Efficacy of phosphine and insect penetration ability in ZeroFly® bags

Journal of Stored Products Research 82 (2019) 81e90

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Efficacy of phosphine and insect penetration ability in ZeroFly® bags Grace O. Otitodun a, Moses O. Ogundare a, Shekinat K. Ajao b, Samuel I. Nwaubani a, Grace I. Abel a, George P. Opit c, *, Georgina Bingham d, Mobolaji O. Omobowale e a

Durable Crop Research Department, Nigerian Stored Products Research Institute, P.M.B. 1489, Ilorin, Nigeria Department of Zoology, University of Ibadan, Ibadan, Nigeria c Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK, 74078-3033, USA d Vestergaard Frandsen SA, Chemin Messidor 5-7, 1006, Lausanne, Switzerland e Department of Agricultural and Environmental Engineering, University of Ibadan, Ibadan, Nigeria b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 November 2018 Received in revised form 18 April 2019 Accepted 27 April 2019 Available online 17 May 2019

The deltamethrin incorporated woven polypropylene ZeroFly® storage bag is a promising novel technology for grain storage. However, if grain stored in ZeroFly bags gets infested and has to be fumigated using phosphine (PH3), data on the effectiveness of such treatments are needed. Additionally, obtaining field data on ability of stored-product insect pests to breach ZeroFly bags would facilitate insect management. Therefore, efficacy of PH3 in immature and adult Sitophilus zeamais (Motschulsky), Prostephanus truncatus (Horn), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) in experimental cages in maize stored in 100-kg polypropylene (PP), jute and ZeroFly bags was investigated. Postfumigation mortality of adults was recorded after 7 d, and after 7 wk for immatures. The ability of either S. zeamais or P. truncatus to penetrate fabric of PP, jute and ZeroFly bags was assessed. Phosphine efficacy was good in all the three types of bags and resulted in complete mortality of adults and immatures of the four species tested. Sitophilus zeamais and P. truncatus were more successful in penetrating the PP bag fabric and on average made 84 and 780 holes per bag over a 4 mo-period, respectively; this was followed by jute with 37 and 614 holes. The ZeroFly bag was harder to breach and
3 holes per bag were made for both species. This study shows that PH3 is highly efficacious in insects that infest maize stored in ZeroFly bags, and that these bags are not easily penetrated by stored product insect pests. Hitherto, ZeroFly bags are a good technology for storing grain that is not infested, and fumigation using PH3 can be effectively conducted if infestation occurs. Therefore, ZeroFly bags can be incorporated in integrated stored product insect management (IPM) programs for bagged grains. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Bag fumigation Sitophilus zeamais Prostephanus truncatus Rhyzopertha dominica and Tribolium castaneum

1. Introduction Various methods are used to prevent insects from attacking stored products. Phosphine or hydrogen phosphide (PH3) gasproducing metal phosphides are widely used for fumigation of stored grain against insect pest infestation throughout the world. Metal phosphides react with moisture to produce PH3 and are available in solid formulations of aluminum phosphide (AlP) or Magnesium phosphide (Mg3P2). Because PH3 is highly toxic, it is a restricted-use pesticide that should be used only by trained and certified applicators in accordance with label instructions. Maize (Zea mays L.) is a major cereal that is cultivated and

* Corresponding author. E-mail address: [email protected] (G.P. Opit). https://doi.org/10.1016/j.jspr.2019.04.006 0022-474X/© 2019 Elsevier Ltd. All rights reserved.

consumed in Nigeria. Initially it was a subsistence crop but has gradually become a more important crop, now rising to the category of a commercial crop on which many agro-based industries depend on as a raw material. In Nigeria, insect pests associated with maize losses are Sitophilus zeamais (Mostchulsky) (Coleoptera: Curculionidae), Sitophilus Oryzae (L.) (Coleoptera: Curculionidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae), Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), Sitotroga cerealella (Olivier) (Lepidoptera: Gelechidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloidae). Several measures are used for insect pest management but each comes with its own challenges and limitations. For example, use of synthetic insecticides is associated with hazards and limitations such as contamination of the environment, creation of toxic residues in stored grains, development of

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resistance in target species, lethal effects on non-target organisms, direct toxicity to users, health hazards, high cost of the insecticides and application of insecticides by people with inadequate skills (Adedire and Lajide, 2003; Adedire et al., 2011). Storage of maize in bags such as jute or polypropylene (hereafter referred to as PP) bags is common in many developing countries. Jute bag is made from jute fibre obtained from stems of jute plant (kenaf); it is 100% bio-degradable. Jute is one of the most versatile natural fibers that has been used in raw materials for production of good quality fabrics and sacks (Pan et al., 1999). Woven polypropylene is created when strips or threads of polypropylene are woven together in two directions (warp and weft) resulting in a lightweight but strong and durable material suited for holding and storing agricultural commodities for extended periods of time. In Nigeria, polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC) and jute materials are frequently used for packaging agricultural products, whereas the Zerofly bag is being gradually introduced into the market. Ease of PH3 entry into these materials is critical in ensuring successful insect control. Permeability and dispersal of PH3 can vary depending on the material and its thickness. Several studies on the permeability of PH3 have been conducted (Allahvaisi and Safaralizade, 2010; Hassan et al., 2016; Kengkanpanich et al., 2018). Allahvaisi et al. (2010) investigated PH3 permeability in PP, PE and PVC at two thickness levels (16.5 and 29.0 mm) and found that polypropylene (PP) was best for PH3 permeability, while PE with thickness of 16.5 mm performed worst as it had highest insect infestation level. Similarly, Hassan et al. (2016) found that insect mortality during PH3 fumigation occurred more in PP and PE packaging and least in PVC packaging, though package thickness levels of 0.02 and 0.04 mm examined were said to play a marginal role in PH3 permeability. Contrastingly, Marouf and Momen (2004) showed that package thickness had a significant role in mortality of insects and insect mortality was higher in thinner packaging due to enhanced PH3 penetration. Generally, for the studies conducted, there seems to be unanimity regarding the fact that an ideal polymer packaging should be one which is mostly resistant to penetration and invasion by different storage insect pests as well as allow maximum fumigant gas penetration for high mortality of storage insect pests. Polypropylene packaging appears superior to the rest of the fabrics in this respect (Hassan et al., 2016). The rampant use of jute and PP bags for storage in most developing countries prompted Vestergaard SA, Lausanne, Switzerland to develop a Deltamethrin incorporated polypropylene bag called the ZeroFly® storage bag (hereafter referred to as ZF) (Vestergaard, 2014). When used according to manufactures recommendations, the bag can reduce postharvest losses of cereal grains and grain legumes stored in it (Anankware et al., 2014). Deltamethrin is incorporated in the individual yarns of the ZF bag fabric and is slowly released onto the surface of the yarn in a sustained manner (Vestergaard, 2014). Recent studies show that ZF bags exhibit complete protection against insect pests during paddy storage at ambient conditions (Wasala et al., 2016). Also, Okonkwo et al. (2016) reported that ZF bags were more effective in reducing infestation by stored product insect pests and maintaining grain quality than untreated PP bags containing permethrin-treated grain. Studies conducted by Kavallieratos et al. (2017) showed that more than 98% insect knock down occurred after 1 h of exposure to ZF bags, and significantly higher insect mortality was observed on the outside of the ZF bags compared to the inside. Therefore, ZF bags effectively limit penetration by stored product insects. Paudyal et al. (2017) showed the ZF bag fabric was effective against stored product insect pests and caused 99% knockdown within 3 h, increased adult insect mortality, significantly suppressed progeny production and no insects were

able to penetrate the fabric. Furthermore, the ZF bag was effective in suppressing insect population levels, reducing insect damaged kernels (IDK) and maize weight loss compared to the control, hence beneficial for grain storage (Paudyal et al., 2017). However, there is a dearth of information on efficacy of PH3 against insects in commodities stored in ZF bags. Additionally, there is lack of quantitative field data on the ability of insects to penetrate ZF bags. Therefore, we evaluated efficacy of PH3 in insects that infest maize stored in ZF bags and the ability of insects to penetrate these bags. 2. Materials and methods 2.1. Evaluating efficacy of PH3 in insects that infest maize stored in ZF bags 2.1.1. Experimental sites This experiment was conducted during the period MarcheJune 2017 at Nigerian Stored Products Research Institute Headquarters (NSPRI HQ), Ilorin, Kwara State, Nigeria. Maize for this experiment was stored in 100-kg jute, PP and ZF bags. Stack fumigation was conducted inside a building with four large windows (1.1 m  1.1 m) and two doors (0.90 m  2.0 m) to facilitate safety during fumigation. 2.1.2. Maize Freshly harvested dried maize used for this experiment on efficacy of PH3 in insects that infest maize stored in ZF bags was obtained from Ijaye Farm Settlement in Akinyele Local Government Area, Ibadan Oyo State, Nigeria. The SWAN 2 maize variety was used; this variety is widely grown in the Southwest of Nigeria. Moisture content of the maize used was determined on-farm as 10.5% using a John Deere moisture meter (Manufactured by AgraTronix™; Moisture Check Plus™, USA for Deere and Company; Batch SW08120). The maize was properly cleaned but not fumigated or treated with any protectant insecticide prior to use. 2.1.3. Insects To assess the efficacy of PH3 in insects that infest maize stored in ZF bags, P. truncatus, R. dominica, S. zeamais and T. castaneum were cultured on maize in the Entomology Laboratory at NSPRI HQ. For each species, 50 live adults of mixed sex that were ~1 mo-old were placed on 100 g of diet in cylindrical-shaped plastic dishes (6.5 cm height x 6.5 cm diameter) to mate and lay eggs, i.e. to initiate new infestation (immatures). The diet for S. zeamais, R. dominica and P. truncatus was whole maize kernels while cracked maize was used as diet for T. castaneum. The plastic dishes were then kept inside a Gallenkamp cooled incubator (Model: IR211GA, marketed by Heath Works Beasley'sr, Sunbury on Thames TW16 6AS, United Kingdom) for 14 d. The 50 adult insects placed inside each plastic dish were the F1 generation of insects obtained from laboratory insect cultures maintained at 30 ± 2  C and 56 ± 3% RH at NSPRI HQ. For each species, 18 plastic dishes were set up per replication, and hence 72 containers per replication for the four species tested. Therefore, in total, for the three replications of the experiment, there were 216 plastic dishes. Relative humidity and temperature in the incubator were 58.2 ± 5% and 30 ± 2  C, respectively (GENERAL wireless digital thermo-hygrometer, Model d EMR963HG, 433 MHz, General Tools Instruments, Secaucus, NJ 07094). All adult insects placed in plastic dishes to initiate infestations in them were sieved out after 14 d. Sieving was conducted using a US Standard #10 sieve (2.0-mm openings; Seedburo Equipment Chicago, IL). Thereafter, 30 live insects out of the 50 adults sieved out from each plastic dish were placed on a new 100-g maize or cracked maize portion (depending on species) in a labeled cylindrical cage (19 cm height x 9 cm

G.O. Otitodun et al. / Journal of Stored Products Research 82 (2019) 81e90

diameter) (referred to as cages hereafter) made of flexible copper wire mesh with 150-mm openings. The cages were sealed at the open end using long twine ropes that were 110 cm long to allow for easy retrieval from 100-kg bags of maize. In total, there were 216 cages for adult insects. For each species, 18 cages were set up per replication, and hence 72 cages per replication for the four species tested. Therefore, in total, for the three replications of the experiment, there were 216 cages used for adult insects. In the case of testing immatures of the four insect species used for this experiment, diet used was the 100-g of maize, where the 50 adults had previously been placed for 14 d. The diet of each species was accordingly poured into the copper wire cages from the plastic dishes, and each of these cages too had a 110-cm twine rope tied to the mouth. In total, there were 216 cages for immatures. 2.1.4. Insect cages Flexible copper wire mesh was cut with scissors and then folded accordingly to make each cylindrical cage (19 cm  9 cm) to hold 100 g of maize. The mesh openings of the copper wire mesh were 150 mm and small enough to prevent escape of insects but large enough to allow free movement of air. To assess efficacy of PH3 in immatures and adults of the four species of insects that infest maize stored in ZF bags, 144 cylindrical-shaped cages were used for each replication, i.e. 72 cages for adult insects and the same number for immatures. 2.1.5. Experimental set up To assess efficacy of PH3 in insects that infest maize stored in ZF bags, 100-kg maize-filled jute, PP and ZF bags were used. There were two treatments, a stack of bags that was fumigated and one that was not fumigated (the control). In this study, applying or not applying PH3 is referred to as “treatment”. The stack of 100-kg bags that was fumigated had 12 bags as did the control. Each stack comprised six jute bags, three PP bags and three ZF bags. In the case of jute bags, despite each stack having six bags, insects to be tested were placed in only three of these bags, i.e. in each of the stacks, three bags of each type had insects to be tested d the extra three jute bags maintained the shape and stability of the stack. During stacking, the bags were randomly arranged on wooden pallets using the randomization tool in Microsoft Excel (2013 Version). Four insect cages were placed in each bag and each cage contained 30 live adult insects of just one species. Insect species tested were P. truncatus, R. dominica, S. zeamais and T. castaneum. For assessing efficacy of PH3 in immature insects that infest maize stored in the three types of bags, the set up used was similar to that for adults except that the cages contained 100-g lots of diet of the four species that was presumed to harbor immature stages d refer to description above for how the 100-g lots of diet were obtained. Therefore, for each replication, 72 cages were used for live adults and 72 for immatures. This means that for adults and immatures, there were a total of 216 cages in each case. This experiment had three temporal replications which were set up at 3-wk intervals. Each replication had three sub-replications. Data on insect infestation level, insect damaged kernels (IDK) per 250-g maize samples, moisture content and seed germination were collected before and after fumigation. 2.1.6. Fumigation procedures The twelve 100-kg-bag stack that was fumigated was first covered with thick tarpaulin sheets (12 m length x 2.1 m width) that had 56-m long strong nylon ropes (ID: 6756390648, Shree Ram Poly Packs, Karnataka, India) attached to their ends to facilitate pulling-off of tarpaulins and uncovering of the stack after fumigation. The ropes were pulled from outside the building where fumigation was conducted, through the windows. The stacked bags were fumigated using aluminium phosphide (AlP) tablets

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(Celphos®) (Excel Crop Care Limited, Kutch, Gujarat, India) under sealed tarpaulin covers as already stated. Number of tablets used was calculated based on manufacturer's recommendation of 2e3 tablets/m3 for bagged grains. Given that the volume of the twelve 100-kg-bag stack was 3.146 m3, seven tablets were placed singly inside disposable Petri dishes and distributed under the stack. Small plastic vials (5 cm height x 5 cm diameter) containing water were also placed in different locations under the stack to increase relative humidity in the fumigation enclosure and hence ensure prompt decomposition of the tablets. Assuming complete decomposition and no leakage, the estimated highest concentration of PH3 in the fumigated stack is 1575 ppm. The edges around the tarpaulin cover were then properly secured using sand snakes (plastic bags filled with sand and were 170 cm length x 10 cm diameter) to minimize escape of PH3 gas from the fumigation enclosure. Fumigation lasted 7 d after which the tarpaulins were pulled off from outside the building through windows, using ropes that were attached to the tarpaulin covers to uncover and aerate the stack. Aeration of the stacks lasted 2 d. 2.1.7. Post-fumigation procedures After aeration of the fumigated stack, all cages in both the bags that were fumigated and those in the control stack were retrieved and adult insect mortality was determined by counting live and dead insects within 24 h. Live adults were kept inside white cylindrical-shaped plastic dishes (6.5 cm height and 6.5 cm diameter) covered with muslin cloth and were again checked after 7 d for delayed mortality count. In the case of cages that contained immatures, 100-g diet contents of each cage from the fumigated and control bags was poured into dishes (6.5 cm height and 6.5 cm diameter) and incubated for 7 wk at 30.8 ± 2  C and 66.8 ± 5% RH to determine survival of immatures after the of fumigation. After 7 wk, the number of adults in each dish was counted. In order to assess the viability of seeds at the beginning and end of the experiment, a germination test was conducted for each of the six bags, of each type, in the fumigated and control stacks that had insect cages. From each 100-kg bag, a 1-kg portion of maize was taken and divided into quarters. Samples were obtained using a 1.2m open-ended Trier (grain probe) (Seedburo Equipment, Chicago, IL). Out of each quarter, a small lot of maize was taken to obtain 250 g from which 120 seeds were randomly selected for the germination test. Twenty seeds were placed on moistened filter paper (Whatman No.1) in each of six 9-cm disposable Petri dishes that were arranged on a laboratory bench and moistened every 3 d with 10 ml of water. The number of seeds that germinated was recorded after 7 d. Percentage seed germination was calculated using the formula of Adedire and Akinkurolere (2005) provided below.

Germinationð%Þ ¼

Number of germinated seeds  100 Total number of seeds planted

2.1.8. Statistical analyses Statistical analyses were performed with SAS Version 9.4 (SAS Institute, Cary, NC). Data analyzed were numbers of adult S. zeamais, R. dominica, P. truncatus, and T. castaneum from cages that had contained immatures after 7 wk of incubation, in only the control stack., i.e. numbers of adults that were found in white cylindrical-shaped plastic containers after 7 wk of incubation in the stack that was not fumigated. The contents of these plastic dishes were from cages containing diets that had immature insects and had been placed inside jute, PP and ZF bags that were not fumigated. Analysis of data for only bags in the control was justified by

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the fact that in bags that were fumigated, no live adults of all the four species were found in all cages when final insect counts were conducted 7 d after fumigation, and similarly no adults were found in plastic containers after 7 wk of incubation. Effects of bag type on number of adults were assessed using Analysis of Variance methods (ANOVA) conducted by PROC MIXED. Data were also analyzed to determine effects of fumigation treatment and bag type on germination. The design used for analysis was a Randomized Complete Block Design with sub replication. PROC MIXED was used for ANOVA to determine the effects of fumigation and bag type on germination. Where necessary, analyses of the numbers of insects were conducted with the use of a square root transformation. A square root transformation was used to correct for heterogeneous variances and the lack of normality of the count response variable. 2.2. Evaluating the ability of insects to penetrate ZF bags 2.2.1. Experimental sites This second experiment was conducted in three different buildings (hereafter referred to as locations) that were no closer than 180 m from each other on the premises of NSPRI HQ. A single replication of the experiment was set up in each location. This experiment was conducted during the period JuneeSeptember 2017. A single replication was set up in each location, and there was no subreplication. 2.2.2. Maize The maize for this experiment was also obtained from Ijaye Farm Settlement in Akinyele Local Government Area, Oyo State, Nigeria. The variety, moisture content, and preparation of the maize before use were similar to what is described above for the experiment to evaluate efficacy of PH3 in insects that infest maize stored in ZF bags. 2.2.3. Insects To assess the ability of adult insects to penetrate ZF bags, S. zeamais and P. truncatus were cultured on maize inside 300 ml kilner jars (culture jars). For each species, 100 live adults (~1 moold) of mixed sex were placed on 250 g of maize in individual kilner jars to mate and lay eggs to initiate infestation, the kilner jars were covered with 150-mm mesh muslin cloth to allow air movement and prevent insects from escape. Thereafter, the culture jars were placed on metal trays (42 cm  31.6 cm) that had a thin layer of oil to prevent cross infestation. There were 12 jars for each insect species and these were reared in the Entomology Laboratory maintained at 30 ± 2  C and 60 ± 5%. After 14 d, all adult insects of each species were sieved out of each jar using a US Standard #10 sieve and the contents of the jars (media) kept to obtain F1 progeny used for this study. 2.2.4. Experimental set up To assess the ability of S. zeamais and P. truncatus adults to penetrate ZF bags, 100-kg maize-filled jute, PP, and ZF bags were used. This experiment consisted of three replications which were set up the same day. There was no subreplication. Nine 100-kg capacity bags filled with maize were used for each replication of this experiment. Each replication consisted of maize stored in three jute bags, three PP bags and three ZF bags. For the three bags of each type, one out of the three bags contained maize infested with 100 live unsexed S. zeamais, the second bag of maize was infested with 100 live unsexed P. truncatus and the third bag was not artificially infested with any insects. Each bag of maize was inserted in a 120-kg capacity white muslin cloth bag sewn at NSPRI HQ. The outside muslin bag permitted easy detection and monitoring of the number of holes made by insects every month. For each replicate,

the nine bags were randomly arranged in horizontal manner on wooden pallets with a space of about 25 cm between bags. The number of holes made by insects was determined every month by counting and marking all holes made during that particular month with a marker of a specific color as a way of making sure the holes were not counted again in future. Marking was accomplished by drawing a small circle around the hole on the muslin cloth. The number of holes made by insects, percentage of insect damaged kernels (%IDK) per 250 g maize, number of live and dead insects per 250 g maize, grain moisture content, temperature and relative humidity inside the bags were all monitored every month.

2.2.5. Maize sampling and data collection During this experiment to assess the ability of S. zeamais and P. truncatus adults to penetrate ZF bags, maize samples were obtained using a 1.2-m open-ended Trier (grain probe) (Seedburo Equipment, Chicago, IL). Samples were taken twice from each 100kg bag. Each trier sample weighed ~350 g hence a total of 700 g was taken from each bag during each sampling event. A 3-cm wide opening was made at seam area of each bag to facilitate insertion of the trier during sampling. The incision made on the bag was sealed using tape (Duct Tape™, distributed by ShurTech Brands, LLC Avon, OH 44011, USA) and this facilitated easy opening and closing of the bags during subsequent sampling events.

2.2.6. Insect damaged kernels Percentage of insect damaged kernels (IDK) by numerical basis (IDKnb) was calculated based on 250-g maize sub-samples that were obtained from 700-g samples taken from each bag every month. The 250-g sub-sample was poured on a tray and all kernels were examined using a hand lens (10 magnifications). Kernels with insect exit holes were separated from the undamaged kernels and the numbers and weight of kernels in each category was recorded. Insect damaged kernels were weighed using an electronic top loading balance (Accuris Instruments, Model: W3300e10K; S/N: 10000103, 10000103, Kingwood, Texas, U.S.A). The percentage of insect damaged kernels was calculated using the method of Quitco and Quindoza (1986):

%IDKðnbÞ ¼

Nd  100 Total grain count

Where, Nd ¼ Number of damaged grain

2.2.7. Weight loss From each 250-g sub-sample previously referred to above, percentage weight loss (%WL) was determined. Damaged kernels with insect exit holes and undamaged kernels were sorted out separately, weighed and their weights recorded. Percentage weight loss was calculated using the count and weigh method (FAO, 1992):

%Wtloss ¼

ðWu  NdÞ  ðWd  NuÞ  100% Wu  ðNd þ NuÞ

Where. Wu ¼ Weight of undamaged grains Nu ¼ Numbers of undamaged grains Wd ¼ Weight of damaged grains Nd ¼ Numbers of damaged grains

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2.2.8. Seed germination The procedures for assessing the viability of seeds at the beginning and end of the experiment were similar to those described above except that the 120 randomly selected seeds were from the 700-g samples taken from a single bag.

2.2.9. Statistical analyses To assess ability of insects to penetrate ZF bags, data were analyzed by type of initial infestation, i.e. initial infestation using S. zeamais, P. truncatus or the control (bags not artificially infested at the start of the experiment). Effects of bag type were assessed using PROC MIXED for ANOVA. A repeated measures model in a randomized complete block design was utilized, with location as the blocking factor and month as the repeated factor. An autoregressive covariance structure was used to model the correlations within bag type and across months. Analyses of the numbers of live insects and puncture holes made by insects were conducted with untransformed data because a correction for heterogeneous variances and the lack of normality of the count response variable was not warranted. The simple effects of bag type given month were assessed with protected planned contrasts (SLICE option in an LSMEANS statement). In the case of percent IDK by number (% IDKnb) and percent weight loss (%WL), data analyses were conducted with the use of an arcsine transformation to stabilize variances but untransformed percentages are reported. Data were also analyzed to determine effects of bag type and initial infestation on germination. The design used for analysis was a RCBD with sub-replication. PROC MIXED was used for ANOVA to determine the effects of bag type and initial infestation on germination. Correlations between number of live insects and number of puncture holes found in 100-kg jute, polypropylene, and ZeroFly bags, were done using the Correlation Procedure of SAS, at P < 0.05.

Table 1 Analysis of variance (ANOVA) for effect of bag type (jute, polypropylene and ZeroFly® storage bags) on the number of adult Sitophilus zeamais, Rhyzopertha dominica, Prostephanus truncatus, and Tribolium castaneum in plastic containers after 7 wk of incubation for data from the control treatment (bags that were not fumigated). Mean number ±SE

Species

ANOVA F

P

Jute

Polypropylene

ZeroFly

S. zeamais R. dominica P. truncatus T. castaneum

1.0 0.9 1.5 1.1

0.46 0.49 0.33 0.42

102.22 ± 5.2 104.4 ± 2.5 94.7 ± 7.1 94.1 ± 6.8

87.3 ± 9.8 93.2 ± 8.8 109.4 ± 6.2 107.4 ± 3.5

87.7 ± 6.2 103.7 ± 3.3 109.9 ± 5.5 99.2 ± 6.2

In all cases df ¼ 2,4. For each species, the number of insects in bags did not differ among the three different types of bags.

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3. Results 3.1. Evaluating efficacy of PH3 in insects that infest maize stored in ZF bags 3.1.1. Cages with adult insects No live adult S. zeamais, R. dominica, P. truncatus, and T. castaneum were found in cages with adult insects inside jute, PP and ZF bags that were fumigated. However, in all bags of the three types that were not fumigated (control treatment), all 30 adult insects of each species that were in individual cages were found alive after fumigation. 3.1.2. Cages with immature insects In the case of immatures, no S. zeamais, R. dominica, P. truncatus, and T. castaneum adults were found in plastic containers associated with cages placed inside jute, PP and ZF bags that were fumigated, after 7 wk of incubation. For each of the four species, the number of adults from cages with immatures that had been placed in bags that were not fumigated did not differ among the three different types of bags (Table 1). Adult insects counted were from plastic containers that were incubated for 7 wk. 3.1.3. Germination In the evaluation of PH3 efficacy, the main effects fumigation treatment and bag type, and their interaction were not significant for germination (Table 2). Germination values (mean ± SE) in the un-fumigated (control) and fumigated stacks were 99.5 ± 0.2 and 99.6 ± 0.2, respectively. These values in jute, PP, and ZF bags were 99.6 ± 0.2, 99.2 ± 0.2 and 99.8 ± 0.1, respectively. 3.2. Evaluating the ability of insects to penetrate ZF bags 3.2.1. Temperature, moisture content and relative humidity Maize temperatures in replications 1, 2, and 3 during the 4 mo of the experiment ranged between 26.5 and 28.8, 26.5e28.8, and 28.8e29.0  C, respectively. This corresponded to means of 27.8, 27.4, and 27.9  C, respectively. In the case of moisture content these values were 13.5e14.6, 13.4e14.5, and 13.2e14.0%, respectively. This corresponded to means of 14.0, 14.0, and 13.6%, respectively. For relative humidity (r.h.), values ranged between 68.9 and 73.5, 66.8e72.3, and 66.1e72.3%, respectively. This corresponded to means of 70.5, 70.3, and 69.9%, respectively. 3.2.2. Bags initially infested with Sitophilus zeamais In bags initially infested with S. zeamais, S. zeamais (total 462 adults), P. truncatus (total 347 adults), Liposcelis spp. (total 112 adults), C. ferrugineus (total 80 adults), T. castaneum (total 69 adults) and R. dominica (total 21 adults) were found in all the three types of

Table 2 Analysis of variance (ANOVA) for main effects fumigation treatment, bag type and interactions (*) for germination in the experiment on efficacy of PH3 in insects that infest maize stored in jute, polypropylene, and ZeroFly bags, and also ANOVA for bag type, initial infestation, and interactions (*) for germination in the experiment on ability of insects to breach the three types of storage bags. Experiment

Germination test time

Source

df

PH3 fumigation and bag type

Post- experiment

Breaching of bags by insects

Pre-experiment

Treatment Bag type * Bag type Initial infestation * Bag type Initial infestation *

1, 2, 2, 2, 2, 4, 2, 2, 4,

Post-experiment

10 10 10 16 16 16 16 16 16

F

P

0.1 2.3 0.8 2.1 0.6 0.2 2.8 2.4 3.1

0.81 0.15 0.48 0.16 0.58 0.95 0.09 0.12 0.05

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Table 3 Analysis of variance (ANOVA) for main effects bag type and month when sampling occurred (Month), and interactions (*) for number of Sitophilus zeamais, Prostephanus truncatus, Liposcelis spp., Cryptolestes ferrugineus and number of insect puncture holes (Holes) in 100-kg jute, polypropylene, and ZeroFly bags initially infested with S. zeamais, P. truncatus and the control (bags not infested at the start of the experiment). Initial infestation

Species

Source

df

S. zeamais

S. zeamais

Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type *

3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6,

P. truncatus.

Liposcelis spp.

Holes

P. truncatus

S. zeamais

P. truncatus.

Liposcelis spp.

Holes

Control

S. zeamais

P. truncatus.

C. ferrugineus.

Holes

11.1 6.0 11.5 13.2 4.73 13.4 14.5 5.78 14.7 12.5 3.56 12.5 15.1 8.08 15.4 15.4 5.59 15.4 14.7 4.86 14.6 12.8 4.58 12.9 14.4 7.71 14.7 15.4 7.13 15.5 17.3 6.23 17.4 14.4 4.42 14.4

F

P

43.5 20.5 7.6 47.2 16.5 12.4 2.9 0.8 1.5 2.9 1.6 1.1 21.9 13.4 5.9 29.6 22.4 6.4 4.1 1.2 1.8 13.0 10.8 3.3 27.8 22.6 7.3 27.8 20.3 7.6 5.3 4.9 2.0 2.0 0.9 0.9

< 0.01 0.01 0.01 < 0.01 0.01 < 0.01 0.07 0.48 0.24 0.08 0.32 0.41 < 0.01 0.01 0.01 < 0.01 0.01 0.01 0.03 0.38 0.16 < 0.01 0.02 0.03 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.05 0.12 0.16 0.46 0.52

bags. The first three species were the most abundant and recovered from all types of bags, and therefore were the only species analyzed separately. For the overall analyses, the main effects month and bag type, and their interaction were significant (P < 0.05) for S. zeamais and P. truncatus, but not for Liposcelis spp. (Table 3). Expectedly, insect numbers increased with time during the JuneeSeptember period in which the study was conducted (Table 4). In August, after 3 mo of storage, numbers of S. zeamais in jute and PP bags were similar but higher than in ZF bags. There were significantly more S. zeamais in jute bags than PP bags in September but numbers were much lower in ZF bags. In relation to P. truncatus, the highest numbers of insects found were in jute and PP bags in September; there was no difference in the numbers of insects in these two types of bags in September. For Liposcelis spp., the numbers were generally low and did not exceed 5.7 ± 3.5 in any of the three types of bags during the 4 mo of storage. In September, psocid numbers were similar in all three types of bags (Table 4). In relation to the number of insect puncture holes, the main effects month and bag type, and their interaction were not significant (Table 3). However, the highest numbers of holes was found in jute and PP bags in September; these numbers were higher than the very low number of holes in ZF bags in September (Table 4).

3.2.3. Bags initially infested with Prostephanus truncatus In bags initially infested with P. truncatus, S. zeamais (total 309

adults), P. truncatus (total 691 adults), Liposcelis spp. (total 150 adults), C. ferrugineus (total 79 adults), T. castaneum (total 122 adults), and R. dominica (total 21 adults) were found in all the three types of bags. The first three species were the most abundant and recovered from all types of bags, and therefore were the only species analyzed separately. For the overall analyses, the main effect month was significant for S. zeamais, P. truncatus, and Liposcelis spp. (Table 3). The main effect bag type and interaction of month and bag type were significant for S. zeamais and P. truncatus, but not for Liposcelis spp. (Table 3). Expectedly, numbers of insects of different species increased with time to a varying degree during the JuneeSeptember period in which the study was conducted. The numbers of S. zeamais in September were higher in jute and PP bags, and were significantly lower in ZF bags (Table 4). The numbers of P. truncatus in September were higher in jute and PP bags, but were significantly lower in ZF bags compared to jute and PP bags (Table 4). The number of Liposcelis spp. in September were higher in jute bags than in PP and ZF bags, but numbers in the latter two types of bags were similar (Table 4). In relation to the number of insect puncture holes, the main effects month and bag type, and their interaction were significant (Table 3). The numbers of holes in jute and PP bags increased significantly over time and were highest in September (Table 4). The number of holes in jute and PP bags were similar in September but were significantly higher than the number of holes found in ZF bags (Table 4). 3.2.4. Bags initially not infested In bags initially not artificially infested, S. zeamais (total 333 adults), P. truncatus (total 508 adults), C. ferrugineus (total 141 adults), Liposcelis spp. (total 110 adults), T. castaneum (total 96 adults), and R. dominica (total 16 adults) were found in all the three types of bags. The first three species were the most abundant and recovered from all types of bags, and therefore were the only species analyzed separately. For the overall analyses, the main effect month was significant for S. zeamais, P. truncatus, and C. ferrugineus (Table 3). The main effect bag type and interaction of month and bag type were significant for S. zeamais and P. truncatus, but not for C. ferrugineus (Table 3). Expectedly, numbers of insects of different species increased with time to a varying degree during the JuneeSeptember period in which the study was conducted. The numbers of S. zeamais in September were higher in jute than PP bags, and were significantly lower in ZF bags compared to jute and PP bags (Table 4). The numbers of P. truncatus in September were highest in jute and PP bags but numbers of insects in these two types of bags were similar; no P. truncatus were found in ZF bags in September. The number of C. ferrugineus in September were highest in jute and ZF bags but numbers of insects in these two types of bags were similar; numbers in PP bags were lower than in jute bags but similar to those in ZF bags (Table 4). In relation to the number of insect puncture holes, the main effects month and bag type, and their interaction were not significant (Table 3). However, the highest numbers of holes were found in jute and PP bags in September; these numbers were higher than the very low number of holes in ZF bags in September (Table 4). 3.2.5. Correlation between insect numbers and puncture holes Across all jute bags initially infested with S. zeamais, P. truncatus, and those that were uninfested, the only significant correlations were between numbers of P. truncatus, S. zeamais, and total number of all live insects with number of puncture holes (Table 5). Total number of live insects comprised the sum of P. truncatus, S. zeamais, T. castaneum, R. dominica and C. ferrugineus numbers. For all PP bags initially infested with S. zeamais, P. truncatus, and those that were

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Table 4 Number (mean ± SE) of Sitophilus zeamais, Prostephanus truncatus, Liposcelis spp., Cryptolestes ferrugineus and puncture holes found in 100-kg jute, polypropylene, and ZeroFly bags initially infested with S. zeamais, P. truncatus and the control (bags not infested at the start of the experiment) in the months of JuneeSeptember 2017. For each species, differences among months within each bag type are denoted by lower-case letters, and significant differences within each bag type by month are denoted with different uppercase letters (P < 0.05, SLICE option in an LSMEANS statement). If there are no letters, there were no significant differences among means associated with the species (P  0.05). Initial infestation

Species

Bag Type

June

July

August

September

S. zeamais

S. zeamais

Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF

3.3 ± 3.3a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0a 0.0 ± 0.0a 0.0 ± 0.0a 5.3 ± 0.7a 1.7 ± 1.6ab 1.7 ± 1.6a 6.7 ± 3.4a 0.7 ± 0.3a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 3.3 ± 3.3a 11.0 ± 10.5a 0.3 ± 0.3a 0.0 ± 0.0a 1.0 ± 0.6a 3.0 ± 2.5a 38.7 ± 8.1a 46.3 ± 34.8a 0.0 ± 0.0a 0.7 ± 0.3a 0.0 ± 0.0a 0.0 ± 0.0a 1.7 ± 1.6a 0.0 ± 0.0a 0.0 ± 0.0a 0.3 ± 0.3a 0.0 ± 0.0a 0.0 ± 0.0a 3.7 ± 2.7a 0.3 ± 0.3a 0.0 ± 0.0a

5.0 ± 3.2a 3.4 ± 3.7a 5.3 ± 2.7a 0.0 ± 0.0a 0.3 ± 0.3a 1.0 ± 1.0a 2.7 ± 1.2a 3.0 ± 2.5ab 4.0 ± 1.5ab 12.7 ± 9.8ab 1.7 ± 1.2a 0.0 ± 0.0a 3.7 ± 3.2a 2.0 ± 0.6a 0.3 ± 0.3a 3.3 ± 2.8a 4.7 ± 4.7a 0.3 ± 0.3a 2.0 ± 1.0a 0.3 ± 0.3a 0.7 ± 0.7a 151.3 ± 61.7aB 263.0 ± 19.0bB 0.0 ± 0.0aA 1.0 ± 0.6a 0.0 ± 0.0a 0.3 ± 0.3a 0 ± 0a 4.0 ± 3.1a 1.0 ± 1.0a 3.0 ± 0.6a 2.0 ± 1.0a 2.7 ± 1.2a 8.7 ± 4.5a 1.0 ± 0.6a 0.0 ± 0.0a

20.0 ± 6.4bB 28.7 ± 4.1bB 4.3 ± 1.9aA 10.0 ± 5.8a 0.0 ± 0.0a 0.0 ± 0.0a 2.7 ± 2.6a 0.7 ± 0.3a 2.0 ± 2.0a 7.0 ± 4.7a 18.7 ± 9.4a 0.0 ± 0.0a 18.0 ± 9.9bB 0.3 ± 0.3aA 1.3 ± 0.9aA 40.7 ± 6.4bB 42.7 ± 6.4bB 2.7 ± 1.8aA 5.0 ± 4.0a 2.3 ± 2.3a 4.7 ± 1.8a 117.7 ± 28.8aB 209.3 ± 72.6bB 0.0 ± 0.0aA 19.3 ± 5.2bB 17.0 ± 3.5bB 0.3 ± 0.3aA 33.3 ± 3.3bB 23.3 ± 8.8bB 0.0 ± 0.0aA 13.3 ± 6.8bB 0.3 ± 0.3aA 2.7 ± 1.5aA 10.7 ± 6.4a 3.7 ± 3.2a 0.0 ± 0.0a

44.0 ± 5.6dC 31.0 ± 4.6bB 8.7 ± 2.6aA 57.7 ± 6.7bB 46.7 ± 12.0bB 0.0 ± 0.0aA 3.3 ± 3.3a 4.7 ± 2.9b 5.7 ± 3.5b 37.0 ± 22.5bAB 84.3 ± 56.3bB 1.7 ± 0.9aA 35.7 ± 4.9cB 40.0 ± 9.8bB 1.7 ± 1.6aA 64.0 ± 8.7cB 52.3 ± 6.2bB 5.0 ± 2.9aA 20.7 ± 10.4bB 3.7 ± 2.3aA 6.7 ± 0.3aA 614.0 ± 211.0bB 780.0 ± 215.1cB 2.7 ± 1.2aA 41.7 ± 4.4cC 30.3 ± 9.7cB 0.3 ± 0.3aA 49.3 ± 11.5cB 56.7 ± 8.8cB 0.0 ± 0.0aA 12.0 ± 2.5bB 4.7 ± 2.0aA 6.0 ± 2.9abAB 15.3 ± 8.4aAB 26.3 ± 21.1bB 1.0 ± 1.0aA

P. truncatus

Liposcelis spp.

Holes

P. truncatus

S. zeamais

P. truncatus

Liposcelis spp.

Holes

Control

S. zeamais

P. truncatus

C. ferrugineus

Holes

Table 5 Correlation between number of live insects and number of puncture holes found in 100-kg jute, polypropylene, and ZeroFly bags. Bag type

Species

Pearson correlation coefficient (r)

t

P

Jute

Sitophilus zeamais Prostephanus truncatus Tribolium castaneum R. dominica C. ferrugineus All species Sitophilus zeamais Prostephanus truncatus Tribolium castaneum R. dominica C. ferrugineus All species Sitophilus zeamais Prostephanus truncatus Tribolium castaneum R. dominica C. ferrugineus All species

0.40 0.34 0.23 0.36 0.03 0.36 0.30 0.35 0.16 0.05 0.26 0.37 0.19 0.04 0.01 0.05 0.35 0.24

2.55 2.09 1.39 2.21 0.18 2.27 1.83 2.17 0.92 0.28 1.60 0.22 1.15 0.24 0.04 0.28 2.17 1.41

0.02 0.04 0.17 0.03 0.86 0.03 0.08 0.04 0.37 0.78 0.12 0.03 0.26 0.81 0.97 0.78 0.04 0.17

Polypropylene

ZeroFly

In all cases df ¼ 2,4.

uninfested, the only significant correlations were between numbers of P. truncatus and total number of all live insects with number of puncture holes (Table 5). In the case of ZF bags, the only significant correlation was between numbers of C. ferrugineus with number of puncture holes (Table 5). There was good correlation between P. truncatus and insect puncture holes for jute (r ¼ 0.34,

P < 0.04) and PP (r ¼ 0.35, P < 0.04) bags. No correlation existed between P. truncatus and puncture holes in ZF bags (r ¼ 0.04, P < 0.81). In the case of total number of insects, there was good correlation with the number of puncture holes for jute (r ¼ 0.36, P < 0.03) and PP (r ¼ 0.37, P < 0.03) bags but not for ZF bags (r ¼ 0.24, P < 0.17).

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Table 6 Analysis of variance (ANOVA) for main effects bag type and month when sampling occurred (Month), and interactions (*) for percent insect damaged kernels (IDK) computed by number (%IDKnb) and percent weight loss (%WL) in 100-kg jute, polypropylene, and ZeroFly bags initially infested Sitophilu zeamais, Prostephanus truncatus and the control (bags not infested at the start of the experiment). Initial infestation

Variable

Source

df

S. zeamais

%IDKnb

Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type * Month Bag type *

3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6, 3, 2, 6,

%WL

P. truncatus

%IDKnb

%WL

Control

%IDKnb

%WL

14.9 8.0 15.3 14.9 8.16 15.2 12.9 5.92 13.1 13.8 5.77 13.9 17 6.44 16.9 13.6 6.2 13.7

F

P

6.3 0.9 0.9 0.9 0.4 0.3 2.9 0.7 2.4 2.2 0.9 0.6 5.2 1.8 1.2 3.3 1.3 0.6

< 0.01 0.43 0.55 0.48 0.67 0.94 0.08 0.53 0.08 0.13 0.46 0.70 < 0.01 0.23 0.35 0.05 0.34 0.76

3.2.6. Damage variables %IDKnb and %WL Across all jute bags initially infested with S. zeamais, P. truncatus, and those that were uninfested, mean %IDKnb and %WL values were 2.36% and 0.08%, respectively. These values for PP bags were 5.72 and 0.24%, respectively. In ZF bags, these values were 2.46 and 0.08%, respectively. For the overall analyses in the case of bags initially infested with S. zeamais, the main effect month was significant for %IDKnb but not for %WL (Table 6). The main effect bag type and interaction of month and bag type were not significant for both of the variables (%IDKnb and %WL) (Table 6). Expectedly, % IDKnb increased with time but the highest value was found in ZF bags in September (Table 7). The mean value of %IDKnb in ZF bags in September was 4.8 ± 1.6. For all bag types in the 4 months, %WL values were similar (Table 7). For overall analyses in the case of bags initially infested with P. truncatus, the main effects month and bag type, and their interaction were not significant for %IDKnb and %WL (Table 6). The highest values for %IDKnb and %WL were found in PP bags in September and were 41.47 ± 18.6 and 1.54 ± 0.7, respectively

Table 8 Post-experiment germination (%) values (mean ± SE) for the interaction between bag type and initial infestation for the experiment on ability of insects to breach jute, polypropylene and ZeroFly storage bags. Initial infestation

Bag type

S. zeamais P. truncatus Control

Jute 91.7 ± 2.2bc 90.6 ± 6.1bc 94.2 ± 1.9c

Polypropylene 97.5 ± 0.8c 83.9 ± 3.74 ab 95.8 ± 0.00c

ZeroFly 94.7 ± 2.0c 97.2 ± 1.1c 97.2 ± 1.0c

(Table 7). In the case of bags initially not infested, overall analyses showed that the main effect month was significant for %IDKnb but not for % WL (Table 6). The main effect bag type and the interaction of month and bag type were not significant for both variables (%IDKnb and % WL) (Table 6). The highest values for %IDKnb and %WL were found in jute bags in September and were 4.13 ± 2.0 and 0.1 ± 0.03, respectively (Table 7). 3.2.7. Germination The main effects bag type and initial infestation, and their interaction were not significant for germination pre-experiment (Table 2). Germination values (mean ± SE) in jute, PP, and ZF bags were 96.02 ± 1.85, 99.54 ± 0.15 and 98.52 ± 0.41, respectively. These values in bags initially infested with P. truncatus, S. zeamais, and the control were 97.04 ± 1.86, 98.80 ± 0.64 and 98.24 ± 0.60, respectively. Post-experiment, the main effects bag type and initial infestation were not significant for germination, but their interaction was significant (Table 2). In all cases, germination exceeded 90% with the exception of PP bags that were initially infested with P. truncatus (Table 8). 4. Discussion The use of PH3 to effectively control stored products insect pests is widely popular and reported worldwide. Data from this study showed good efficacy of PH3 in P. truncatus, R. dominica, S. zeamais and T. castaneum that infest maize stored in jute, PP and ZF storage bags and results in complete immature and adult insect mortality if fumigations are conducted according to label recommendation. According to Nath et al. (2011), the ease of application, together

Table 7 Values (mean ± SE) of percent insect damaged kernels (IDK) computed by number (% IDKnb) and percent weight loss (%WL) in 100-kg jute, polypropylene, and ZeroFly bags initially infested with S. zeamais, P. truncatus and the control (bags not infested at the start of the experiment) in the months of JuneeSeptember 2017. For each response variable, differences among months within each bag type are denoted by lower-case letters, and significant differences within each bag type by month are denoted with different upper-case letters (P < 0.05, SLICE option in an LSMEANS statement). If there are no letters, there were no significant differences among means associated with the variable (P  0.05). Initial infestation

Response variable

Bag type

June

July

August

September

S. zeamais

%IDKnb

Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF Jute PP ZF

0.53 ± 0.3a 0.53 ± 0.4a 0.30 ± 0.3ab 0.02 ± 0.01 0.01 ± 0.01 0.05 ± 0.05 0.53 ± 0.3a 0.93 ± 0.1a 0.40 ± 0.2a 0.00 ± 0.00a 0.03 ± 0.02a 0.03 ± 0.03a 0.53 ± 0.4a 0.27 ± 0.1a 3.47 ± 3.1a 0.05 ± 0.03ab 0.04 ± 0.02a 0.02 ± 0.01a

0.93 ± 0.7a 1.10 ± 0.5a 1.70 ± 1.0bc 0.05 ± 0.05 0.07 ± 0.03 0.11 ± 0.06 4.13 ± 2.6a 1.73 ± 0.6a 1.33 ± 0.6a 0.02 ± 0.01a 0.06 ± 0.03a 0.08 ± 0.02a 0.67 ± 0.4a 0.13 ± 0.1a 1.47 ± 1.3a 0.05 ± 0.03ab 0.00 ± 0.0a 0.00 ± 0.00a

0.53 ± 0.3aa 0.40 ± 0.2a 0.40 ± 0.2ab 0.03 ± 0.03 0.03 ± 0.01 0.03 ± 0.01 5.2 ± 2.8a 20.27 ± 15.7b 2.53 ± 0.7a 0.23 ± 0.15a 1.06 ± 1.0ab 0.15 ± 0.04a 0.93 ± 0.6a 0.00 ± 0.0a 2.13 ± 2.13a 0.04 ± 0.04a 0.00 ± 0.0a 0.04 ± 0.04a

2.00 ± 1.2aAB 1.60 ± 0.6aA 4.80 ± 1.6cB 0.07 ± 0.04 0.03 ± 0.03 0.06 ± 0.05 8.13 ± 3.4aA 41.47 ± 18.6cB 8.53 ± 4.6aA 0.31 ± 0.2a 1.54 ± 0.7b 0.29 ± 0.15a 4.13 ± 2.0b 0.30 ± 0.3a 2.40 ± 2.4a 0.10 ± 0.03b 0.04 ± 0.04a 0.07 ± 0.07a

%WL

P. truncatus

%IDKnb

%WL

Control

%IDKnb

%WL

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with its effectiveness, lack of residues, and low cost of the fumigant, have resulted in PH3 use on nearly all internationally traded grain for human consumption. Seed germination is of great importance to Nigerian farmers and under normal conditions they primarily apply PH3 to protect seed for future planting. In this study, seed germination was not affected by PH3 fumigation, and it was ~100% in jute, PP and ZF bags. Similar germination results were obtained in the control (unfumigated bags). Data from this study corroborate those from previous studies which indicate PH3 does not interfere with the germination of seeds (White and Jacobson, 1972; Sittisuang and Nakakita, 1985; Krzyzanowski et al., 2013). According to Vijayanna (2006), high seed moisture content increases seed respiration leading to increased absorption of PH3 by the seeds and this may cause adverse effects on chemical balance of the seed (Harrington and Douglas, 1970), changes in enzyme activity during storage (Srinivasan, 1939) and changes in membrane permeability (Hebbard and Miller, 1928). High fat content in grain kernels too result in increased PH3 absorption (Vijayanna, 2006). In this study, the fact that maize moisture content was low (10.45%) and it is not a crop grown mainly for oil, may have mitigated PH3 absorption and its toxic effects on seed embryos resulting in the preservation of seed and high germination rates. Data from this study show that P. truncatus and S. zeamais are able to penetrate jute, PP, and ZF bags, but to varying extent. Jute and PP bags were more easily breached by P. truncatus and S. zeamais than ZF bags. On average over the 4-mo period, the number of puncture holes in ZF bags did not exceed 9 compared to up to 614 and 780 found in jute and PP bags, respectively. These data indicating a low number of holes in ZF bags corroborate the findings of Anankware (2014), Paudyal et al. (2017) and Kavallieratos et al. (2017) which showed that no insect holes were found in ZF bags exposed to P. truncatus, S. zeamais and T. castaneum. These data indicate that the use of deltamethrin incorporated ZF bags can greatly reduce insect entry into and exit out of the bags through breaching the bag fabric. According to Kavallieratos et al. (2017), insect pests could potentially absorb enough deltamethrin insecticide to cause their death when they attempt to penetrate ZF bags. However, stored-product insects that are resistant to deltamethrin may be able to penetrate the bag fabric (Paudyal et al., 2017). Prostephanus truncatus and S. zeamais are major threats to maize storage in Africa (Holst, 2000). In particular, there have been concerns that P. truncatus can breach storage bags and drums made of plastic material (Hodges and Stathers, 2015). This means that adults of P. truncatus may be able to breach bags with fibers that are not treated with insecticide and cause serious infestations (Kavallieratos et al., 2017). In fact, Hell et al. (2014) showed that P. truncatus is capable of breaching PICS bags. Compared with S. zeamais, the greater ability of P. truncatus to cause more damage to stored products and also penetrate storage materials can be attributed to its feeding behavior and type of mouthparts (Ragumamu, 2005). Prostephanus truncatus feeds voraciously on stored grains with strong and powerful mandibles which also enables it to penetrate into storage fabrics (Popoola, 2012), while S. zeamais mandibles are less adapted for such damage (Ragumamu, 2005). This demonstrates that the behavior of P. truncatus is related to the morphology of its mouthparts (Chapman, 1982; Vendi et al., 2018; Stejskal et al., 2018). Although both P. truncatus and S. zeamais possesses strong mandibular molars for trituration of dense materials, a typical feature of insects feeding on hard substances (Vendi et al., 2018), it appears these molars are better developed in P. truncatus. This can be seen in the quantity of dust generated in infested commodities (Kumar, 2002) and the number of perforations created on storage materials. Prostephanus truncatus incisors are long and blunt, and are adapted

89

for scraping and penetration of hard materials such as seed, wood and food packages (Vendi et al., 2018). These morphological features of P. truncatus seem to play a significant role in the breaching of storage bags. In this study, we also showed that insect populations in jute and PP bags that were not artificially infested with S. zeamais and P. truncatus were quite high. This increase in population during the 4 mo storage period was possible in jute and PP bags possibly because these types of bags allow insect pests to move readily in and out of the bags which results in exacerbating insect population levels in bags and spread of infestation, respectively. Ng'ang'a et al. (2016) also found that PP and jute bags permit build-up of storedproduct insect populations hence corroborate findings of this study. For ZF bags, the deltamethrin insecticide incorporated in fibers of its fabric greatly reduces entry of insect into the bag and high populations found inside bags was most likely due to multiplication of insects introduced with maize that was bagged at the start of the study. Generally, lower insect numbers were found in ZF bags and this could be partly due to increased mortality from exposure to deltamethrin. Paudyal et al. (2017) indicated that there was increased mortality in insects that infest maize stored in ZF bags. Expectedly, other secondary pests such as Liposcelis spp., C. ferrugineus and T. castanuem were found in high numbers after S. zeamais and P. truncatus damaged maize kernels in bags. Ng'ang'a et al. (2016) observed a similar trend when T. castaneum was found in jute and PP bags after initial infestation by S. zeamais. Mean temperatures of 27.8, 27.4, and 27.9  C, respectively, existed in buildings where the study to assess ability of insects to penetrate ZF bags was conducted; these temperatures are favourable for the development of S. zeamais and P. truncatus and other external feeding insects for the entire 4 mo storage period. Similarly, MC of maize was also favorable and mean values in the buildings were 14.0, 14.0, and 13.6%, respectively. Ditto for relative humidity where mean values were 70.5, 70.3, and 69.9%, respectively. Therefore, environmental conditions in the buildings where this study was conducted most likely contributed to high insect populations recorded. Insect infestation resulted in kernel damage and weight loss in jute, PP and ZF bags. The PP bag had the highest IDK level followed by jute and ZF bag. The PP and jute bags had higher insect populations and expectedly also had high kernel damage and weight loss; the converse was true for ZF bags. There was good correlation between P. truncatus and insect puncture holes for jute bags. This relationship is expected because P. truncatus is quite destructive and is even known to easily penetrate plastic materials used for storage (Hodges and Stathers, 2015). No correlation existed between P. truncatus and puncture holes in ZF bags. In the case of total number of insects, there was good correlation with the number of puncture holes for jute bags but not for ZF bags. This may be because S. zeamais and P. truncatus are the predominate species in total numbers of insects and they are the species more prone to boring holes in bags. Data from this study showing fewer puncture holes in ZF bags are corroborated by Kavallieratos et al. (2017) who opined that the use of treated bags for storage purposes may provide a satisfactory level of control for storage insect pests. At the end of the study to assess ability of insects to penetrate ZF bags, germination rates of maize in jute and ZeroFly bags initially infested with P. truncatus, S. zeamais and the control (uninfested) were >90%. These high germination rates indicate that maize viability was maintained after 4 mo of storage. However, in the case of maize contained in PP bags that was infested with P. truncatus, germination rate was reduced to 83%. This reduced viability is most likely a result of P. truncatus infestation causing substantial damage to kernels.

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This study has shown that jute and PP bags can be readily breached by P. truncatus and S. zeamais thereby leading to infestations, relatively quicker spread of infestations, kernel damage and grain weight loss. Importantly, this study has demonstrated that when applied according to label recommendations, PH3 is highly effective against all stages of P. truncatus, R. dominica, S. zeamais and T. castaneum regardless of whether maize under fumigation is stored in jute, PP or ZF bags. ZeroFly bags have great potential to protect grains placed in them from external insect infestation as long as insect-free grain is used. In case infestation occurs in grain in ZF bags, then fumigation can be conducted without transferring maize to jute or PP bags. In a nutshell, we recommend that infested grain inside ZF bags be fumigated so that it can be safe for longer periods inside the bags. Acknowledgements We thank the sponsors of this project, Vestergaard Frandsen SA, Lausanne, Switzerland for the funding. Any mention of trade names or commercial products in this publication is only for the purpose of providing specific information and does not imply recommendation or endorsement by Nigerian Stored Products Research Institute, University of Ibadan, Oklahoma State University, and Vestergaard SA. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jspr.2019.04.006. References Adedire, C.O., Akinkurolere, R.O., 2005. Bioactivity of four plant extract on coleopterous pests of stored cereals and legumes in Nigeria. Zool. Res. 26, 243e249. Adedire, C.O., Lajide, L., 2003. Ability of extracts of ten tropical plant species to protect maize against infestation by the maize weevil, Sitophilus zeamais during storage. Niger. J. Exp. Biol. 2, 175e179. Adedire, C.O., Obembe, O.O., Akinkurolere, R.O., Oduleye, O., 2011. Response of Callosobruchus maculatus (Coleoptera: chrysomelidae: Bruchidae) to extracts of cashew kernels. J. Plant Dis. Prot. 118 (2), 75e79. Allahvaisi, S., Pourmirza, A.A., Safaralizade, M.H., 2010. Study on polymers permeability for foodstuffs packaging by some serious species of stored pest insects and phosphine gas. J. Agric. Sci. Technol. 6 (4), 747e759. Anankware, J.P., Hadi, M., Bingham, G., 2014. Deltamethrin contact bioassay and boring/chewing tests with the maize weevil, Sitophilus zeamais (Mot). Int. J. Agric. Res. Rev. 1, 133e142. Chapman, R.F., 1982. The Insects: Structure and Function. The English University Press, London. Food and Agriculture Organisation FAO, 1992. Maize in Human Nutrition. FAO Food and Nutrition Series No. 25. Rome, Italy. Harrington, J.F., Douglas, J.E., 1970. Seed Storage and Packaging: Application for India. National Seed Corporation and US AID, New Delhi, pp. 3e22. Hassan, M.W., Ali, G.U., Rahman, F.U., Najeeb, H., Sohail, M., Irsa, B., Muzaffar, Z., Chaudhry, M.S., 2016. Evaluation of standard loose plastic packaging for the management of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Herbst) (Coleoptera: tenebriondiae). J. Insect Sci. 16 (1), 91. Hebbard, R.P., Miller, R.V., 1928. Biochemical studies on seed viability: I. Measurement of Conductance and Reduction. Plant Physiol. 3 (3), 335e352. Hell, K., Ognakossan, E., Lamboni, Y., 2014. PICS hermetic bags ineffective in controlling infestations of Prostephanus truncatus and Dinoderus spp. in traditional cassava chips. J. Stored Prod. Res. 58, 53e58. Hodges, R., Stathers, T., 2015. Summary Report on a Survey of Grain Storage Option. Natural Resources Institute (NRI) of University of Greenwich, Chatham, U.K, p. 51. Holst, N., Meikle, W.G., Markham, R.H., 2000. Grain injury models for Prostephanus

truncatus (Coleoptera: Bostrichidae) and Sitophilus zeamais (Coleoptera: Curculionidae) in rural maize stores in West Africa. J. Econ. Entomol. 93, 1338e1346. Kavallieratos, N.G., Athanassiou, C.G., Arthur, F.H., 2017. Effectiveness of insecticideincorporated bags to control stored-product beetles. J. Stored Prod. Res. 70, 18e24. Kengkanpanich, R., Suthisut, D., Sitthichaiyakul, S., 2018. Application of ECO2FUME®Phosphine fumigant for the complete control of major stored product insect pests in milled rice in Thailand. In: Adler, C.S., Opit, G., Furstenau, B., Muller-Blenkle, C., Kern, P., Arthur, F.H., et al. (Eds.), Proceedings of the 12th International Working Conference on Stored Product Protection, 7e11, October 2018, Berlin, Germany. Julius Kuhn Institute, Berlin, Germany, pp. 618e624. Krzyzanowski, F.C., Lorini, I., Franca-Neto, J., Henning, A.A., 2013. Effects of phosphine fumigation on the quality of soybean seeds. J. Seed Sci. 35 (2). Kumar, H., 2002. Resistance in maize to the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). J. Stored Prod. Res. 38, 267e280. Marouf, A., Momen, R.F., 2004. An evaluation of the permeability to phosphine through different polymers used for the bag storage of grain. In: Proceedings of 8th International Conference on Controlled Atmosphere and Fumigation in Stored Product, 8e13th August 2004, Gold-Coast Australia. FTIC Ltd, Publishing, Israel (xxxexxx). Nath, N.S., Bhattacharya, I., Tuck, A.G., Schlipalius, D.I., Ebert, P.R., 2011. Mechanisms of phosphine toxicity. J. Toxicol. 2011 (3), 1e9. Ng’ang’a, J., Mutungi, C., Imathiu, S.M., Affognon, H., 2016. Low permeability triplebag layer plastic bags prevent losses of maize caused by insects in rural on-farm stores. Food Security 8, 621e633. Okonkwo, E.U., Nwaubani, S.I., Otitodun, O.G., Ogundare, M.O., Bingham, G.V., Odhiambo, J.O., Williams, J.O., 2016. Evaluation of efficacy of a long lasting insecticide incorporated polypropylene bag as stored grain protectant against insect pests on cowpea and maize. J. Stored Prod. Postharvest Res. 8 (1), 1e10. Pan, N.C., Day, A., Kumar, K.M., 1999. Chemical composition of jute and its estimation. Man Made Text. India 9, 467e473. Paudyal, S., Opit, G.P., Osekre, E.A., Arthur, F.H., Bingham, G.V., Payton, M.E., Danso, J.K., Manu, N., Nsiah, E.P., Guatam, S.G., Noden, B., 2017. Field evaluation of the long-lasting treated storage bag, deltamethrin incorporated (ZeroFly® Storage Bag) as a barrier to insect pest infestation. J. Stored Prod. Res. 70, 44e52. Popoola, K.O.K., 2012. Hermetic control treatment of P. truncatus (Horn) (Coleoptera: Bostrichidae) infestation on stored dried cassava chips in polythene bags. Adv. J. Food Sci. Technol. 4 (3), 118e120. Quitco, R.T., Quindoza, N.M., 1986. Assessment of Paddy Loss in Storage. Unpublished Terminal Report. NAPHIRE, p. 46. Ragumamu, C.P., 2005. Influence of simultaneous infestations of Prostephanus truncatus and Sitophilus zeamais on the reproductive performance and maize damage. Tanzan. J. Sci. 31 (1), 65e72. Sittisuang, P., Nakakita, H., 1985. The effect of phosphine and methyl bromide on germination of Rice and Corn seeds. J. Pestic. Sci. 10 (3), 461e468. Srinivasan, A., 1939. Studies on quality in rice IV. Storage changes in rice after harvest. Indian J. Agric. Sci. 9, 208e222. Stejskal, V., Vendi, T., Aulicky, R., 2018. Biological abilities of storage pests required for the successful penetration of food packages or seeds. In: Adler, C.S., Opit, G., Furstenau, B., Muller-Blenkle, C., Kern, P., Arthur, F.H., et al. (Eds.), Proceedings of the 12th International Working Conference on Stored Product Protection, 7e11, October 2018, Berlin, Germany. Julius Kuhn Institute, Berlin, Germany, pp. 94e97. Vendi, T., Aulicky, R., Stejkal, V., 2018. Comparison of the mandible morphology of two stored product bostrichid beetles Rhyzopertha dominica and Prostephanus truncatus. In: Adler, C.S., Opit, G., Furstenau, B., Muller-Blenkle, C., Kern, P., Arthur, F.H., et al. (Eds.), Proceedings of the 12th International Working Conference on Stored Product Protection, 7e11, October 2018, Berlin, Germany. Julius Kuhn Institute, Berlin, Germany, pp. 208e211. Vestergaard, 2014. ZeroFly® Storage Bag by Vestergaard. https://www.vestergaard. com/zerofly-storage-bags. (Accessed 26 March 2019). Vijayanna, S.V., 2006. Effect of Fumigation on Seed Quality during Storage of Groundnut (Arachis hypogaea Gaertn). A Thesis Submitted to the University of Agricultural Sciences, Dharwad in Partial Fulfillment of the Requirements for the Degree of Master of Science (Agriculture). Department of Seed Science and Technology College of Agriculture, Dharwad, India. Wasala, W.M.C.B., Dissanayake, C.A.K., Gunawaradhane, C.R., Wijewardhane, R.M.N.A., Gunathilake, D.M.C.C., Thilakarathne, B.M.K.S., 2016. Efficacy of insecticide incorporated bags against major pests of Stored paddy in Sri Lanka. Proceedia of Food Science 6, 146e169. White, G.D., Jacobson, E.T., 1972. Phosphine fumigation: effects on the germination of grass seed. J. Econ. Entomol. 65, 1523e1524.