the australian sheep blowfly ( lucilia cuprina ) is an important ecto - parasite that causes fly strike , which has significant health and welfare , as well as economic , impacts on the sheep industry in australia ( sandeman et  al . , 2014 ) . the female blowfly is attracted to the sheep by odours , particularly those associated with bacterial infections in damp fleece , and lays eggs ( tellam and bowles , 1997 ) . the developing larvae feed on the sheep , causing severe tissue damage , toxaemia , and in some cases , death . the consequent loss of livestock , costs of preventative and curative chemical treatments , and animal welfare issues place significant economic burdens on livestock enterprises ( lane et  al . , 2015 ) . the blowfly has developed resistance to various classes of chemical insecticides used for its control , including organochlorines , organophosphates , the benzoyl - phenyl urea diflubenzuron ( levot , 1995 , sandeman et  al . , only two preventative blowfly control chemicals , the macrocyclic lactone ivermectin and the cyanopyrimidine dicyclanil , remain effective with no resistance yet reported . there is therefore a need to identify new chemical classes of insecticides , preferably with different target proteins , to control this important parasitic insect . histone deacetylase inhibitors ( hdaci ) have been recognised as therapeutic targets in cancer for many years ( cairns , 2001 ) , with a number in clinical use or clinical trials as anti - cancer drugs . they have also been studied extensively over recent years for their potential in chemotherapy for parasitic diseases of humans , including malaria , toxoplasmosis , trypanosomiasis , schistosomiasis and leishamaniasis ( andrews et  al . , 2012a , andrews et  al . , 2012b ; marek et  al . , hdac enzymes have been studied extensively in the model dipteran insect drosophila with respect to their roles in longevity and memory formation ( fitzsimons et  al . , 2013 , 2016 ) , with a drosophila model providing experimental evidence to highlight hdaci as potential therapeutics for the treatment of huntington 's disease ( sharma and taliyan , 2015 ) . however , only a single study has reported the insecticidal activity of an hdaci against this fly species , with pile et  al . ( 2015 ) who showed that trichostatin and suberoylanilide hydroxamic acid ( saha ) were able to inhibit the development of sheep blowfly larvae in  vitro . that report also highlighted similarities and differences in amino acid sequences of blowfly and human hdac enzymes , with differences particularly noted between species for the class ii enzymes hdac4 and 6 , and the class iv hdac11 , raising the possibility of identifying insect - specific inhibitors . the present study expands on our earlier report of insecticidal activity for trichostatin and saha ( kotze et  al . , 2015 ) by examining other hdaci with different chemical structures and mechanisms of action . we focus on hydroxamic acids since these are the best known group of hdaci , but also include inhibitors with different chemical components , such as benzamides , thioesters , thiolates , disufides , cyclic depsi- and tetra - peptides , fatty acids , and sesquiterpene lactones ( table  1 ) . we measure the effects of these hdaci on the development of blowfly larvae ( larval growth rate and pupation rate ) and on the hdac enzyme activity of nuclear protein extracts prepared from blowfly eggs . we also compare these results with reported inhibitory activities against human hdac enzymes as an initial step towards identification of insect - specific inhibitors . the l.  cuprina used in this study were from the laboratory reference drug - susceptible ls strain , derived from collections made in the australian capital territory ( canberra , australia ) over 40 years ago . this strain has been maintained in a laboratory since that time ( in canberra for 30 years , and then at csiro and university of queensland laboratories in brisbane for the last 10 years ) , and has no history of exposure to insecticides . adult flies were maintained at 28  c and 80% relative humidity with a daily photoperiod of light 16  h and dark 8  h. adults were fed a diet of sugar and water , while larvae were raised on a wheatgerm culture medium ( tachibana and numata , 2001 ) . protein meals ( bovine liver ) were provided on days 4 and 8 after adult eclosion in order to prime adult flies for subsequent egg - laying . for provision of eggs for bioassays , liver was placed into cages of gravid flies for a period of two hours ( 12 p.m. until 2 p.m. ) . the liver was then removed and kept at room temperature overnight . at 10 a.m. the next morning hdaci were synthesized by reported procedures or obtained from commercial sources ( table  1 ) . stock solutions for use in larval bioassays were prepared in ethanol at a concentration of 1  mg / ml . in cases where the compound did not dissolve at this concentration the solutions were further diluted 2-fold with ethanol until no precipitate was evident ( to give stocks at 0.5 or 0.25  mg / ml ) . exceptions were cudc-907 and mc1568 which required dilution to a concentration of 0.05  mg / ml . the commercial insecticide stocks used as controls were prepared at 1  mg / ml in water ( cyromazine and dicyclanil ) or acetone ( diflubenzuron ) . stock solutions of hdaci for use in nuclear extract hdac enzyme assays were prepared at 1  mg / ml in dmso . the effects of hdaci on the growth of blowfly larvae was assessed using a bioassay system in which larvae were allowed to develop on cotton wool impregnated with the compounds at various concentrations ( modified slightly from kotze et  al . , 2014 ) . briefly , 4  ml aliquots of hdaci or commercial insecticide solutions were added to cotton wool plugs and the solvent ( 4  ml of either ethanol , acetone , or water ) was allowed to evaporate overnight . control containers were prepared by addition of 4  ml of the relevant solvent to the cotton wool . the next day ( day 0 of the assay ) , a sheep serum - based medium ( 80 g / l yeast extract ( merck ) , 1.6  mg / ml tylosin ( sigma ) in lamb serum ( life technologies ) buffered with 35  mm kh2po4 , ph7.5 ) was added to the cotton wool , and groups of 50 freshly - hatched larvae ( prepared as described in section 2.1 , above ) were placed onto the cotton wool . the assay pots were placed at 28  c . in order to calculate mean larval weight at the beginning of the drug exposure period , two groups of 100 larvae were collected , blotted dry on paper towel , weighed and discarded on day 0 . after 24  h ( day 1 ) , 3 larvae were removed from each container , weighed , and discarded . the remaining larvae were fed with 1  ml of nutrient medium on day 1 , and then 2  ml on each of days 2 and 3 . late on day 4 , the containers were placed into larger pots with a layer of sand at the base to serve as a medium for pupation , and returned to the incubator . each experiment consisted of a single container at each concentration of hdac inhibitor or insecticide , alongside 4 control assays . the effect of the compounds on larval development was defined in two ways : i)larval weight gain in first 24  h ; the total weight gain of the 3 larvae sampled on day 1 was expressed as a percentage of the mean of the weight gain of the 3 larvae sampled from each of the 4 control containers ( weight gain was calculated by difference using weight on day 1 and the mean weight of larvae on day 0);ii)pupation rate ; the number of pupae in each drug - treated container was expressed as a percentage of the mean number of pupae in the 4 control containers . larval weight gain in first 24  h ; the total weight gain of the 3 larvae sampled on day 1 was expressed as a percentage of the mean of the weight gain of the 3 larvae sampled from each of the 4 control containers ( weight gain was calculated by difference using weight on day 1 and the mean weight of larvae on day 0 ) ; pupation rate ; the number of pupae in each drug - treated container was expressed as a percentage of the mean number of pupae in the 4 control containers . the larval weight and pupation rate dose - response data were analysed with graphpad prism software using non - linear regression , with the variable slope option selected , in order to calculate ic50 values ( with 95% confidence intervals ) representing the concentration of inhibitor required to reduce the larval weight gain or pupation rate to 50% of that measured in control ( no drug ) treatments . nuclear extracts were prepared from blowfly eggs ( 0.5  g ) using a nuclear extraction kit ( millipore , usa ) following the manufacturer 's protocol with some modifications . the chorion was removed by soaking for 80  s in a solution of bleach ( 2% v / v ) , followed by centrifugation to sediment the eggs . complete mini protease inhibitor ( roche , basel switzerland ) in pbs was added to the washed eggs before disrupting them by hand with a plastic pestle . the disrupted eggs were centrifuged at 250 g for 1  min at 4  c , and supernatant removed . the egg cell pellet was washed with 1000  l of ice cold pbs , resuspended by inversion , centrifuged at 1000 g  for  5  min at 4  c , and the supernatant removed . the cells were then disrupted by drawing 5 times through a 21  g needle fitted to a 1  ml syringe . the suspension was centrifuged at 8000 g for 20  min at 4  c , the supernatant removed and discarded , and the pellet retained ( nuclear portion ) . the nuclear pellet was resuspended in 2/3 of the original cell pellet volume of ice cold nuclear extraction buffer ( containing 0.5  mm dtt and protease inhibitor cocktail , millipore , temecula ) . the solution was placed on low speed roller for 1  h at 4  c , then centrifuged at 16000 g for 5  min at 4  c , and the supernatant ( the nuclear extract ) transferred to a new tube . the protein concentration was measured by the method of bradford ( 1976 ) using the bio - rad protein assay reagent , and bovine serum albumin as a standard . the extract was then aliquoted into separate tubes , snap - frozen in liquid nitrogen , and stored at  80  c . a fluorometric assay kit ( sigma - aldrich , usa ) was used to measure hdac enzyme activity in blowfly nuclear extracts , as described in the kit instructions , except that the volumes of all reagents were reduced to give a total assay volume of 27.5  l . control assays were also run in the presence of 1.25  m trichostatin in order to calculate the amount of fluorescent product that was derived from a trichostatin - inhibitable reaction , that is , the amount of product derived from the action of hdac enzymes alone . the assay was performed using a series of at least 4 serially - diluted working solutions of each hdaci . the fold dilutions used to generate each working solution series varied from 2fold to 10-fold , and were set ( based on initial dose - finding experiments ) in order to provide a dose response curve consisting of 46 data points . the % inhibition of hdac activity was calculated for each concentration of hdaci added to the reaction . the enzyme assay dose - response data were analysed with graphpad prism software using non - linear regression , with the variable slope option selected , in order to calculate ic50 values ( with 95% confidence intervals ) representing the concentration of inhibitor required to reduce the hdac activity of the nuclear extract by 50% . we performed a non - parametric ( spearman ) correlation analysis in graphpad prism in order to examine the relationship between the effects of hdaci in inhibiting blowfly larval development and inhibiting nuclear extract hdac enzyme activity . in addition , in order to examine the relationship between the blowfly bioassay data and the reported inhibitory effects of the hdaci against specific human hdac enzymes , we performed a correlation analysis using the bioassay data and ic50 values reported in the scientific literature for the hdaci against human hdac enzymes ( see supplementary table  1 ) . while blowflies are known to possess hdac1 , 3 , 4 , 6 and 11 , ( kotze et  al . , 2015 ) , the analysis was only performed with human hdac1 , 3 , 4 and 6 as insufficient inhibition data was available for an analysis of inhibitory effects on human hdac11 . for the correlation analysis , we grouped hdac 1 and 3 together as class i hdac enzymes , and hdac4 and 6 together as class ii hdac enzymes . forty hdaci compounds were investigated for inhibition of the growth of blowfly larvae , with their activities reported in table  2 as inhibition of larval weight gain and pupation ( g / assay ) . for comparison , the most potent inhibitor of blowfly larval growth was the depsipeptide romidepsin , which was more potent , or as potent as , the commercial insecticides : 10-fold more potent than cyromazine , 2-fold more potent than diflubenzuron , and equipotent with dicyclanil ( table  2 , fig .  1 , fig .  2 ) . the most potent hydroxamic acids were trichostatin , cudc-907 , al179 - 3b and ar-42 : approximately 10-fold less potent than cyromazine , and approximately 50100fold less potent than diflubenzuron and dicyclanil . also showing marked activity ( ic50  <  100  g / assay ) were the thioester compound kd5170 , the disulfide compound psammaplin a ( which is a prodrug that forms a thiolate much like romidepsin ) , and the cyclic tetrapeptide apicidin . many of the compounds , including 13 of the hydroxamic acids , the two fatty acids ( valproic acid and an-9 ) , and the single sesquiterpene lactone ( parthenolide ) showed little or no insecticidal activity ( ic50  >  1000  g / assay ) . comparisons between the larval weight gain and pupation ic50 for the commercial insecticides showed that the two values were within 2-fold of each other . for 7 of the 8 most active hdaci ( larval ic50  <  100  g / assay , fig . 2 ) , the variation between the larval and pupation ic50 values was also within a 2-fold range . the two values were approximately equal for cudc-907 and ar-42 , while within 2-fold for trichostatin , al1179 - 3b , romidepsin and kd5170 . on the other hand , the hdaci were also investigated for inhibition of hdac activity in nuclear extracts from blowfly eggs ( table  3 ) , with representative dose - response curves shown in fig .  3 ( some of the compounds shown in table  1 , table  2 were not examined in nuclear extract assays as insufficient material was available ) . as with the insecticidal assays , romidepsin was the most potent inhibitor of hdac activity . this compound was approximately 600-fold more potent than the second most - active compound , quisinostat , and about 1000-fold more potent than trichostatin . the hydroxamic acids that were the most active in the blowfly larval bioassay were among the most potent enzyme inhibitors ( ic50 0.0160.212  m for trichostatin , cudc-907 and ar-42 ) . a number of hydroxamic acids that were significant hdac enzyme inhibitors in the nuclear extracts ( ic50  <  0.3  m ) had low potency in the larval bioassay ( e.g. panobinostat , givinostat , belinostat : larval ic50 295 , 477 , and 740  g / assay , respectively ) . among the other compounds highlighted above for their insecticidal activity ( from fig . 2 ) , all showed significant potency in inhibiting the hdac enzyme activity of the nuclear extract ( all ic50  <  1  m ) . the relationship between larval bioassay ic50 and nuclear extract hdac inhibition ic50 is shown in fig .  4 ( fig . 4b shows data points with extract hdac inhibition ic50  <  2.0  m only ) . 4a ) , revealed that the two assay parameters were significantly correlated ( spearman correlation coefficients shown on figure  panels ) . despite this , some differences between the two measurements were apparent , with larval weight ic50 values of 1000 ( n  =  14 ) corresponding to a range of nuclear extract activities from 0.032  m ( cudc-101 ) to  >  100  m ( six compounds ) . importantly , low larval weight ic50 values ( < 100 g / assay ) did not occur alongside high nuclear extract ic50 . 4b illustrates this , with the most active insecticidal compounds all being potent inhibitors of hdac activity in blowfly nuclear extracts ( ic50  <  0.5  m ) . we also examined the relationship between published ic50 values for inhibition of human hdac enzymes by the hdaci used in this study with their activity in inhibiting blowfly larval development . the analysis was restricted to just the human hdacs that corresponded to the class i and class ii hdac enzymes present in the blowfly , namely hdac1 and 3 ( class i ) and hdac4 and 6 ( class ii ) . the published data on the inhibition of human hdac11 ( corresponding to the other hdac present in the blowfly ) was not extensive enough with respect to the hdaci examined in the present study ( see supplementary table  1 ) to allow for a separate analysis of this class iv hdac . the relationship between the blowfly bioassay data for each hdaci and the reported enzyme inhibition ic50 values against the class i and ii human hdac enzymes are shown in fig . the two parameters were significantly correlated for the class i enzymes , but not for the class ii enzymes . however , even though a significant correlation existed for class i enzymes across the whole data set , a number of compounds that were potent inhibitors of the human class i enzymes showed no insecticidal activity ( ic50  >  1000 g / assay ) . the present study has examined the ability of a number of known hdaci to inhibit the growth and development of blowfly larvae , and correlated this effect with their ability to inhibit the hdac activity of nuclear extracts prepared from blowfly eggs . there was a significant correlation , suggesting that their insecticidal activity was likely due to the inhibition of blowfly hdac enzymes . romidepsin was a very potent inhibitor of both blowfly larval growth and blowfly hdac activity , the potency being equivalent to or greater than commercial blowfly insecticides . in addition , we have shown that a number of other hdaci have significant insecticidal activity against blowfly larvae , including hydroxamic acids ( trichostatin , cudc-907 , al1179 - 3b , ar-42 ) , a thioester ( kd5170 ) , a disulphide ( psammaplin a ) and a cyclic tetrapeptide with a zinc - binding ketone ( apicidin ) . while these hdaci validate the concept of a potentially valuable new target for insecticides , we are not advocating the use of the particular compounds reported herein as commercial insecticides . they would be too expensive to be economically viable for any livestock or agronomic production setting . moreover , most of the more potent hdaci described are also potent inhibitors of human hdaci ( ic50 nm - m ) and might prove cytotoxic in sheep and unacceptable in terms of human consumption of sheepmeat . hence , while our demonstration of the potent insecticidal activity of a number of hdaci helps to prove the concept that hdaci may be effective insecticides , issues associated with cost of production and target pest selectivity need to be solved next . romidepsin is a prodrug that is first activated by reduction of its disulfide to the free thiol that can then bind to the catalytic zn in hdac enzymes . . the higher potency of romidepsin involves either a highly complementary fit of the conformationally constrained cyclic depsipeptide component of romidepsin with the enzyme , or higher metabolic stability than the hydroxamates . apicidin is another compound with significant insecticidal activity ( table  2 ) which also has a rigid cyclic tetrapeptide component that adds affinity to the relatively weak interaction between its ketone component and zinc . ( 2015 ) found that romidepsin inhibited the growth of asexual stage plasmodium falciparum ( ic50 0.1  m ) , the bloodstream form trypanosoma brucei parasites ( ic50 0.035  m ) , and was a potent inhibitor of hdac enzyme activity in p.  falciparum nuclear extracts ( ic50 0.9  nm ) . in contrast , most hydroxamic acid based inhibitors derive their affinity from zinc chelation which sometimes compensates for a suboptimal fit between the remaining features of the inhibitor and the enzyme active site . the 31 hydroxamic acids examined here have considerable structural diversity and are mostly potent inhibitors of human hdacs . they show quite a range of inhibitory potencies against blowfly larval growth over two log units ( table  2 ) . most of the hydroxamate - based inhibitors were derived from 4-aminopyrimidine or 4-aminobenzene hydroxamic acids , which confer an extended linear shape to the fragment projecting towards zn in the enzyme . other active inhibitors with a linear structure due to an aromatic group in conjugation with a double bond and hydroxamate are the cinnamic acid hydroxamates , panobinostat & pracinostat . the potent suberoylhydroxamates ( al1179 - 3b & pg50 ) have a more flexible linear zinc - binding extension like the similarly flexible but simpler parent compound saha , but exhibit superior activity attributed to their branched capping group that likely makes multiple interactions with the enzyme . pg50 was developed as a selective inhibitor of human hdac6 ( gupta et  al . , 2010 ) , however it seems to be a class i hdaci in the blowfly possibly suggesting its capping groups are too small to influence selectivity as the other hydroxamate inhibitors known to specifically inhibit human hdac6 ( tubacin & tubastatin a ) were inactive in the blowfly bioassay . the reasons why other hydroxamates were inactive is not clear , but they do show how selectivity between highly homologous enzymes can be achieved , in this case away from blowfly and towards human . in principle clues derived from the capping cyclic peptide groups away from the zinc - binding moieties of romidepsin and apicidin may steer the development of new compounds with greater potency and selectivity for the target enzyme to make better and safer insecticides . firstly , effective insecticides must kill , or inhibit the growth of early stage larvae before they can damage the host . secondly , where the initial effects are inhibitory rather than lethal , they must persist over at least several days and then kill the larva to prevent it recovering and developing to damage the host . a comparison of the two bioassay ic50 values is informative with respect to these time course considerations . the commercial insecticides show a pupation ic50 that is similar ( within two fold ) to the 24  h weight gain ic50 , consistent with the larvae not recovering from an initial growth inhibition phase . this was also observed for seven of the eight hdaci highlighted in fig .  2 . apicidin on the other hand showed a pupation ic50 value almost 6-fold greater than the weight gain ic50 , indicating some recovery of larvae after the initial inhibitory effects on growth . a number of compounds showed potent inhibition of the nuclear extract hdac activity , but only low or no activity in the larval bioassay ( for example : nuclear enzyme assay cay10603 ic50 0.165  m , cudc-101 0.0317  m vs larval bioassay ic50 this is likely due to poor uptake or low stability of the compounds in the larval assay . there are likely to be differences between the various compounds examined in terms of uptake across the larval cuticle ( trans - cuticular uptake ) and across the intestinal membranes ( following ingestion ) , as well as access to the cellular target following uptake . some of the compounds are likely to be metabolised to a greater degree than others by the blowfly xenobiotic - detoxification systems , which include esterases ( campbell et  al . , 1997 ) , cytochromes p450 ( kotze , 1993 ) and glutathione transferases ( kotze and rose , 1987 ) . potency against human class i hdac enzymes generally correlated with insecticidal activity , but some potent inhibitors of human class i hdac ( ic50  <  0.10  m ) showed no insecticidal activity . this may be due to factors associated with uptake and stability of the compounds in the bioassay , as well as differences in the intrinsic level of interaction of the compounds with the human enzymes compared to the equivalent blowfly hdac enzymes . ( 2015 ) described some differences in the amino acid residues between the human and blowfly class i hdacs , with catalytic domain amino acids showing 86% and 73% identities between human and blowfly hdac1 and 3 , respectively . the relationship between inhibitory effects of hdaci on human class ii hdacs and their insecticidal activity was poor , with no significant correlation between the two parameters . the catalytic domain amino acids differ to a much greater extent between the human class ii hdacs and their blowfly equivalents compared to the class i comparisons , with % identities of 61% , 47% and 50% for hdac 4 and the two catalytic domains of hdac6 , respectively , between the human and blowfly ( kotze et  al . , 2015 ) . hence , hdaci of human and blowfly class ii enzymes may show a lower level of relatedness than among inhibitors of class i enzymes from the two species . the lack of correlation for class ii hdacs may be favourable for potential identification of more insect - specific hdaci that interact specifically with the blowfly class ii enzymes , while showing less inhibition of the human class ii enzymes . however , more information on the different roles played by the blowfly class i and ii hdac enzymes is required before a preferred target hdac class or individual enzyme can be determined . ( 2006 ) found that rnai mediated silencing of drosophila hdacs 1 and 3 resulted in inhibitory effects on growth curves for drosophila schneider ( s2 ) cell lines , whereas silencing of hdacs 4 , 6 and 11 did not inhibit cell growth , suggesting more important roles for the two class i enzymes in cell viability . ( 2010 ) reported that drosophila hdac6 loss - of - function mutant flies were viable and fertile , suggesting that this enzyme may not be essential for the development of this fly species . in conclusion , the present study shows that hdaci from various chemical groups can substantially inhibit the development of blowfly larvae . in particular , romidepsin was at least equipotent with the major commercial blowfly insecticides , supporting the concept of inhibiting blowfly hdac enzymes to produce new insecticides for preventing infection by sheep blowfly , and to potentially control other insects . there is a great deal of interest currently in developing hdac inhibitors for use in chemotherapy against other human parasitic disease  malaria , toxoplasmosis , trypanosmiasis , schistosomiasis and leishmaniasis ( andrews et  al . , 2014 , kelly et  al . , 2012 , hansen et  al . , 2014 , engel et  al . , 2015 , marek et  al . , 2015 ) a focus of these studies is the identification of hdaci that show selectivity for the parasite hdac enzymes over the human enzymes . similarly , further work on developing hdac inhibitors as potent insecticides could focus on identifying insect - specific inhibitors , but at the very least should focus on producing hdaci that are cheap to manufacture and market as prospective insecticides .
histone deacetylase inhibitors ( hdaci ) are being investigated for the control of various human parasites . here 
 we investigate their potential as insecticides for the control of a major ecto - parasite of sheep , the australian sheep blowfly , lucilia cuprina . 
 we assessed the ability of hdaci from various chemical classes to inhibit the development of blowfly larvae in  vitro , and to inhibit hdac activity in nuclear protein extracts prepared from blowfly eggs . 
 the hdaci prodrug romidepsin , a cyclic depsipeptide that forms a thiolate , was the most potent inhibitor of larval growth , with equivalent or greater potency than three commercial blowfly insecticides . 
 other hdaci with potent activity were hydroxamic acids ( trichostatin , cudc-907 , ar-42 ) , a thioester ( kd5170 ) , a disulphide ( psammaplin a ) , and a cyclic tetrapeptide bearing a ketone ( apicidin ) . on the other hand , no insecticidal activity was observed for certain other hydroxamic acids , fatty acids , and the sesquiterpene lactone parthenolide . 
 the structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity ; for example , among compounds with flexible linear zinc - binding extensions , greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly hdac enzymes . 
 the insecticidal activity correlated with inhibition of hdac activity in blowfly nuclear protein extracts , indicating that the toxicity was most likely due to inhibition of hdac enzymes in the blowfly larvae . 
 the inhibitor potencies against blowfly larvae are different from inhibition of human hdacs , suggesting some selectivity for human over blowfly hdacs , and a potential for developing compounds with the inverse selectivity . in summary , these novel findings support blowfly hdac enzymes as new targets for blowfly control , and point to development of hdac inhibitors as a promising new class of insecticides .