Influence of low temperature on lethal time extension for different life stages of Cryptolestes ferrugineus(Stephens)with strong resistance to phosphine fumigation

Dianxuan Wang*,Judong Hou,Yilin Huang,Jianhua Lyu

Collaborative Innovation Center of Henan Grain Crops,Henan Collaborative Innovation Center of Grain Storage and Security,Henan University of Technology,Zhengzhou 450001,China

Keywords:

Cryptolestes ferrugineus

Phosphine

Stages

Low temperature

Mortality

Lethal time extension

ABSTRACT

Knowing the time extension degree of full mortality in phosphine fumigation at low temperature significantly contributes to successful insect pest control,especially for reducing fumigation failure and inhibiting resistance development.The comparison of mortality and lethal time on eggs,larvae,pupae and adults of Cryptolestes ferrugineus(Stephens)was conducted,and the strain with 1043 times of resistance factor to phosphine was assayed during fumigation with 300 mL/m3 of phosphine concentration at 18,23 and 28°C.The LT50 values to eggs,larvae,pupae and adults of C.ferrugineus at 18°C were postponed by 2,2,1 and 5 d compared with that at 23°C,and 5,4,5 and 7 d compared with that at 28°C,respectively.The LT99 values to eggs,larvae,pupae and adults at 18°C were 5,1,2 and 7 d longer than that at 23°C,and 6,5,5 and 10 d longer than that at 28°C,respectively.The lethal time of different life stages of the C.ferrugineus strain significantly increased with temperature decreasing.The exposure time of full mortality on pupae at 300 mL/m3 of phosphine at 18°C reached 38 d,which was 12 d longer than that of larvae.The order of tolerance of different life stages of C.ferrugineus exposed to 300 mL/m3 phosphine from high to low was pupae,eggs,adults,larvae at tested temperatures.

1.Introduction

As a number of fumigants in stored product protection were abandoned in the past decades,phosphine has been used in the globe for disinfestation of stored grains and other durable commodities for several decades[1-3].Phosphine generated from solid formulations or cylinder is extremely important in world trade and food security due to lack of other available practical substitutes[4].Many factors related to environmental conditions may cause the fumigation failures[5],such as temperature,sealing condition,dosage,concentration,distribution,and so on.Temperature is a key factor to influence the effect of phosphine on killing insects existing in chosen enclosure storage.The exposure periods of full mortality for mixed-age populations ofCryptolestes ferrugineusin 1.0 mg/L of phosphine were 20,15 and 15 d at 25,30 and 35°C,respectively[6].The respiration rate of insects increased significantly with temperature rising.The temperature rising increased the uptake of phosphine and led to higher mortality[7].Insect resistance is another serious problem that affects the effectiveness of phosphine fumigation.It has been reported and quantified for some species of stored insects in many countries of the world[8-14].A number of storedproduct insect species or strains were called“strong resistance”who have been measured and reported,such asRhyzopertha dominica[8],Sitophilusoryzae[15],C.ferrugineus[12],Tribolium castaneum[16]andOryzaephilus surinamensis.The resistance factor ofC.ferrugineusto phosphine had been reported exceeded 1000 folds evaluated by FAO recommended methods in China.The development of insect resistance to phosphine was mostly relative to the failures in practical fumigations[17]due to unsuitable condition control,such as short exposure period,poor sealed enclosures,and so on[18,19].Phosphine resistance in stored grain insects has been reported continually due to lack of proper fumigation operation [20,21], which mainly included insufficient exposure time in a controlled concentration of phosphine.It means that long exposure time is necessary for successful phosphine fumigation on stored-product insects[22].Insects may survive after fumigation for unsuitable exposure time which varies according to temperature,insect tolerance or resistance and other factors.A successful fumigation should aim at killing all life stages of insects with enough exposure time and specific concentration of fumigant[23].Usually,eggs and pupae are more tolerant to phosphine than adults and larvae.Furthermore,a lower temperature may postpone the exposure time of full mortality due to lower metabolism of insects.Pieces of literature about phosphine fumigation against different stages of insects under special conditions have been reported[24].There are still lacks of reports on phosphine fumigation at low temperature,especially for all life stages ofC.ferrugineusbelonging to high resistant strain to phosphine.The economic significance of resistant strains ofC.ferrugineusincreased in the last several decades due to the special development of extremely high resistance to phosphine (resistance factor ＞1000)[12].The difficulty to controlC.ferrugineusincreased absolutely through the development of strong resistance to phosphine[25].A major issue to deal with phosphine resistance ofC.ferrugineuswas understanding the movement of the insects relative to the spatio-temporal distribution of bulk stored grain and any other breeding habitats, where the temperatures varied in different parts of large scale grain mass [25].Some insects can survive during and after fumigation,which may be caused by lacking of enough exposure time at effective concentration and low temperature.A low temperature of grain storage has been developed and used more and more in the last several years in many places,for example,grain was stored at a temperature ＜20°C in China.Knowing the extension degree of exposure time on full insect killing in phosphine fumigation at low temperature could significantly contribute to successful insect pest control and resistance development suppression.The comparison and influence on lethal time for different stages ofC.ferrugineuswith 1043 times of resistance factor at different temperatures of 18, 23 and 28 °C were assayed during fumigation with 300 mL/m3phosphine concentration.

2.Materials and methods

2.1.Test insects

The strain ofC.ferrugineuswas collected from a grain depot located in Xiaoshan,Zhejiang Province,China.They were cultured for many generations in the lab of Henan University of Technology,China with a diet of whole wheat flour,oat,broken wheat and yeast(weight ratio of 5:3:1:1)at 30±1°C and 70%±5%RH.Before the assay,the resistance factor of this strain was 1043 times determined with a modified version of the FAO recommended method.

2.2.Fumigation chamber

The fumigation was carried out in a rectangle chamber(60×35×40 cm)made of metal armor plate(2 mm in thickness)and a transparent top lid(5 mm in thickness)made from Plexiglas.A rubber glove operating opening was set on one vertical side of the chamber.A sampling steel cylinder was inserted from another vertical side of the chamber,and had an open mouth could be sealed by two screwed caps with 80 mm in diameter and 200 mm in length.The insect cages could be taken out through this cylinder during fumigation, thereby avoiding fumigant leaking.Glass cages with 10 mm in diameter and 70 mm in length were used for accommodating insects and diets.While checking mortality at each interval,we could open the screwed cap in the chamber through the rubber glove operation opening,send the insect cages to the chamber and close the screwed cap.Then the cages could be taken out of the cylinder through the screwed cap outside the chamber.The airtightness of the chamber was guaranteed by an airproof mat and a bolt between the rectangle bin and the transparent top during fumigation.The pressure half time at 500 Pa was more than 2 min for the chamber.The phosphine in the chamber could be recirculated and monitored by an electronic phosphine monitor with a pump and two plastic pipes that controlled by valves.The phosphine monitor could detect phosphine concentration in a range of 0-2000 mL/m3and in precision of 1 mL/m3(model HL-210,Jialiang Co.,Ltd.,Beijing,China).Supersaturated solution of sodium chloride was loaded in a 200 mL beaker for maintaining 70%RH on the bottom of the chamber.

2.3.Insect setting and checking

Adults(14-d-old)and last instar larvae were transferred to the cages with 5 g of diet.24-h-old eggs were obtained from the diet that was gotten by adding 1000 adults to wheat folur,culturing for 1 d,and sieving through 80-mesh sieve to remove the adults.The pupae were obtained from last instar larvae cultured after 24 h in a 48-well cell culture plate.Fifty individuals of all test life stages were put into the cages separately.Three replicates were used for each treatment and control.

Insect cages were taken out from the chambers every 5 d and disposed at 30 ± 1 °C and 70% ± 5% RH.The adults and eggs were examined after 14 and 10 d to calculate the mortality,respectively.The last instar larvae and pupae were examined every 5 d until there was no emergence of new adults.

2.4.Operation

The fumigation chamber was kept at 18,23 and 28°C in the lab,respectively.The test insect cages were maintained in the fumigation chamber to adapt the environment for 24 h in advance.After sealing,phosphine gas was injected to the chamber and maintained at 300 mL/m3of phosphine.The controls were treated with no phosphine in same environment.

2.5.Statistical analysis

All experimental data were performed at least three independent replicates and results were presented as mean and standard deviation.One-way analysis of variance with Duncan's post-test was performed using SPSS(SPSS Inc.,Chicago,USA)to evaluate differences between samples at a significance level of 0.05.Excel was used to generate graphs.

3.Results

3.1.The mortality of C.ferrugineus at different fumigation time and temperatures

In the first 5 d,the mortality of adults ofC.ferrugineus,which were 1%at 18°C,7%at 23°C and 15%at 28°C,had significant difference among the three tested temperatures during fumigation with 300 mL/m3of phosphine(Fig.1).Adults ofC.ferrugineuscould be killed easily at high temperature due to higher level of physiological respiration.The mortality of adults increased with fumigation time extending at all tested temperatures.In period of the 20-d fumigation,the mortality of adults reached 41%at 18°C,75%at 23 °C and 98%at 28 °C.After 25 d fumigation,the mortality of adults reached 100%at 28°C,but some survivals existed at 23 and 18°C.After 30 d fumigation,the adults were killed completely at 23 and 28°C while the mortality of adults was 91%at 18°C.The full mortality of adults was found after 35 d fumigation at all three temperatures.

According to Fig.2,the mortality of larvae increased gradually with the extension of fumigation time.The mortalities ofC.ferrugineuslarvae after 5 d fumigation with 300 mL/m3phosphine were 20%at 28°C,5%at 18°C and 11%at 23°C,which showed significant difference among the tested temperatures.The mortality of the larvae at 18°C was significantly lower than that at 23 and 28°C after fumigation for 10 and 20 d.After 20 d fumigation,all tested larvae could be killed at 28°C,and the mortality was 91%and 97%at 18 and 23°C,respectively.Some larvae survived to 25 d at 18°C owing to low temperature.The larvae ofC.ferrugineuscould be killed completely after 30 d fumigation at all tested temperatures.The full mortality of the larvae at 18°C occured 5 and 10 d later than that at 23 and 28°C,respectively.

Fig.1.The mortality of adults of C.ferrugineus at different temperatures after fumigation for different days.

Fig.2.The mortality of larvae of C.ferrugineus at different temperatures after fumigation for different days.

As shown in Fig.3,the mortality ofC.ferrugineuseggs was 3%at 18°C,4%at 23°C and 7%at 28°C after 5 d fumigation with 300 mL/m3of phosphine,indicated that some sensitive eggs could be killed by the fumigation in 5 d.The immobile eggs had lower physiological respiration and less inhaling of phosphine.After 10 d fumigation, the mortality of eggs were 10%at 18°C,and 11%at 23°C,which were significantly lower than that(32%)at 28°C.Phosphine fumigation at higher temperature could result in a significant increase on mortality of the insect in same fumigation time.After 30 d fumigation,eggs ofC.ferrugineuscould be completely killed at 28°C,and the mortalities of eggs were 88%at 18°C and 97%at 23°C.The full mortality time at 18 and 23°C were 35 d,which were 5 d longer than that at 28°C.

According to Fig.4,the mortality ofC.ferrugineuspupae after 5 d fumigation with 300 mL/m3of phosphine was 1%at 18°C,3%at 23°C and 9%at 28°C,respectively.A few of the pupae could be killed after 5 d fumigation,and the pupae mortality was obviously influenced by temperature in a very short fumigation time.The mortality difference influenced by temperature also appeared in the 10,15,20,25 and 30 d of fumigation.The mortality of pupae increased significantly with fumigation time at all tested temperatures,indicated that phosphine could be inhaled by pupae and kill pupae with the fumigation time going on.Except for 35 d and 40 d fumigation,the mortality of pupae ofC.ferrugineusincreased significantly with temperature increasing at 18-28°C in any fumigation duration.All pupae could be killed after fumigation for 35 d at 23 and 28°C and for 40 d at any temperature,but some of them survived at 18°C after 35 d fumigation.

Fig.3.The mortality for eggs of C.ferrugineus at different temperatures after fumigation for different days.

Fig.4.The mortality for pupae of C.ferrugineus at different temperatures after fumigation for different days.

3.2.The LT50 and LT99 values of C.ferrugineus at different fumigation time and temperatures

According to Table 1,the regression equation of mortality(y)on adults at different fumigation time(x)wasy=ax+b,whereinawas 3.844 at 18°C,4.613 at 23°C and 5.680 at 28°C;the mortality went up faster at a higher temperature than that at a lower temperature;the LT50and LT99value of adults increased at a lower temperature;and the LT99value of adults at 18 °C was 1.2 times that at 23 °C and 1.4 times that at 28 °C,respectively.

In the regression equation for larvae,awas 4.054 at 18°C, 5.747 at 23°C and 6.533 at 28°C,larger than that of adults,pupae and eggs.This indicated that the mortality of larvae increased faster than that of adults,pupae and eggs at the same temperature.However,theavalues of pupae and eggs had little difference at same temperature.With the temperature increasing,larvae ofC.ferrugineuswere killed more quickly than adults,pupae,and eggs.The LT50value for larvae at 18°C was 1.1 times that at 23°C and 1.4 times that at 28°C.The LT99value at 18°C was 1.0 times that at 23°C and 1.2 times that at 28°C,respectively.

The order of LT99value for different life stages ofC.ferrugineusat three tested temperatures was pupae ＞eggs ＞adults ＞larvae.The LT99values of all life stages ofC.ferrugineusat 18°C were the largest,indicated that low temperature could indeed extend the full mortality time of phosphine fumigation.This may attribute to low respiration rate of the insects and small amount of phosphine intake at low temperatures.

4.Discussion

Residue survivor of the insect after fumigation was the most possible factor of resistance development to phosphine in stored product pests[14].At a lower temperature environment,it was more difficult to control the insect completely due to the low level of physiological respiration of insects and lower toxicity with a smaller amount of phosphine intake for insects[6].Besides the base of air tightness,insect resistance,species and life stage,extend in full lethal time at low temperature should be considered in phosphine fumigation.The results indicated that all adults were killed after 30 d fumigation with 300 mL/m3of phosphine at the temperature above 23°C,but still 9%of insects could survive at 18°C.More survival at lower temperatures may be a reason for the fumigation failure and resistance development.

The results showed that the survival rate of all stages ofC.ferrugineusat 18 and 23°C was higher than that at 28°C,separately.The order of tolerance to phosphine of different stages ofC.ferrugineuswas pupa,egg,adult and larva,from high to low.The eggs and pupae may have more survival opportunities in fumigation and become the dominant power of insect resistance development.The survival in phosphine fumigation at a lower temperature increases the probability of resistance development,especially for eggs and pupae.

Table 1 The LT50 and LT99 values of C.ferrugineus at different temperatures.

In all resistant strains of six beetle species, includingO.surinamensis,T.castaneum,C.ferrugineus,R.dominica,S.oryzaeandS.grunarius,the most tolerant stage to phosphine was pupae[24].It was consistent with our result that the pupa was the most tolerant stage to be controlled by phosphine.The top target ofC.ferrugineuscontrol by phosphine fumigation should be killing all pupae.In practical fumigation,the fumigation time should be postponed for several days at low temperature even through the full mortality was checked during the fumigation by means of insect cage monitoring and checking.Taking this strain ofC.ferrugineus(1,043 of resistance factor)for example,the LT99results showed that the full mortality time of pupae at 18°C should be prolonged for at least 6 d than that at 28°C.

5.Conclusions

The mortality ofC.ferrugineusfumigated with 300 mL/m3phosphine at three temperatures was different significantly.The tolerance order of different stages ofC.ferrugineuswas pupa,egg,adult and larva,from high to low.The full mortality time of the high resistance strain ofC.ferrugineusat 18°C should be prolonged for at least 2 d and 6 d than that at 23°C and 28°C,respectively.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by National Key Research and Development Program of the 13th Five-Year Plan(No.2017YFC1600800).