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Donahay e, E.J., Nav arro, S. and Leesch J.G. [Eds.] (2001) Proc. Int. Conf. Controlled Atmosphereand Fumigation in Stored Products, Fresno, CA. 29 Oct. - 3 Nov . 2000, Executive PrintingServices, Clovis, CA, U.S.A. pp. 79-89 METABOLISM OF CARBONYL SULFIDE TO HYDROGEN SULFIDE IN
INSECTS IS CATALYSED BY CARBONIC ANHYDRASE
Stored Grain Research Laboratory, CSIRO Entomology, Canberra, ACT 2601, Australia Carbonyl sulfide (COS) is a new fumigant under development as a methyl bromidereplacement for fumigation of durable commodities. COS has been shown to be relativelyfast acting and toxic to a broad range of stored-product pests but little is known of themechanism of toxicity of COS to insects. In rats, COS is metabolised to hydrogen sulfideby the enzyme carbonic anhydrase, a widely distributed family of enzymes that usuallycatalyse the reversible hydration of carbon dioxide. The present study investigated whetherhydrogen sulfide was the toxic agent of COS in insects. Firstly, adult stored-productinsects Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica(F.) were fumigated with hydrogen sulfide for 6 h at a range of concentrations from 1-50mg L-1. The percentage mortality of insects was ≥84% at concentrations of 5 mg L-1 andabove suggesting hydrogen sulfide is potently toxic to insects. To determine the role ofmetabolism in COS toxicity, 17-21 day old larvae of T. castaneum were raised on culturemedium containing carbonic anhydrase inhibitors, acetazolamide or methazolamide, atconcentrations up to 20 mg g-1 media. Larvae raised on inhibitors and untreated larvaewere then fumigated with 60 mg L-1 COS for 5 h, a concentration - time product that hadbeen shown to produce approximately 90% mortality in untreated larvae. The mortality oflarvae raised on acetazolamide-containing medium (20 mg g-1) was much lower thanuntreated larvae (40% versus 95%) after fumigation. Similarly, mortality was reduced ininsects administered methazolamide (20 mg g-1) resulting in 53% mortality.
Administration of carbonic anhydrase inhibitors to insects protected them from the acutetoxicity of COS. This work demonstrates that the acute toxicity of COS to insects isdependent on carbonic anhydrases metabolism to hydrogen sulfide, the toxic agent ofCOS.
Carbonyl sulfide (COS) is a new fumigant under development as a methylbromide replacement for fumigation of durable commodities (Desmarchelier,1994). COS is relatively fast acting toward a broad range of stored-product andtimber pests (Desmarchelier, 1994; Plarre and Reichmuth, 1997; Zettler et al.
1997). These authors also report that the egg stage is the most tolerant stage ofthe common stored product insects to COS. The lethal concentration - timeprofile for COS fumigation over a range of time, concentration and temperature conditions has been characterised for both adult and egg stages of the rice weevil,Sitophilus oryzae (L.) (Weller and Morton, 2001).
Although the effective concentration range for fumigation of stored-product insects has been established, little is known of the mechanism of toxicity of COSin insects. In rats, COS administered by injection of the gas into the peritoneumwas lethal at doses of 20 mg kg-1 and above; COS was converted to hydrogensulfide which was detected in the blood (Chengelis and Neal, 1980). However,when rats were pretreated with the enzyme inhibitor acetazolamide (at 200 mg kg- 1) prior to administration of COS, they were protected from the lethal effects ofCOS. Acetazolamide is a specific inhibitor of carbonic anhydrases - an enzymegroup that catalyses the reversible hydration of carbon dioxide (CO ) (Maren and Sanyal, 1983). Therefore it appeared that the acute toxicity of COS in the rat wasa result of its metabolism to hydrogen sulfide and by inhibiting its metabolismwith acetazolamide, COS toxicity was prevented.
Carbonic anhydrases are ubiquitous enzymes. They are found in vertebrates, plants, microorganisms and insects (Tashian, 1989). The usual substrate forcarbonic anhydrases is CO , which is metabolised with the addition of water to bicarbonate and hydrogen ion; an essential reaction for maintaining pH balance,transport of CO and production of ions for secretory fluids in animals (Tashian, 1989). The presence of the same family of enzymes in rats and insects impliesthat a similar metabolic pathway for COS to that in rats may occur in insects.
Support for this notion includes the finding that whole body homogenates ofadult S. oryzae and larval Ephestia kuehniella (Zeller) catalysed the formation ofhydrogen sulfide when exposed to COS (Haritos, unpublished data). However, itis not clear whether the toxicity of COS to insects is due to its metabolism tohydrogen sulfide. This paper describes the role of carbonic anhydrases in thetoxicity of COS and the toxicity of the proposed metabolite, hydrogen sulfide tostored-product insects.
Chemicals
COS (97.5%) and hydrogen sulfide (99%) were supplied by BOC Gases Australia
Ltd. (Chatswood, Australia). The purity of COS with respect to hydrogen sulfide
was checked using a GowMac gas density balance combined with a Tracor
MT150 gas chromatograph and found to be <1%. Acetazolamide and
methazolamide were purchased from Sigma Chemical Co. (St Louis, MO, USA).
Insect culturing
Tribolium castaneum (Herbst) were cultured on wholemeal flour containing
brewers yeast (10% w/w) and maintained in controlled temperature rooms at 30°C
and 60% r.h. Early 3rd-4th instar larvae (17-21 d) or adult insects were used in
experiments. Adult S. oryzae were obtained from cultures reared on soft wheat,
and Rhyzopertha dominica (F.) from cultures raised on soft wheat containing
flour at 25°C and 65% r.h.
Hydrogen sulfide toxicity to insects
Fifty adult T. castaneum, R. dominica and S. oryzae were placed in separate glass
jars without media in a 2.5 L glass desiccator fitted with a gas sampling port and
septum. Hydrogen sulfide was added by gas-tight syringe, after removal of the
corresponding volume of air, to achieve a final concentration of 0, 1, 5, 25 or 50
mg L-1. Nominal concentrations of hydrogen sulfide were used in the experiment.
There was a single desiccator for each hydrogen sulfide concentration. After a 6
h exposure, the desiccators were aired in a fume cabinet. Culturing medium was
added to each jar, then the jars were covered and returned to culture rooms.
Mortality was assessed 7 d after fumigation.
Determination of COS toxicity to larval T. castaneum
The acute toxicity of COS to T. castaneum larvae (approximately 3rd-4th instar)
was tested over the concentration range of 50 to 80 mg L-1 for a duration of 3 to
5 h. Larvae (50) and culture medium were added to glass jars and placed in glass
desiccators (2.5 L). The required volume of COS was added by gas-tight syringe
through a septum port in the lid. The concentration of COS in each desiccator
was measured by gas chromatography at the beginning and end of the exposure
period. Each concentration/time interval for COS was tested in duplicate. After
fumigation, the culture medium and insects were aired for at least one hour in a
fume cabinet and then returned to culture rooms at 30°C, 60% r.h. Insect
mortality was assessed 1, 7 and 21 d after fumigation.
Effect of dietary enzyme inhibitors on COS toxicity
In the initial screening experiment, 100 T. castaneum larvae per treatment were
placed on culture medium (11 g) containing acetazolamide at 0, 1, 2.5 or 5 mg
g-1 medium. Acetazolamide had been mixed by hand as a dry powder with flour
and yeast. The insects were raised on the medium for 4 d at 30°C and 60% r.h.
Each acetazolamide concentration was tested in duplicate. All cultures werefumigated with COS at 80 mg L-1 for 4 h in glass desiccators fitted with gassampling ports, and each desiccator held control and inhibitor-fed insects. Gasconcentrations were measured by gas chromatography at the start and end of theexperiment. One treatment group of larvae (2.5 mg acetazolamide g-1 medium)was placed on fresh media without inhibitors before fumigation to determinewhether the inhibitors themselves had any direct effect on mortality duringfumigation. After fumigation the jars were aired, insects placed on fresh mediaand returned to culture rooms. Mortality was assessed 7 d and 21 d followingfumigation.
In the second experiment, 100 T. castaneum larvae per treatment were placed in glass jars containing culture medium (11 g) and acetazolamide (0, 0.5, 1, 2 and4 mg g-1 medium) for 3 d at 30°C, 60% r.h., in duplicate. The untreated and inhibitor-fed insects were exposed to 60 mg L-1 COS for 5 h. Gas concentrationswere measured by gas chromatography at the start and end of the experiment.
After fumigation the jars were aired and insects placed on fresh media andreturned to controlled temperature and humidity rooms. Mortality was assessed 5d following fumigation.
In the final acute exposure, 100 T. castaneum larvae were raised on medium containing 0, 10 or 20 mg g-1 of acetazolamide or methazolamide for 36 h mixed in half the original amount of medium (5.5 g medium). The cultures were thenfumigated with 60 mg L-1 COS for 5 h. The fumigated containers were aired andthe larvae were placed on fresh medium (11 g) and returned to the culture room(30°C, 60% r.h.) for 5 d after which mortality was assessed.
In a separate experiment designed to investigate the effect of carbonic anhydrase inhibitors during a longer fumigation period (24 h), 100 T. castaneumlarvae per treatment were cultured on media containing acetazolamide (0 or 2 mgg-1 medium) for 2 or 3 d. This was followed by fumigation with 15, 20 or 25 mgL-1 COS in replicate experiments. Gas concentrations were monitored at the startand end of the experiment. The mortality of larvae was assessed 4 d after airingand removal of insects to fresh media.
Analysis of COS by gas chromatography
The concentration of COS in the vessels was measured by gas chromatography
using a Tracor MT-220 instrument fitted with a flame photometric detector. The
samples were injected onto a 6 ft glass column packed with HayeSepQ 80/100
mesh operated at an oven temperature of 100°C, with the injector and detector
temperature at 150°C. The concentration of COS in each vessel was calculated
from a calibration curve prepared over the range 40-80 mg L-1 (typically
achieving an r2 = 0.989) or between 1-30 mg L-1 for the lower exposure groups
(r2 = 0.992). According to this analysis, the loss of COS from the fumigation
chambers during the exposure periods was negligible.
Hydrogen sulfide toxicity
Hydrogen sulfide was found to be acutely toxic and fast acting towards adult
stored-product insects as most were killed at concentrations between 5 and 25 mg
L-1 for 6 h. The percentage mortalities of the insects exposed to hydrogen sulfide
over the concentration range of 0-50 mg L-1 are given in Table 1. S. oryzae was
the most tolerant insect among the three species tested. There were no signs of
agitation from the insects treated with hydrogen sulfide and they appeared
sedated. By observation of the insects through the glass chamber, the 50 mg L-1
concentration appeared to kill the insects very quickly, within 2 min of addition
of gas. At 25 mg L-1 hydrogen sulfide, the insects had ceased moving within 5
min and at 5 mg L-1, appeared to be dead within 15 min.
Adult insect mortality (percentage) following exposure to various concentrations of hydrogen sulfide for 6 h. Mortality assessed 7 d after fumigation Mortality (%) R. dominica T. castaneum Determination of COS toxicity to larval T. castaneum
The mortality of T. castaneum larvae exposed to COS was determined at a range
of concentrations and short exposure times to find a concentration and time (Ct)
product of COS that would result in approximately 90% mortality. These
findings were used in subsequent experiments. In Fig. 1, the percentage mortality
of larvae exposed to COS at a range of Ct products from 162 to 365 mg h L-1 are
given by a sigmoidal-shaped curve. The Ct product that gave 86% mortality was
315 mg h L-1. Fumigation of 60 mg L-1 COS for 5 h (300 mg.h L-1) was used in
successive experiments.
COS (mg.l/h)
Fig. 1. The relationship between mortality of T. castaneum larvae and the concentration-timeproduct of COS; concentrations between 50 and 80 mg L-1 and exposure periods of 3 to 5 h.
Effect of dietary enzyme inhibitors on COS toxicity
T. castaneum larvae were raised on a diet containing acetazolamide at
concentration levels up to 5 mg g-1 for 4 d with no mortality or obvious
detrimental effect on the insects. In the initial COS fumigation, untreated larvae
exposed to COS at 80 mg L-1 for 4 h resulted in 99% mortality. Larvae raised on
a diet containing acetazolamide at 1 to 5 mg g-1 media had reduced mortality(73-79%) compared with untreated larvae. In one treatment group, larvae wereraised on acetazolamide (2.5 mg g-1) for 4 d then placed on fresh media for thefumigation. The mortality following COS fumigation in this sample was 77%,close to the mortality obtained for other acetazolamide treated groups.
After exposure to 60 mg L-1 COS for 5 h the percentage mortality of untreated larvae was 95% but the mortality of the acetazolamide-treated larvaewas reduced by the presence of inhibitor in the diet (Fig. 2). The reduction inmortality was proportional to the amount of inhibitor at concentrations of 1 mgg-1 and below but appeared to level off at the higher concentrations. Mortalityfollowing fumigation with COS was reduced by approximately one third in larvaefed with acetazolamide-containing diet (> or equal to 1 mg g-1) compared withlarvae raised on a normal diet. This result was reproduced in replicatedexperiments (data not shown). A group of T. castaneum larvae that were raisedon acetazolamide in the diet (2.5 mg g-1, 3 d) but not fumigated with COS,showed no signs of toxicity and developed normally after returning to culturemedium.
Acetazolamide (mg/g diet)
Fig. 2. Effect of dietary administration of acetazolamide (0.5-4 mg g-1) on the percentagemortality of T. castaneum larvae exposed to COS (60 mg L-1, 5 h) compared with a controldiet.
In a further experiment, T. castaneum larvae were raised on a diet containing higher concentrations of carbonic anhydrase inhibitors (10 or 20 mg g-1) and themortality after exposure to COS was compared with larvae raised on a controldiet. Larvae raised on either acetazolamide or methazolamide were protectedfrom the lethal effects of COS fumigation as shown in Fig. 3. Fifty-five percent of the T. castaneum larvae raised on the highest dietary level of acetazolamidewere protected from a lethal COS exposure. In a similar result, up to 42% of Inhibitor (mg/g diet)
methazolamide (20 mg g-1) treated larvae were protected from COS toxicity.
Fig. 3. Protection of T. castaneum larvae from the acute toxicity of COS (60 mg L-1, 5 h) byadministration of carbonic anhydrase inhibitors, acetazolamide and methazolamide in the dietcompared with larvae raised on a control diet.
The effect of dietary acetazolamide on the toxicity of COS during a 24 h fumigation was investigated in T. castaneum larvae. The larvae were raised oncontrol medium or medium containing acetazolamide (2 mg g-1) for 2 or 3 dprior to COS fumigation at 15 or 20 mg L-1. The mortality of larvae raised onuntreated media and then exposed to COS (15 mg L-1) was 87% but foracetazolamide-treated larvae, mortality was reduced by 17-23% (Table 2). Larvaeraised on acetazolamide-containing medium for 3 d experienced slightly highermortality when fumigated with COS in comparison with those raised onacetazolamide medium for 2 d.
In all experiments where T. castaneum larvae were raised on a diet containing carbonic anhydrase inhibitors and then fumigated with COS, the surviving larvaewere placed on culture medium and appeared to recover fully and developnormally into adult insects.
Mean mortality of T. castaneum larvae fumigated with COS for 24 h and the effect of dietary exposure to acetazolamide Dietary acetazolamide COS is highly toxic to all stages of stored-product insects with the exception ofyoung eggs which appear to be the most tolerant (Desmarchelier, 1994). As afumigant COS can be effectively used in short-term fumigation using highconcentrations or lower concentrations for longer exposure periods (Weller,1999). The current work examines the basis for the toxicity of COS to insects.
Homogenates of stored-product insects have been shown to metabolise COS tohydrogen sulfide in vitro but it was not known whether hydrogen sulfide was thetoxic agent of COS and to what degree metabolism was involved in COS toxicity.
This question was approached by first confirming that hydrogen sulfide was toxicto stored-product insects (Table 1). Hydrogen sulfide is highly toxic to animalsas it has a high affinity for metallic ion-containing proteins, particularlycytochrome c oxidase, a key component of cellular respiration. It is thought thatthe inhibition of this enzyme is the main acute toxic action of hydrogen sulfide(Beauchamp Jr et al. 1984). It is highly likely that hydrogen sulfide is acting bya similar toxic mechanism in insects.
The next step was to determine the role of metabolism in COS toxicity to insects, particularly the role of carbonic anhydrase which was known from thework of Chenglis and Neal (1980) to catalyse the conversion of COS to hydrogensulfide in rats. This question was approached by administering inhibitors ofcarbonic anhydrase to insects, followed by fumigation with COS, and monitoringthe effect on mortality or development. Acetazolamide, a crystalline sulfonamidederivative, is one of the most widely used inhibitors of carbonic anhydrases but itis only moderately diffusible through tissues and has low water solubility (Marenand Sanyal, 1983). A structurally related compound, methazolamide, isconsidered a superior inhibitor for experimental work, as it is more diffusiblethrough tissues, it inhibits two major forms of carbonic anhydrase equally and isslowly excreted from animals compared with acetazolamide (Maren and Sanyal,1983; Maren, 1991). In homogenised tissues of tobacco hornwormacetazolamide is a potent inhibitor of carbonic anhydrase activity at concentrations in the nanomolar range (Jungreis et al. 1981). Drosophilamelanogaster and D. hydei administered acetazolamide by injection or mixedwith the diet showed a significant decrease in potassium, magnesium and chloridein the cytoplasm of Malpighian tubule cells, a function that is supported bycarbonic anhydrases (Wessing et al. 1997).
There are several methods of introducing inhibitors of carbonic anhydrase into insects such as through incorporation with food or water or by injection of asolution into the body or by inhalation of a gas or aerosol. One benefit of thedietary route of administration is that the cuticle remains intact unlikeadministration by injection where it is punctured. This is of particular relevancefor fumigation where disruption of the insect cuticle may give rise to higher gaspenetration and toxicity. In the present study, larval T. castaneum were selectedfor the dietary administration of enzyme inhibitors as the larvae are activelyfeeding and the inhibitor was easily mixed into the culture medium. No obviousadverse effects were observed in the larvae raised on carbonic anhydraseinhibitors in the diet although prolonged exposure to the inhibitors maydetrimental to the insects.
T. castaneum larvae that were given carbonic anhydrase inhibitors in the diet were protected against the acute lethality of COS; the percentage mortality ofuntreated larvae fumigated with COS was more then double that of larvae givendietary acetazolamide (20 mg g-1) (Fig. 3). This result suggests that the mainmechanism of COS toxicity is through the toxic action of the metabolitehydrogen sulfide. Complete protection of insects from toxicity of COS by dietaryadministration of inhibitors would be difficult to achieve because individualinsects probably consume different amounts of food containing inhibitor and theamount required to inhibit the carbonic anhydrases of insects is not known.
Dietary administration of methazolamide was not as effective as acetazolamide inprotecting larvae from the toxic effects of COS (Fig. 3). The reason for this resultis not clear. Methazolamide would be expected to diffuse more easily in theinsect and cause more potent, longer lasting inhibition of carbonic anhydrasesbased on its action in mammals (Maren and Sanyal, 1983).
In longer exposures to COS, larvae fed on a diet containing acetazolamide were moderately protected from the toxicity (Table 2). The level of protectionobserved was lower than for insects fed a similar amount of inhibitor in the dietbut fumigated with 60 mg L-1 COS for 5 h (Fig. 2). The difference could be dueto the larvae becoming moribund or ill from COS exposure and no longerconsuming the food containing the inhibitor, and as acetazolamide is rapidlyeliminated from the body of animals (Maren 1991), protection was not continuedthroughout the exposure period. Alternatively, carbonic anhydrase metabolism ofCOS to hydrogen sulfide may not be the only mechanism of toxicity to insects inlonger-term exposures.
The knowledge of the mechanism of COS toxicity can be used to explain aspects or make some predictions of insect response to the fumigant. Insectspecies or life stages that are tolerant to COS toxicity, such as the egg stage ofS.!oryzae, may have a reduced capacity to metabolise COS to hydrogen sulfide.
The probability of resistance forming in insects to hydrogen sulfide (from COS)is expected to be low because of the known mechanism of toxicity of hydrogensulfide. Its action is to potently inhibit metabolic respiration and this mechanismis likely to require a major metabolic shift to result in resistance. Deletion ofcarbonic anhydrases in mutant insects, as a possible mechanism of resistance, would most likely be non-viable as these enzymes perform essential endogenousfunctions in most organisms. The target site of COS toxicity in insects is differentto that of protectants and phosphine. In the case of phosphine, the formation ofreactive oxygen radicals as a result of its interaction with the electron transportchain is the likely mechanism of toxicity (Chaudhry, 1997; Hsu et al. 1998). Themechanism of phosphine resistance in insects is thought to be via active exclusionfrom the respiratory system combined with a detoxication process (Chaudhry,1997). These resistance mechanisms appear to be exclusive to phosphine ininsects as Chaudhry (1997) found methyl phosphine to be more toxic to resistantthan to susceptible insects. Also, from what is known of the resistancemechanisms of protectants, for example increased esterase activity or alteredacetylcholinesterase in organophosphate resistance (Guedes et al. 1997;Hemingway, 2000) or altered sodium channel structure in pyrethroid resistance(Williamson et al. 1996), it is unlikely that cross-resistance would occur betweenCOS and protectants.
In summary, when fed enzyme inhibitors in their diet T. castaneum larvae were protected from the acute lethal effects of COS in vivo and this findingsupports the view that the metabolism of COS to hydrogen sulfide forms theultimate toxic agent of COS, hydrogen sulfide. Future efforts will be focussed onimproving the understanding of COS tolerance in young insect eggs with anunderstanding of the mechanism of COS toxicity to insects.
The technical assistance of Greg Dojchinov and Gaye Weller from the StoredGrain Research Laboratory is gratefully acknowledged, as is the financialassistance of the Grains Research and Development Corporation and the Partnersto the SGRL Agreement. The author is grateful to Peter Annis and Gaye Wellerfor their comments on the manuscript.
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