the blowflies ( c. putoria ) were reared at the laboratory for the study of diptera , department of microbiology and parasitology , universidade federal do estado do rio de janeiro .
adults were obtained from a stock colony originated from insects collected at the rio de janeiro zoo , quinta da boa vista park , city of so cristvo , rj . three traps following the model of mello et  al .
adults and larvae of muscoid flies were taken to the laboratory , where they were sorted and identified following mello ( 2003 ) .
experimental flies were reared in plastic cages ( 40 by 30 by 20  cm ) with an opening at the top for aeration and an anterior opening to allow access to the inside of the cage , which was covered with escaline - coated fabric .
each cage received water , honey and water ( 50% ) solution , and chicken gizzard or beef ( which are protein sources and serve as substrate for oviposition and ovary maturation ) .
first - instar larvae of the third laboratory generation were transferred with the help of a fine paintbrush to 100-ml beakers containing 60  g gizzard / agar 65% homogenate ( ferraz et  al . 2012 ) .
this diet was selected because it is practical and sterile ( autoclaved ) , allowing the antibiotic to act fully and only on the larvae .
each repetition received 1  ml of the antibiotic , resulting in the following three different diet concentrations : 4.44  m / ml , 13.33  mg / ml , and 66.66  mg / ml . the concentrations were chosen from the serum gentamicin concentration ( established according to the dosage indicated in the prescription advisor ) , as follows : 23 daily intravenous or intramuscular doses ; maximum concentration 30  min/1  h after administration : 46  g / ml . avoid concentrations above 12  g / ml for extended periods ( gentamicina 2011 ) .
the first concentration ( t1 ) is close to the intravenous serum concentration ; the second ( t2 ) is close to the maximum intravenous serum concentration ; the third ( t3 ) is approximately five times the maximum serum intravenous concentration .
each antibiotic concentration tested and the control were replicated four times using 40 larvae in each repetition .
each beaker was put into a larger beaker containing sterilized sawdust , which was sealed with elastic escaline .
the sawdust serves as a pupation substrate for the larvae after they abandon the diet .
larvae were kept in a climatic chamber at 30c per day and 28c per night , 70  +  10% relative humidity ( rh ) , and 14-h photoperiod .
observations were made daily , always at the same time ( 12  h ) . as a precaution
, all treatments in this study ensured that each individual larva had > 1  g of food available .
we ensured this quantity because aguiar - coelho and milward - de - azevedo ( 1996 ) had previously suggested that this is the optimal amount of meat for rearing calliphoridae . ensuring that larvae have the optimal amount of food prevents exploitative competition and the stress associated with it , and with the chemical changes in the food substrate resulting from larval metabolism ( khazaeli et  al .
the body mass after diet abandonment was recorded in batches of five larvae , with the help of an analytical scale .
larvae were then stored in test tubes sealed with nylon and elastic fabric for further observation .
the dates of pupation and emergence , as well as the sex ratio and morphological abnormalities in the adults , when present , were recorded .
the program microsoft excel was used to analyze the raw data , and further analyses were conducted in the program past .
variations in the mean body mass of larvae and the duration of larval and pupal stages and total development ( from neolarvae to adult ) were analyzed using the student s t - test (   =  5% ) .
the four treatments did not differ significantly in the following parameters : body mass of c. putoria larvae ( table 1 ) ; mean duration of larval inoculation to abandonment ; and larval , pupal , and total development times ( d ) ( table 2 ) . 
 table 1.body mass of larvae ( g ) of the blowfly c. putoria ( wiedemann , 1830 ) ( diptera , calliphoridae ) after four treatments with different concentrations of gentamicin , reared in a climatic chamber ( 30c day and 28c night , 70  +  10% rh , and 14-h photoperiod)treatmentmean individual mass ( g )    standard deviationvariation intervalp  =  significance value ( t - test)controlt1t2t3control0.049a    0.0070.0350.0590.5690.2390.483t10.053a    0.0120.0210.0680.5690.9090.974t20.054a 
  0.0030.0410.0620.2390.9090.826t30.053a    0.0080.0370.0640.4830.9740.826means followed by same letter in same column do not differ significantly by the t - test , 5% confidence . control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  g / ml ; t2  =  gizzard homogenate agar  +  gentamicin 13.33  g / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  g / ml . 
 table 2.average duration of the postembryonic development stages ( d ) of larvae of the blowfly c. putoria ( wiedemann , 1830 ) subjected to four treatments with different concentrations of gentamicin and reared in a climatic chamber ( 30c day and 28c night , 70  +  10% rh , and 14-h photoperiod)daysvariation intervalstandard deviationp  =  significance value ( t - test)controlt1t2t3duration until abandonmentcontrol4.478a450.5170.2140.4880.418t14.114a450.0890.2140.6450.150t24.225a450.4500.4880.6450.231t34.989a461.0560.4180.1500.231larval stagecontrol5.477a560.5160.5280.5730.494t15.270a560.3400.5280.9860.264t25.275a560.4410.5730.9860.287t35.845a570.8710.4940.2640.287pupal stagecontrol3.874a350.2140.4060.1810.652t13.610a360.5510.4060.1630.741t24.057a450.1140.1810.1630.278t33.741a250.5180.6520.7410.278total stagecontrol9.339a9100.3220.1240.9540.532t18.785a8110.5300.1240.1680.139t29.323a9100.4350.9540.1680.531t39.642a9110.8540.5320.1390.531means followed by same letter in same column do not differ significantly by the t - test , 5% confidence .
control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  g / ml ; t2  =  gizzard homogenate agar  +  gentamicin 13.33  g / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  g / ml
. body mass of larvae ( g ) of the blowfly c. putoria ( wiedemann , 1830 ) ( diptera , calliphoridae ) after four treatments with different concentrations of gentamicin , reared in a climatic chamber ( 30c day and 28c night , 70  +  10% rh , and 14-h photoperiod ) means followed by same letter in same column do not differ significantly by the t - test , 5% confidence .
control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  g / ml ; t2  =  gizzard homogenate agar  +  gentamicin 13.33  g / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  g / ml . average duration of the postembryonic development stages ( d ) of larvae of the blowfly c. putoria ( wiedemann , 1830 ) subjected to four treatments with different concentrations of gentamicin and reared in a climatic chamber ( 30c day and 28c night , 70  +  10% rh , and 14-h photoperiod ) means followed by same letter in same column do not differ significantly by the t - test , 5% confidence .
control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  g / ml ; t2  =  gizzard homogenate agar  +  gentamicin 13.33  g / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  g / ml . the rates of diet abandonment , pupation , and emergence ( % ) obtained for c. putoria in the four different treatments ( control  =  gizzard and agar homogenate ; t1  =  gizzard and agar homogenate  +  gentamicin 3.33  mg / ml ; t2  =  gizzard and agar homogenate  +  gentamicin  +  13.33  mg / ml ; t3  =  gizzard and agar homogenate  +  gentamicin 66.66  mg / ml ) is shown in fig .
diet abandonment peaked in the control group between 5 and 6  d , and predominantly on the fifth day in the three treatments .
in t3 , and only in this treatment , larval abandonment lasted until the seventh day .
the pupation peak of control flies happened between the sixth and seventh days , whereas in the other treatments , it happened on day 6 , extending up to day 8 in t3 . in all treatments ,
the emergence peak occurred on day 10 , lasting to day 12 only in t1 and t3 . 

1.rate of abandonment of the diet , pupation , and emergence ( % ) of c. putoria ( wiedemann , 1830 ) subjected to four treatments : control = gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / ml . 
  rate of abandonment of the diet , pupation , and emergence ( % ) of c. putoria ( wiedemann , 1830 ) subjected to four treatments : control = gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / ml . the emergence rate of c. putoria males and females was similar in the control and t3 ( fig .
2 ) . in t1 , males emerged first ( beginning on day 9 ) and continued to do so up to day 12 .
females , in contrast , began to emerge a day later ( day 10 ) and continued up to day 11 .
in t2 , all female flies emerged on day 10 , whereas males emerged from days 10 to 11 .
the sex ratio for each treatment was as follows : control , males  =  47% , females  =  53% ; t1 , males  =  48% , females  =  52% ; t2 males  =  50% , females  =  50% ; and t3 , males  =  51% , females  =  49% .
the chi - square tests revealed that the sex ratios obtained for the four treatments did not differ from the expected ( control : =  0.398 , t1 : =  0.221 , t2 : =  0.006 , t3 : =  0.081 ; tabulated =  3.84 , df  =  1 ,   =  5% ) . 

2.rate of emergence , in days , of males and females c. putoria ( wiedemann , 1830 ) subjected to four treatments : control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / ml
. 
  rate of emergence , in days , of males and females c. putoria ( wiedemann , 1830 ) subjected to four treatments : control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / ml . all adults in the control and in the other treatments were normal .
as for larval viability , only t2 differed significantly from the control ( table 3 ) . 

table 3.anova comparison of the viability of larvae , pupae , and total ( neolarvae to adult ) of c. putoria ( wiedemann , 1830 ) subjected to four treatments : control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / mlviability ( % ) p  =  significance value ( anova)controlt1t2t3larvalcontrol81.88a0.1960.0060.874t190.63ab0.1960.3260.419t296.88b0.0060.3260.185t380.00ab0.8740.4190.185pupalcontrol93.60a0.2630.7390.228t175.40a0.2630.2870.468t292.26a0.7390.2870.234t386.85a0.2280.4680.234totalcontrol76.88a0.7170.0730.517t170.63a0.7170.2840.948t289.38a0.0730.2840.093t369.38a0.5170.9480.093anova , analysis of variance .
anova comparison of the viability of larvae , pupae , and total ( neolarvae to adult ) of c. putoria ( wiedemann , 1830 ) subjected to four treatments : control  =  gizzard homogenate agar ; t1  =  gizzard homogenate agar  +  gentamicin 3.33  mg / ml , t2  =  gizzard homogenate agar  +  gentamicin 13.33  m / ml , t3  =  gizzard homogenate agar  +  gentamicin 66.66  mg / ml anova , analysis of variance . means followed by the same letter do not differ at 5% significance .
previous studies have detected toxins and controlled substances in samples collected from carrion in advanced stage of decomposition and the insects that visit them ( goff and lord 2001 ) . in some studies , decreased larval mass gain , and larval growth , as well as reduced larval activity , were correlated with the presence of certain drugs ( soto 2008 ) .
( 2012 ) , who reared experimental larvae on meat , gizzard , and gizzard and agar homogenate , the mean larval mass obtained in our study was similar in all treatments . in the treatments , the total duration of development increased with increased antibiotic concentrations
after all treatments in our study , the larval stage lasted longer than in the study of ferraz et  al .
this was expected , because meat is the natural diet of this species ( leal et  al .
( 2006 ) found that another blowfly , cochliomyia macellaria ( f. ) ( diptera : calliphoridae ) , when reared under sterile conditions , developed faster and survived longer than flies reared in the presence of bacteria . in this study ,
no significant differences in the rate of diet abandonment , pupation , and adult emergence were found between the control and each of the treatments . according to our results
( 2009 ) , who tested the effect of morphine on calliphora stygia ( diptera : calliphoridae ) .
however , in a study on the effects of cocaine on the development of c. putoria ( reared on liver containing the substance ) , treatment larvae developed significantly faster , pupated , and emerged sooner than control larvae ( carvalho 2004 ) .
other substances such as diazepam and anfepromona also accelerated the development of c. putoria larvae ( carvalho et  al .
in contrast , the painkiller scopolamine slowed down larval development in treatments containing higher concentrations of the substance ( grella et  al .
2007 ) , and buscopan accelerated the rate of growth of chrysomya megacephala ( diptera : calliphoridae ) ( oliveira et  al .
2009 ) . additionally , a study on the effect of malathion on c. megacephala revealed that this insecticide can alter ipm estimates in up to 36  h ( liu et  al .
2009 ) . in this study , the only statistically significant effect of gentamicin was on the viability of t2 larvae .
larvae were more viable after this treatment than the others , although viability after all treatments was high .
in diptera , viabilities above 60% are considered promising ( loureiro et  al . 2005 ) . in a study using artificial diet
plus scopolamine to rear c. putoria flies , mortality was higher with increased drug concentrations ( grella et  al .
it is also possible that , in our study , the drug was not metabolized by the larvae .
this is consistent with the results of grella and thyssen ( 2008 ) , who reared chrysomya albiceps , c. megacephala , and c. putoria on artificial diet plus oxycodone hydrochloride and failed to find significant differences in development of the control and the experimental groups .
larvae are able to efficiently eliminate various toxic substances during development ( nuorteva and nuorteva 1982 ) .
also , at least in two other studies , treatments containing low drug concentrations paradoxically resulted in higher survival rates than the control ( goff et  al .
this percentage is considered appropriate and is higher than the values obtained by soto ( 2008 ) for c. putoria larvae reared on artificial diet plus chicken heart , both for the control and the treatments with barbiturates .
total survival was above 69% in all treatments , being higher than in the control and other treatments in the results of soto ( 2008 ) .
( 1995 ) for several different larval densities of chrysomya ranged between 60 and 98% , also consistent with the findings of this study .
the sex ratio found in our study , after chi - square analysis , was 1:1 , indicating that the population is stable ( fisher 1930 ) . additionally , 100% of the control and treatment flies were normal , indicating that the flies metabolic adjustments buffered or neutralized the effects of the antibiotics ( lomnaco and germanos 2001 ) .
this result is consistent with the effects of method on the sex ratio of lucilia sericata ( diptera : calliphoridae ) .
however , this substance delayed the development of those flies and altered their mortality rates ( gosselin et  al .
entomotoxicology is the analysis of substances in necrophagous arthropods to identify drugs and toxins present in dead tissues , their effects on the development of arthropods , and how they interfere with ipm estimates ( goff and lord 2001 ) .
such analyses are particularly relevant when blood , urine , and the internal organs of a body are no longer available . according to liu et  al .
( 2009 ) , understanding the effects of drugs and toxins on the development of flies is very important to make accurate ipm estimates . though laboratory studies to ascertain how certain drugs affect the development of flies can be made using rodents , rats , and rabbits ( george et  al .
2012 ) , artificial diets containing these substances are a viable alternative . in the study of gosselin et  al .
( 2011 ) , 100  g of bovine heart plus 8% gelatin was given to every 50 larvae .
/ agar homogenate , this diet is low cost and is very rich in nutrients . because the final product is liquid , it can be easily mixed with different concentrations of gentamicin .
two grams of diet in four repetitions were made available to the larvae , because this quantity is ideal for the development of this species and results in normal body mass , growth rate , viability , and sex ratio ( ferraz et  al .
even though forensic entomologists have been conducting ipm calculations in different countries , a standard practice has not been adopted yet .
united states statistics show that overdosing or drug incompatibility may lead to death , especially in older individuals .
therefore , studies such as ours may foster the precision of pmi estimates using c. putoria , especially when antibiotics have been previously used and/or have been the cause of death .
gentamicin did not alter the parameters tested for c. putoria in this work : body mass , larval , pupal , and total development .
therefore , ipm calculations based on the biological parameters of this fly will not be affected when this antibiotic is present .
 we evaluate the effects the antibiotic gentamicin on the development of chrysomya putoria ( wiedemann , 1818 ) . 
 third - generation , first - instar larvae were reared in a climatic chamber on 60  g of homogenate  +  agar 65% and were treated with three concentrations of gentamicin : 4.44  mg / ml , 13.33  mg / ml , and 66.66  mg / ml . the control consisted of distilled water . the relationships between mean body mass of mature larvae ( measured after diet abandonment , in batches of five individuals ) , duration of larval and pupal stages , and overall duration of development were analyzed . 
 the actual sex ratio was compared against the expected using the chi square . 
 none of the parameters measured differed significantly among the four treatments , with one exception : when gentamicin concentration was 13.33  mg / ml , larval viability differed significantly from the control . 
 all larvae from all treatments were considered normal . 
 we conclude that the antibiotic did not significantly alter the development of c. putoria ( wiedemann ) ( diptera : calliphoridae ) . 
Materials and Methods
Results
Discussion