Studies on the toxicity of phosphine to tolerant stages of Trogoderma granarium Everts (Coleoptera: Dermestidae)

Tests were conducted at 20 and 30°C at 60% r.h. to find the dosages of phosphine needed for control of diapausing larvae and eggs of Trogoderma granarium. At 20°C, 60% r.h., some larvae from each of four recently acquired field strains of T. granarium survived a 5-day exposure period at a concentration-time (Ct) product of 164 g hr/m³. Under these conditions laboratory stock larvae were killed by a 4-day exposure period of about 120 g hr/m³, and eggs by a 3-day exposure of about 50 g hr/m³. Eggs, aged 0–1 days, proved the most tolerant stage at 30°C, 60% r.h., surviving a Ct-product of 16 g hr/m³ over a 2-day exposure period. At 30°C the diapause of larvae became unstable and their tolerance of phosphine was low. Adults emerging after fumigation of larvae appeared normal and if sufficient numbers emerged together a second generation was produced.Complete control of larvae of several stocks (about 20 weeks in diapause) was achieved within a 4-day exposure at 20°C with a mixture of methyl bromide (2.0 g/m³) and phosphine (1.4 g/m³), whereas 6 days were required for these stocks using phosphine alone. To ensure elimination of all stages of non-resistant T. granarium, exposures to phosphine should last 7 days at 20°C and 4 days at 30°C.     

The toxic action of phosphine: Role of carbon dioxide on the toxicity of phosphine to Sitophilus granarius (L.) and Tribolium confusum DuVal

Carbon dioxide was found to potentiate the action of phosphine against a normal strain of Tribolium confusum and normal and resistant strains of Sitophilus granarius so that the length of the exposure period could be reduced. Studies on respiration showed that there was a 20 per cent increase in oxygen consumption in the presence of 4% carbon dioxide but no further increase up to 64% carbon dioxide level. Phosphine uptake on the other hand increased steadily with increase in carbon dioxide level and there was a concurrent increase in toxicity up to three-fold. Carbon dioxide enhanced toxicity when applied simultaneously with phosphine but not when applied before or after phosphine treatment.     

Sorption of tritiated phosphine by various stages of Tribolium castaneum (Herbst)

When various stages of Tribolium castaneum were fumigated with 0.14–1.69 mg/l of tritiated phosphine for 5 hr the uptake of the fumigant was found to be considerably greater in larvae and adults than in pupae or eggs. Mature larvae absorbed 2–3 times as much phosphine as pupae at all concentrations tested and mortality was appeciably higher. Uptake by 1-day-old adults was about two fold that of the mature pupal stage from which they had emerged and increased with age so that 14-day adults absorbed about twice as much as newly emerged adults. Uptake by pupae followed a U-shaped curve somewhat similar to the characteristic pattern of oxygen uptake by pupae of different ages. Eggs retained only a small proportion of the radioactive fumigant they absorbed. A considerable amount remained at the surface of the eggs and was converted to soluble compounds that could be washed off with water. The results suggest that the higher tolerance of both eggs and pupae to phosphine may, at least partially, be attributed to the lower uptake of fumigant as compared to larvae and adults.     

The tolerance of developmental stages of four stored product moths to phosphine

The tolerance to phosphine of different stages of Ephestia elutella, E. kuehniella, E. cautella and Plodia interpunctella was followed closely at 10–30°C. Eggs of all species were highly tolerant for the first 30–45% of the developmental period, but all other stages were much more susceptible. Maximum exposure periods permitting some survival over a practical range of concentrations were 1 day at 30°C, 2 days at 25°C, 3–4 days at 20°C, and 5–8 days at 15°C. Some eggs of E. elutella survived the very high CT products of 142 mg h/l. in a 2-day exposure at 25°C, and 288 mg h/l. in an 8-day exposure at 15°C. In a 3-day exposure at 25°C, and a 9-day exposure at 15°C, CT products of about 4 and 20 mg h/l. respectively were sufficient for complete control of all species. Eggs of P. interpunctella were susceptible to low temperature and survived exposures to phosphine at 15°C less well than other species. Survivors from fumigated eggs were of normal fertility.Although much less tolerant than eggs, younger pupae, especially those of E. kuehniella, were more tolerant than larvae or older pupae. The fertility of adults of normal weight emerging from surviving pupae was markedly depressed.In most fumigation experiments, differences in tolerance between laboratory and wild stocks were small. However, wild stocks tended to be more tolerant of cold than laboratory stocks, and survived fumigation better in long exposures at lower temperatures.     

大黑粉盗与赤拟谷盗和锈赤扁谷盗对磷化氢的耐受力比较研究

潜在危害的储粮害虫大黑粉盗Cynaeus angustus研究缺乏,掌握其磷化氢耐受力有助于科学治理。采用快速击倒和FAO推荐方法测定了磷化氢对大黑粉盗的KT50值和毒力方程,并与赤拟谷盗Tribolium castaneum和锈赤扁谷盗Cryptolestes ferrugineus进行了比较,测定了100、200、300、400、500 mL/m3磷化氢模拟熏蒸中3种害虫卵、幼虫、蛹和成虫不同时间的死亡率。磷化氢对大黑粉盗、赤拟谷盗和锈赤扁谷盗的KT50值分别为7、127和3 736 min,相应毒力方程斜率值b为3.67、8.28和9.94,相应LC50值为0.008、1.34和5.88 mg/L。害虫不同虫态在100~500 mL/m3浓度下的半数致死时间LT50值于大黑粉盗卵为4~2 h、幼虫4~1 h、蛹4~1 h、成虫3~1 h,于赤拟谷盗为卵12~5 d、幼虫11~5 d、蛹13~7 d、成虫10~4 d,于锈赤扁谷盗为卵28~13 d、幼虫为18～9 d,蛹26～11 d,成虫17~9 d。相应的完全致死时间（LT100）于大黑粉盗卵为21~6 h、幼虫8~3 h、蛹9~6 h、成虫5~3 h,于赤拟谷盗卵为21~18 d、幼虫21~15 d、蛹27~18 d、成虫21~12 d,于锈赤扁谷盗卵为54~30 d、幼虫42~30 d、蛹48~30 d、成虫36~25 d。所测大黑粉盗为磷化氢敏感品系,其各虫态对磷化氢的耐受力为卵>蛹>幼虫>成虫,其耐受力远小于赤拟谷盗和锈赤扁谷盗磷化氢抗性品系。     

Effect of low temperatures on the rate of respiration and uptake of phosphine in different life stages of the cigarette beetle Lasioderma serricorne (F.)

Studies were undertaken to establish the effects of low temperatures on the rate of respiration in larvae and adults of a susceptible strain of the cigarette beetle Lasioderma serricorne. The results showed that there was a consistent decrease in the rate of respiration in L. serricorne with a decrease in temperature from 25°C to 5°C. The rate of respiration was slightly affected by circadian rhythm in adult beetles, but not in larvae.The levels of phosphine taken up by different life stages of the beetle were also determined using [³²P]-radiolabelled gas. A comparison between susceptible and phosphine resistant strains showed that the mechanism of resistance in L. serricorne involves a reduced uptake of the fumigant.The uptake of [³²P]-phosphine by different life stages of resistant L. serricorne declined with a decrease in temperature from 25°C to 5°C. Similarly, the uptake by susceptible L. serricorne decreased from 25°C to 10°C, but there was a comparatively higher uptake at 5°C and consequently a higher mortality at the end of the exposure. The significance of the findings in relation to control strategies is discussed.     

The toxicity of methyl chloroform to stored product insects

The susceptibility to methyl chloroform of all developmental stages of Cryptolestes ferrugineus, Ephestia kuehniella, Oryzaephilus surinamensis, Rhyzopertha dominica, Sitotroga cerealella, Sitophilus granarius, S. oryzae and Tribolium confusum, diapausing larvae of Ephestia elutella, E. cautella, Plodia interpunctella and Trogoderma granarium and adults of Tribolium castaneum, was studied. Tests were conducted at 15, 20 and 25°C, 55–75% r.h. with concentrations ranging from 25 to 200 mg/l and exposure times from 8 h to 16 days. The toxicity of methyl chloroform was in general similar to that of carbon tetrachloride. Immature stages of R. dominica, S. cerealella and Sitophilus spp, and diapausing larvae of T. granarium, showed the highest levels of tolerance. Methyl chloroform was more effective at the higher temperatures but a concentration of 50 mg/l was below the threshold level for full toxicity against 5 species at 25°C. Depending on the species concerned, the concentration × time (CT) product required for control varied from 3,700 mgh/l at 25°C to 60,000 mgh/l or more at 20°C. At 15°C fewer species were tested but five out of nine survived CT products in the region of 30,000 mgh/l. There was no appreciable difference between the susceptibility of the immature stages of malathion-resistant and susceptible strains of O. surinamensis. Neither were any consistent differences observed between adults of normal susceptibility and those resistant to methyl bromide or phosphine.     

Proteomic assessment of resistance to the fumigant phosphine in the lesser grain borer, Rhyzopertha dominica (F.)

A previous publication compared a strain of the stored-grain pest beetle, Rhyzopertha dominica, resistant to the fumigant phosphine, with a susceptible strain using two-dimensional polyacrylamide gel electrophoresis. Certain proteins appeared to differ and the authors proposed that arginine kinase in particular might be used as a marker for the rapid monitoring of resistance in this species and other pests of stored grain. Here an expanded set of susceptible and resistant strains is surveyed using the two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) system. Of hundreds of spots only two showed a significant difference between the resistant and susceptible strains and the magnitudes of those differences were less than three-fold. Arginine kinase spots were identified but they did not differ significantly. The proposal that certain abundant proteins of R. dominica might be used as markers of phosphine resistance was not supported by the present study.     

Effect of diet on phosphine toxicity,rate of development and reproduction of the rice weevil Sitophilus oryzae (Linnaeus)

One of the loci responsible for strong phosphine resistance encodes dihydrolipoamide dehydrogenase (DLD). The strong co-incidence of enzyme complexes that contain DLD, and enzymes that require thiamine as a cofactor, motivated us to test whether the thiamine deficiency of polished white rice could influence the efficacy of phosphine fumigation against insect pests of stored grain. Three strains of Sitophilus oryzae (susceptible, weak and strong resistance) were cultured on white rice (thiamine deficient), brown rice or whole wheat. As thiamine is an essential nutrient, we firstly evaluated the effect of white rice on developmental rate and fecundity and found that both were detrimentally affected by this diet. The mean time to reach adult stage for the three strains ranged from 40 to 43 days on brown rice and 50–52 days on white rice. The mean number of offspring for the three strains ranged from 7.7 to 10.3 per female over a three day period on brown rice and 2.1 to 2.6 on white rice. Growth and reproduction on wheat was similar to that on brown rice except that the strongly resistant strain showed a tendency toward reduced fecundity on wheat. The susceptible strain exhibited a modest increase in tolerance to phosphine on white rice as expected if thiamine deficiency could mimic the effect of the dld resistance mutation at the rph2 locus. The strongly resistant strain did not respond to thiamine deficiency, but this was expected as these insects are already strongly resistant. We failed, however, to observe the expected synergistic increase in resistance due to combining thiamine deficiency with the weakly resistant strain. The lack of interaction between thiamine content of the diet and the resistance genotype in determining the phosphine resistance phenotype suggests that the mode of inhibition of the complexes is a critical determinant of resistance.     

Comparison of the mitochondrial proteomes of phosphine-susceptible and -resistant Tribolium castaneum

Various lines of evidence suggest that mitochondria might be the site of constitutive differences between coleopteran pests of stored products with and without resistance to the fumigant phosphine. In this study two-dimensional polyacrylamide gel electrophoresis with Differential In-Gel Electrophoresis (DIGE) was used to compare soluble proteins from the mitochondria of phosphine-susceptible and -resistant Tribolium castaneum. Eighty-five spots were aligned across all nine gels and a further 111 across all but one gel. Each gel displayed the proteome from a susceptible strain, a resistant strain and a standard made by mixing aliquots from all experimental samples. No significant differences were observed between resistant strains and strains susceptible to phosphine. However, proteins of lower abundance and membrane proteins were not detected so important resistance-associated differences might yet be detected by more exhaustive techniques.     

Efficacy of phosphine fumigation for different life stages of Trogoderma inclusum and Dermestes maculatus (Coleoptera: Dermestidae)

Despite the importance of dermestid beetles as targets for stored product protection, including the protection of museum artifacts and animal-based products, there are only a few published reports regarding their susceptibility to phosphine fumigation, in contrast with other major stored product insect species. In the current study, we evaluated phosphine against all life stages of Trogoderma inclusum LeConte, the larger cabinet beetle, and Dermestes maculatus (DeGeer), the hide beetle. There were two series of laboratory bioassays; in the first series the concentrations were 0 (control), 50, 100, 200 and 600 ppm, and in the second series the concentrations were 0 (control), 50, 150, 300, 400, 500 and 600 ppm. Both series were carried out on a 5-day insect exposure protocol. The results for both species clearly indicated that eggs were by far the least susceptible life stage, followed by pupae, while most adults and larvae were killed at the 50-ppm concentration. Concentrations between 300 and 400 ppm could be utilized to provide 100% mortality for both species and all life stages. To our knowledge, our results are the first that have provided data regarding efficacy of phosphine for the control of T. inclusum and D. maculatus. Resource managers can utilize our results to more efficiently target these dermestids with specific concentrations of phosphine, depending on the target life stage.     

Response of mixed-age cultures of phosphine-resistant and susceptible strains of lesser grain borer, Rhyzopertha dominica, to phosphine at a range of concentrations and exposure periods

Mixed-age cultures, containing all life stages, of a highly resistant strain (Strong-R) of lesser grain borer, Rhyzopertha dominica (F.), were exposed to a series of fixed concentrations of phosphine at a range of exposure periods at 25 °C. A susceptible strain and a less-resistant strain (Weak-R) were also tested. The aim was to characterise the resistant strain and determine if it could be controlled with phosphine. Times to population extinction (TPE) were recorded and lethal time (LT_99.9) values calculated. The relationship between exposure period t and phosphine concentration C for the resistant strains were: for Strong-R LT_99.9C^0.5457t=3.852 and TPE C^0.6105t=4.0404 and for Weak-R LT_99.9C^0.3553t=3.6521 and TPE C^0.4507t=3.4833. The results were used to define a range of minimum exposure period ×concentration protocols for control of the Strong-R populations. For example, at 1.0, 0.3, and 0.2 mg l⁻¹ complete control can be expected in 5, 10 and 14 days, respectively. This information will be used to recommend phosphine rates for field trial and eventual registration.     

Inheritance of resistance to malathion,DDT and dieldrin in Plodia interpunctella (Lepidoptera: Pyralidae)

Diagnosis of heterogeneous resistance in a field strain of Plodia interpunctella enabled selection to be made of a susceptible and three resistant strains with resistances specific to malathion, DDT or dieldrin respectively. Resistance in F1 larvae from crosses between susceptible and resistant moths was intermediate for each resistance. Diagnostic doses were used to determine the mode of inheritance for the three resistances. Tests on larvae from F2 crosses and repeated back-crosses showed that resistance to malathion was due to a semi-dominant gene. Tests on F2 and first back-cross larvae suggested that resistance to DDT and dieldrin was also explicable in terms of distinct single, semi-dominant genes.     

Variation in the response of Callosobruchus maculatus (F.) to a resistant veriety of cowpea

A Nigerian cultivar of the cowpea has previously been identified as being less suitable than conventional varieties for the development of a local strain of C. maculatus (F.). The survival and duration of development of the immature stages of three strains of C. maculatus in the dried seeds of this cultivar were compared with the performance of the same strains of beetle on a susceptible variety. In all beetle strains, fewer larvae survived to produce adults, and the duration of development was significantly extended, in the resistant cowpea cultivar. A Brazilian strain produced fewest adults and a Nigerian strain had the longest mean development period. Combining all the data, it appeared that a strain from the Yemen Arab Republic was least influenced and that from Brazil most adversely affected by the resistant cowpea. Evidence is presented indicating that factors in both the cowpea seeds and the beetles themselves may contribute to the poorer performance of bruchids in the resistant as compared to the susceptible variety.     

Active exclusion of phosphine as a mechanism of resistance in Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

A strain of Rhyzopertha dominica which is resistant to the fumigant phosphine, was found to absorb less radioactive phosphine than a susceptible strain. The non-absorption of phosphine was enhanced by increased temperature and carbon dioxide. Living resistant insects absorbed less toxin than dead insects, and metabolic detoxication did not appear to contribute to resistance. Since respiratory activity in the presence of the gas is known to be unaffected in the resistant insects, a resistance mechanism based on exclusion of toxic gas is proposed.     

Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic

This study first estimated the current state of phosphine sensitivity (using a knock-down/KT₁₀₀/Degesch kit) in Sitophilus granarius (23 strains) and Tribolium castaneum (8 strains) in Czech Republic grain stores. The resistance of S. granarius (21.7% resistant strains; coefficient of resistance KT₁₀₀ ranged from 0.5 to 2.3 among strains) was substantially lower and less frequent than that of T. castaneum (87.5% resistant strains; coefficient of resistance KT₁₀₀ ranged from 0.9 to 52.5 among strains). The phosphine efficacy of the laboratory and field (i.e., resistant) pest strains was validated during commercial fumigation when suboptimal tarpaulin sealing resulted in low-concentration phosphine exposure (Ct products ranged from 5.9 to 7.4 g*hr/m³). Although even low-dose fumigation led to 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum, the mortality of the field strain of T. castaneum ranged from 47% to 95%. Larval emergence from the fumigated commodity samples with pest eggs was zero or near zero for laboratory strains, while 1.3–6.0 (S. granarius) and 63.7–80.00 (T. castaneum) field-strain larvae emerged per sample (100 g). This study shows that although a high proportion of the tested pest populations were still sensitive, several T. castaneum populations showed an elevated level of resistance that may decrease field fumigation efficacy, especially under suboptimal phosphine dosage conditions.     

The effect of diapause on the tolerance of larvae of Ephestia kuehniella to methyl bromide and phosphine

Larvae from a stock of Ephestia kuehniella Zeller, recently collected from an unheated flour store in southern England, were exposed at 10 and 15°C to two fumigants commonly used for the control of this species in the U.K. Various exposure periods were tested and the mortality of larvae which had entered diapause was compared with that of non-diapausing larvae.

Diapause increased the tolerance of larvae to methyl bromide and phosphine at low temperatures. The minimum dosage of methyl bromide for complete control of diapausing larvae was about 80 mgh/l during a 20-h exposure period at 10°C and less than 70 mgh/l during a 16-h exposure at 15°C. Complete kill of diapausing larvae at 10°C was obtained after a 4-day exposure to phosphine with a CT product of 8.1 mgh/l, but a slighly higher dosage was required in a test at 15°C.     

Variations in the refuge-seeking behaviour of four strains of Cryptolestes ferrugineus (stephens) (Coleoptera: Cucujidae) at different temperatures

The refuge-seeking behaviour of insecticide resistant and susceptible strains of Cryptolestes ferrugineus (Stephens) was investigated in the laboratory by confining adults in arenas containing a refuge filled with wheat. Three strains had been collected from grain stores and mills, and the fourth (Insectary) had been reared in constant conditions for over 25 years. The effect of temperature on behaviour, in the range 15–30°C, was also examined. The Insectary strain was particularly affected by low temperatures with far fewer adults being recorded in the refuge at the end of the 2-week test period compared to other strains. Differences in refuge-seeking behaviour between strains increased with decreasing temperature, and were greatest below 20°C; this corresponds with the temperatures at which grain is usually stored in Britain and in other countries of temperate climate. These differences in behaviour appear to be unconnected with the biochemical/physiological resistance of the strains to malathion.     

Respiration of Tribolium castaneum (Herbst) at reduced oxygen concentrations

Adults, eggs, young and old larvae and pupae of Tribolium castaneum (Herbst) were exposed to atmospheres containing 1%, 2%, 3%, 5%, 10%, and 15% oxygen in nitrogen at 30°C and 70% r.h. Respiration rates were determined with a gas chromatograph. The oxygen intake and carbon dioxide output by insects were expressed in μl/insect/h or μl/mg/h.

Adults exposed to 21% oxygen required an initial acclimatization period of at least 5 h, after which the respiration rate remained stable. Based on this finding, all the respiration measurements were carried out after an initial adaptation of insects to the respirometer conditions for 24 h.

Respiration of eggs, young and old larvae, pupae, and adults at 30°C in normal atmospheric air was at rates of 0.0121, 9.25, 8.45, 1.45, and 4.67 μl CO₂/insect/h, respectively. Respiration rates of the same stages in terms of insect weight were 0.32, 29.08, 3.33, 0.59 and 2.37 μl CO₂/mg insect/h, respectively. At reduced oxygen levels respiration rates of eggs, larvae and pupae were proportional to the oxygen levels. Adult respiration was higher for 3% and 5% oxygen than for normal atmospheric air with rates of 4.77 and 4.98 μl CO₂/insect/h, respectively. In adults, RQ values for the same oxygen levels were also higher than for normal atmospheric oxygen and were 1.07 and 1.18, respectively.     

The effects of high concentrations of carbon dioxide in air on Trogoderma granarium Everts (Coleoptera: Dermestidae)

Several developmental stages of a laboratory strain of Trogoderma granarium Everts were subjected to an atmosphere of 60% carbon dioxide (CO₂) in air at 20 or 30°C and 60% r.h. for varying lengths of time. The eggs, pupae and adults all died within 6 days, but some larvae survived for 16 days or more at both temperatures. Larvae were then exposed at 45, 60 and 75% r.h. to 45, 60 or 75% CO₂ in air at 20 or 30°C and additionally to 30% CO₂ in air at 30°C. Mortality increased with CO₂ concentration and temperature. However, only when a CO₂ concentration of 75% at 30°C was applied did all larvae die and, even then, a 15-day exposure was required. Some survival occurred after 18 days in all other conditions. Exposure times of 10 days or less, at temperatures of 20°C or above, have been recommended for use with high CO₂ atmospheres for the control of stored products pests but this investigation shows that these may be inadequate if T. granarium is present.