess et al., 2002), a physiological process absent in mammals. Insects normally survive the moult, during which time they do not feed, by storing lipid in the fat body during feeding and catabolizing these lipids during the non-feeding period to provide energy for the moulting process. Because both lipid synthesis and catabolism critically require biotin carboxylases, it is possible that due to avidin and streptavidin there was failure to deposit lipid and the inability to use it for energy production which accounted for insect death during moulting and provide an explanation for the heightened sensitivity that insects show to biotin depletion compared with mammals.

This mortality depends on the concentration of biotin binding proteins, avidin and streptavidin which bind to biotin in the insect diet. They may not be able to remobilize the fat during ecdysis process. Similarly the larvae may die as they may not be able to cope with the high energy expenditure in the absence of active feeding. It is clearly known that biotin dependent carboxylases are required for the deposition of fat reserves during active feeding and for subsequent utilization of these reserves.

Besides the above mentioned role, Biotin functions as an enzyme cofactor that acts as a one-carbon transfer agent via carboxybiotin (Attwood and Wallace, 2002; Jitrapakdee and Wallace, 2003). Biotin is covalently bound to various enzymes via a reaction catalyzed by holobiotin carboxylase (Zempleni and Mock 1999). The enzymes that are known to use biotin as a cofactor are acetyl-CoA carboxylase, 3-methylcrotonyl CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase. These are enzymes involved in lipid and amino acid biosynthesis and degradation.

This work is part of thesis work submitted for M.Sc., (Ag) degree to Acharya NG Ranga Agricultural University.