Investigation of biological, ecological characteristics of anisopteromalus calandrae (howard) parasitizing stored product beetle pests in Dong Thap province

harmful insects. However, the use of phosphine is the main method to control insect pests in Viet Nam as well as in Dong Thap, while the Intergrated Pest Management (IPM) has not been properly applied. Thereby, determination of biological characterizations of A. calandrae is necessary to control stored insect pests. This study provides a biological and safe method instead of using insecticides to control these pests. 1.2. Reports in the world relating to this study 1.2.1. Studies of natural enemy species for stored insect pests A number of reports on natural enemy species for harmful insects have been published. For examples, Lebeck (1991) described 22 species of natural enemies for these pests. Sedlacek et al. (1998) recorded 7 parasitoid species parasitizing insects damaging stored argiultural products. A. calandrae is one of 9 parasitic species in 4 families (Encyrtidae, Eulophidae, Bethylidae and Pteromalidae) recorded in Southern Africa (Helbig, 1998). In Thailand, Hayashi et al. (2004) listed 29 prey species and 19 parasitoid species for stored insect pests. 4 1.2.2. Studies of Anisopteromalus calandrae 1.2.2.1. Morphological and biological characteristics of L. serricorne (a host of A. calandrae) Morphological and biological characteristics of L. serricorne have been determined by Visarathanonth (1985), Ryan (1999) and Mahroof and Phillips (2008). The authors also reported that the life cycle, logevity and egg laying of L. serricorne depended on its food types. 1.2.2.2. Morphological characteristics of A. calandrae Morphological characteristics of A. calandrae have been described by some authors (Hayashi et al. 2004; Baur et al. 2014). Some details of Anisopteromalus were described with pictures and keys to its species. 1.2.2.3. Behaviors of A. calandrae Biological Studies of behaviors of A. calandrae such as egg laying on large - sized hosts (usually 4th larvae instar). The behaviors including host searching, host stinging have been described. Moreover, the host searching at different depths (such as 14.5 cm) of the grain mass has been studied. 1.2.2.4. Biological characteristics of A. calandrae Studies of the life cycle and reproduction of A. calandrae show that the egg stage lasts 1 - 1.5 days on average. The life cycle of the parasitoid is from 11.4 days to 26.6 days depending on temperature, host larvae and complementary food, while adult longevity was in a range of 9.6 - 26.6 days. The progeny numbers produced by a female were 80.9 - 240.0. However, sometimes male longevity was only 5.4 - 6.0 days depending on its hosts. The lower developmental threshold 5 and the thermal constant of A. calandrae were determined as 11.5oC and 263.2 degree - days, respectively. 1.2.2.5. Ecological characteristics of A. calandrae Studies of ecology of A. calandrae including the competition with other parasitoid species have been published. Effects of food or food supplement and host density on the development A. calandrae have also been determined. The investigation of temperature influence on its development shows that the life cycles are various at 20, 25, 30°C, but no statistical difference on these at the temperature of 30°C and 35°C has been found. 1.2.2.6. Ability to control stored insect pests of A. calandrae Previous studies showed that A. calandrae effectively controlled some species for stored insect pests. The performance of controlling these species ranged from 32.24% to 85.0% depending on agricultural seeds and preservation methods. 1.3. Research in Vietnam 1.3.1. Research on natural enemies for stored insect pests There are only some studies of natural enemy species for stored insect pests in Viet Nam. Some authors reported these subjects such as Duong Minh Tu (2005) and Tran Van Hai (2008), Nguyen Quy Duong (2010), Nguyen Van Duong and Khuat Dang Long (2017). Most studies reported species compositions in specific places, but no publication showed the presence of A. calandrae. 1.3.2. Studies of morphological and biological characteristics of L. serricorne The investigation of L. Serricorne on tobacco, hot peppers, rice and stored peanuts was carried out by Bui Cong Hien (1995). 6 However, the Studies of morphological and biological characteristics of L. serricorne damaging produced fish food have not been documented yet. 1.3.3. Studies of biological characteristics, behaviors, ecology and the ability to control stored insect pests of A. calandrae No research on biological characteristics, behaviors, ecology and the ability to control stored insect pests of A. calandrae has been carried out in Viet Nam so far. General comments of related studies in Vietnam Overall, natural species of agricultural insect pests in storehouses in Vietnam have been investigated by some authors. For examples, Duong Minh Tu (2005) recorded 2 natural species in Northern Vietnam. 2 species were recorded by Tran Van Hai et al., (2008) in Can Tho and An Giang Province. In 2010, Nguyen Quy Duong described 7 species of natural enemies for stored pests on beans in Vietnam, including 3 parasitic species. Nguyen Van Duong and Khuat Dang Long (2017) recorded 6 species of parasitoids on maize in Son La. These results showed that only some the research on natural enemies of stored - grain pests in the storehouses have been conducted. Most publications have focused on recording the presence and general description of natural enemies species, but have not studied in details. Studies of biology, behaviors and ecology as well as control ability of A. calandrae for stored - grain pests in Vietnam have not been conducted yet. 7 CHAPTER 2. STUDYING SITES AND TIME AND METHODS 2.1. Time and sites for studying 2.1.1. Time for studying The research was conducted from November 2015 to December 2018. The sampling and field survey were carried out one time per month, 5 - 7 days per time, from November 2015 to December 2016. Further experiments were conducted in laboratory from 2016 to 2018. 2.1.2. Sites for researching The investigation of natural enemy species for controlling stored agricultural products was conducted in agricultural storehouses in countryside and town sites in Dong Thap Province, including Cao Lanh district, Thanh Binh district, Lap Vo district, Chau Thanh district, Cao Lanh city and Sadec city. A number of agricultural products and aquafeed types were collected from processing companies, mainly from Sadec city. 2.2. Preparation of insects and scale of the study ❖ Preparation of insects - Natural enemy species for stored insect pests. - Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). ❖ Scale of the study The investigation of natural enemy species for agricultural insect pests in storehouses was conducted in Dong Thap Province. The biological and ecological characteristics of A. calandrae parasitized and grew on larvae of L. serricorne. The ability to control 8 L. serricorne and S. zeamais by A. calandrae was evaluated in laboratories in Dong Thap University. 2.3. Proposal outline - Investigation of natural enemy species stored - product pests in Dong Thap Province. - Study of characteristics of morphology, biology, behaviors and ecology for parasitoid A. calandrae in the laboratory conditions, and some these characteristics of S. zeamais. - Study of the ability to control S. zeamais and L. serricorne by A. calandrae in laboratory conditions. 2.4. Materials 2.4.1. Materials - Catfish feed pellets (8 mm in diameter) were processed from raw materials including rice bran, broken rice, maize, soy, wheat grain and some other essential substances. - Bean seeds (Vigna unguiculata), corn seeds (Zea mays). - Some Coleoptera species are hosts of A. calandrae such as L. serricorne (Anobiidae), S. zeamais (Curculionidae) and C. maculatus (Bruchidae). 2.4.2. Chemicals and laboratory devices - Stainless steel samplers, sieves, insect sweep net samplers, flashlights. - Plastic containers, plastic rectangle boxes (length × width × height of 17 cm × 13 cm × 7 cm), plastic cylinder containers (20 cm in height, 14 cm in diameter of mouth and bottom), and plastic cylinder containers (7 cm in height, 12 cm in diameter of mouth and 9 cm in diameter of bottom); 9 - Petri dishes with 8 cm in diameter; - Carton boxes (length × width × height of 45 cm × 28 cm × 22 cm); - Soft polyethylene bags (55 cm in length, 36 cm in width); - Cover nets with mesh size < 0.01 mm and from 0.40 to 1.20 mm; - A magnifier with 70 times of magnification; - Microscope (Meiji Techno DK3000, Japan) with 1000 times magnification connecting to a computer with capture software of Lumenera INFINITY1 - 3C (Canada); - Camera Sony DSC W - 800 20.1 Mega; - Drying oven Memmert UN55 (Germany); - Sanyo refrigerated incubator, type MIR - 153 (Japan); - Extech temperature meter (Taiwan) and thermometer; - Digital Grain Moisture Meter Tester MD - 7822; - Digital Thermometer and Hygrometer HTC - 2; - And other devices. 2.5. Methods 2.5.1. Investigation of natural enemy species for stored agricultural products. There are two methods used in this experiment. - Investigation of the natural enemy species for stored agricultural products was carried out according to the plant quarantine method (based on Vietnamese standard: TCVN 4731 - 89). The natural enemy species were collected manually and using sampling collector nets. Collected insects were stored in carton boxes, soft polyethylene bags and glass tubes. - Samples with stored - grain possibly infested with pests were taken to the laboratory, stored in plastic containers covered with 10 ventilable textile fabric. The containers were monitored to the appearance of natural enemy species for stored insect pests. These species were collected and then stored in 70% alcohol solution for analysis. - Natural enemies species ware classified according to the documents of Graham (1969), Yoshimoto (1984), Janzon (1986), Boucek and Rasplus (1991), Noyes (2003), Hayashi et al. (2004), Lim et al. (2007), Baur et al. (2014) and Fayaz et al. (2016). Meanwhile, L. serricorne, S. zeamais and C. maculatus were classified based on the description of Haines (1991), Bui Cong Hien (1995) and Chaisaeng (2007). The abundance of a natural enemy species was determined as following equation: a 100 Abundance(%) b  = where a is numbers of sites with the appearance of a species, and b is the total numbers of sites. The appearance ratio of a species was determined as following equation: A 100 Appearance ratio (%) B  = where A is the appearance times of a species, and B is total investigation times. 11 2.5.2. Morphological, biological and ecological characteristics of A. calandrae The morphological characteristics of A. calandrae were measured and recorded to compare with the description of Pteromalidae family by Graham (1969), Yoshimoto (1984), Hayashi et al. (2004), Sureshan (2007), Janzon (1986), Baur et al. (2014). Moreover, the biological, behavioral and ecological characteristics of A. calandrae were also studied. 2.5.3. Determination of the ability to control stored - product pests by A. calandrae This experiment was conducted according to Chaisaeng (2007) and Visarathanonth et al. (2010). 2.5.4. Photographing and data analysis Photographing: A. calandrae was observed using stereomicroscope Meiji Techno DK3000 (Japan) with 70 times magnification. The measurement of specimens and picture processing were conducted using scale ruler Lumenera INFINITY1 - 3C software (Canada). Data analysis: Obtained data are shown as the mean ± one standard deviation. Microsoft Excel 2010 was used to set up grapths, while the SPSS software program version 22.0 was used to analyze variance, and significant differences (p < 0.05) were calculated using Duncan’s test. 12 CHAPTER 3. RESULTS AND DISCUSSION 3.1. Species composition of natural enemies for stored - product insect pests in Dong Thap Province 3.1.1. Species composition of natural enemies for stored - product insect pests 13 natural enemies for stored - product insect pests were recorded during the survey for 12 months. These species were in 10 families and 5 orders. 5 of them were carnivorous species, including Tyrophagus putrescentiae (Acarina), Forficula auricularia (Dermaptera), Xylocoris flavipes and Amphibolus venator (Heteroptera), and Chelifer cancroides (Pseudoscorpionida). 9 species are parasitoids of Hymenoptera. 3.1.2. Appearance ratio of natural enemies depending on types of agricultural products in storehouse The survey results showed that there were 11 species of natural enemies appearing in all samples of rice and wheat grains. 10, 9, 8 and 6 these species were found in aquafeed, maize grains, beans and rice bran, respectively, while only 2 species appeared in cassava samples. A. calandrae was found in 5 types of 7 stored agricultural products with the appearance ratios greater than 80%. 3.2. Morphological, biological and ecological characteristics of A. calandrae 3.2.1. Some morphological and biological characteristics L. serricorne - host of A. calandrae All stages the L. serricorne growing on aquafeed at 30 ± 1.07oC, humidity of 74.64 ± 3.17% were recorded. The results showed that the life cycle of L. serricorne is 41.53 ± 6.21 days on 13 average. The egg stage of the beetle was 7.10 ± 0.76 days, the 5th larvae stage was 26.4 days, pupa stage was 4.23 ± 0.82 days, while adult stage ranged from 33 to 53 days (temperature 30 ± 1.07oC, humidity 74, 64 ± 3.17%). 3.2.2. Morphologcal characteristics of A. calandrae Whole body of A. calandrae is dark - green color (Figure 3.8). Only males have gaster with milky white or opaque yellow colors on the 2nd, 3rd and 4th tergites. When parasitizing L. serricorne, females and males have a body length of 3.5 ± 0.3 mm and 2.4 ± 0.5 mm on average, respectively. The compound and ocellus eyes are reddish brown. An antenna has 13 segments (including scale). Legs with black coxae, black or brown, and yellow tibiae were identified. Each tarsus has 5 segments. Forewings have hard hairs, speculum bare and setea on wing disc dark. A space under forewings mergin has not any hair (Figure 3.8). Figure. 3.8. Adults of A. calandrae 14 3.2.3. Biological characteristics of A. calandrae 3.2.3.1. Activity behaviors of parasitoid adult ❖ The number of eggs in L. serricorne and a host insect On larvae of L. serricorne, A. calandrae females usually laid an egg on a host with 82.47%. Some females laid 2, 3, 4 and 5 eggs on a host with 10.31%, 5.62%, 1.12% and 0.47%, respectively. The ratios of A. calandrae emerged from a pupa with 1, 2, 3, 4 and 5 eggs were 85.45%, 47.27%, 28.33%, 25.0% and 20.0%, respectively. ❖ Behaviors of courtship and ovipositor - The courtship behavior of parasitoid males and the reaction of females When a male of the parasitoid encountered a female, he often run around and flirted with the female. The total time that the male run, flapped their wings and moved around the female was approximately 44.80 ± 10.40 seconds. Thereafter, the time that male jumped on the female’s abdomen and stroked the female’s antennae lasted 16.45 ± 2.21 seconds. - Ovipositor An A. calandrae male jumped on the back of the female, then moved to her abdomen to perform mating if the female agreed. The sex making lasted 22 ± 2.70 seconds. 15 - Successful ovipositor process and ovipositor time of A. calandrae Table 3.6. Successful ovipositor process and ovipositor time of A. calandrae Numbers of mating couples Mating after flirting (%) Mating time (seconds) Lần 1 Lần 2 Lần 3 16 53.33 87.50 ± 3.10 8 26.67 68.25 ± 5.31 6 20.00 56.67 ± 3.98 ❖ Host finding behavior of A. calandrae After ovipositor for about 1 day, females began to probe signals for finding their hosts by moving slowly on the grain mass surface. A female often identified a suitable host by using their mouths and antennae for laying her eggs. ❖ Host selection to lay eggs of a A. calandrae female When exposed to hosts at different age stages (stages of 1 - 5), prepupae and pupae of L. serricorne, a A. calandrae female tended to choose larvae stages of the 2nd, 3rd and 4th or prepupae and pupae. However, females do not lay eggs on the 1st and 5th larvae. 3.2.3.2. Behaviors of larvae A. calandrae laid its eggs on any position on the host body. After hatching, larvae of the 1st stage moved to an appropriate host and clung on the host. They sucked up the nutrient fluid from the host until reaching the 4th stage and becoming pupae. Larvae grew quickly from the first to the 4th stage. 16 3.2.3.3. Life cycle of A. calandrae parasitizing L. serricorne The development of A. calandrae in laboratory conditions (30 ± 1.0oC, 75 ± 3.1% RH) is shown in Table 3.7. Table 3.7. The development of A. calandrae parasitizing L. serricorne Stage N0 samples Time (days) Range Mean Egg 45 1.00 - 2.00 1.22 ± 0.42 1st larvae instar 115 1.00 - 1.40 1.08 ± 0.12 2nd larvae instar 102 1.00 - 1.50 1.21 ± 0.17 3rd larvae instar 104 1.00 - 1.70 1.30 ± 0.16 4th larvae instar 106 1.20 - 2.20 1.80 ± 0.24 Larvae instar stage 427 4.20 - 6.80 5.39 ± 0.69 Prepupae 48 0.30 - 0.70 0.49 ± 0.09 Pupae 45 7.00 - 10.00 8.58 ± 0.75 Prenatal stage 45 1.00 - 4.00 1.64 ± 0.77 Total time of female life 13.50 - 23.60 17.32 ± 2.72 The life cycle of A. calandrae is short and shown in Figure 3.31 with 17.3 ± 2.7 days on average. The time for egg development is 1.2 ± 0.4 days, larval phase was 5.4 ± 0.3 days, prepupa phase is 0.5 ± 0.1 days, pupa phase is 8.6 ± 0.7 days. The time to lay eggs is 1.6 ± 0.8 days. 17 Figure 3.31. Life cycle of A. calandrae 3.2.3.4. Longevity, parasitic ability and egg laying of A. calandrae parasitizing L. serricorne When feeding with honey solution (30%), females and males of A. calandrae could live 27.07 ± 2.89 and 24.33 ± 2.64 days, respectively. A female laid 71.13 ± 4.24 eggs in its life with 2.65 ± 0.28 eggs/day. The number of emerged adults was 55.00 ± 4.94 from a female of which 37.00 ± 4.12 females in its progeny. ❖ Egg laying rhythm of A. calandrae the larvae of L. serricorne During the life time, the average number of a female laid from 0.07 eggs/day on the first day to 7.4 eggs/day on the 9th day and then gradually decreased until the female died (Figure 3.33). 18 Figure 3.33. Egg laying rhythm of A. calandrae 3.2.3.5. Temperature threshold for development In all development phases of A. calandrae, the highest temperature threshold was at 12.96°C, followed by larvae stage (at 12.54°C), egg stage (at 8.77oC), prepupa (at 6.85oC), and emerged from pupae (5.78oC). The threshold for the whole life cycle is at 12.09oC. 3.2.4. Ecological characteristics of A. calandrae 3.2.4.1. Effects of temperature on the development of A. calandrae ❖ Life cycle time of A. calandrae developed at 20oC and 75% RH. When A. calandrae grew at 20oC and 75% RH, its life cycle was 45.38 ± 3.45 days, which was significantly longer than that at room temperature with 17.32 ± 2.72 days. 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 E g g s/ d a y /f em a le Age of female (days) 19 Table 3.10. Development of A. calandrae on larvae of L. serricorne (at 20oC, 75% RH) Stage development N0 samples Development time (days) Range Means Egg stage 45 2.00 - 3.00 2.63 ± 0.34 1st larvae instar 112 1.80 - 2.90 2.32 ± 0.30 2nd larvae instar 106 1.90 - 3.50 2.58 ± 0.41 3rd larvae instar 108 2.40 - 3.50 2.95 ± 0.33 4th larvae instar 109 2.60 - 4.00 3.20 ± 0.31 Larvae instar stage 435 8.70 - 13.90 11.05 ± 0.35 Pupa 55 0.50 - 1.60 0.98 ± 0.23 Prepupa 45 25.00 - 32.00 27.76 ± 1.19 Prenatal stage 45 2.30 - 4.00 2.96 ± 0.34 Total time of female life 38.50 - 54.50 45.38 ± 3.45 ❖ Life cycle time of A. calandrae at 25oC and 75% RH When A. calandrae grew at 25oC and 75% RH, its life cycle was shorter compared to at 20oC and 75% RH. The results are presented in Table 3.11. Table 3.11. Development of A. calandrae on larvae of L. serricorne (at 20oC, 75% RH) Stage development N0 samples Development time (days) Range Means Egg stage 45 1.00 - 2.50 1.82 ± 0.48 1st larvae instar 105 1.00 - 1.60 1.32 ± 0.16 2nd larvae instar 106 1.00 - 2.00 1.37 ± 0.23 3rd larvae instar 108 1.30 - 2.40 1.69 ± 0.22 4th larvae instar 109 1.50 - 2.50 2.09 ± 0.21 20 Larvae instar stage 428 4.80 - 8.50 6.47 ± 0.82 Pupa 51 0.50 - 1.00 0.71± 0.16 Prepupa 45 14.00 - 20.00 16.62 ± 1.50 Prenatal stage 45 1.00 - 4.00 2.19 ± 0.55 Total time of female life 17.50 - 34.50 17.50 - 34.50 The development of A. calandrae was apparently affected by temperature when it parasitized L. serricorne. When it grew at 75% RH and at 20oC, 25oC and 30oC, the life cycles were longer at lower temperatures. The life cycles lasted 45.38 ± 3.45, 27.80 ± 3.51 and 17.32 ± 2.72 days at these temperatures, respectively. 3.2.4.2. Effects of hosts and food supplement on the longevity, and egg laying of A. calandrae Mature females and males could live for 10.73 ± 1.72 and 10.52 ± 1.98 days, respectively, when they were fed on only distilled water. Meanwhile, their logevities were longer when they were fed on honey bee solutions from 10% to 50%. The highest longevity of females and males were recorded when A. calandrae was fed on honey solution at 30%. 3.2.4.3. Effects of host density on egg production of female A. calandrae The experiment showed that the number of L. serricorne larvae suitable for 1 pair of A. calandrae was about 25 - 30 host larvae (Table 3.16). 21 Table 3.16. Correlation between the density of L. serricorne larvae and the number of parasitic hosts, the number of parasitic eggs/hosts Density of larvae (individuals)/box Parasitized larvae (individuals)/day Eggs/host/day 20 3.87 2.67 25 2.64 1.53 30 2.51 1.08 3.2.4.4. Effects of A. calandrae density on the sex ratio of their offspring parasitizing S. zeamais and L. serricorne A. calandrae was released at different densities to control S. zeamais and L. serricorne. The results showed that the increase of released A. calandrae reduced the ratio of male to female in their descendants in general. 3.3. Ability to control insect pests by A. calandrae in the laboratory condition 3.3.1. Ability of A. calandrae to control S. zeamais fed on Vigna unguiculata After releasing 6, 8 and 10 couples of A. calandrae, the appearance of S. zeamais fed on V. unguiculata reduced by 30.0 ± 2.1%, 28.7 ± 1.7% and 23.1 ± 2.0%, respectively. 3.3.2. Ability to control L. serricorne fed on aquafeed by A. calandrae After releasing 10 couples of A. calandrae to control S. zeamais fed on aquafeed for 3 months, the L. serricorne was reduced by nearly 85%. 22 3.3.3. The ability of A. calandrae to control L. serricorne fed on aquafeed The experiment was carried our using 100 g tablets of aquafeed placed in a carton box. After 3 months, the number of L. serricorne was reduced by 72.2 - 80.9%. In another experiment carried out in a carton box containing 5 kg the aquafeed, the number of L. serricorne was reduced by 74.8 - 82.3%. CONCLUSION AND RECOMMENDATION Conclusion 1. 13 species natural enemies of stored - product insect pests in Dong Thap Province were identified. These species were 5 carnivorous species and 8 parasitoid species, and belong to 10 families, 5 orders. 11 species were first recorded in agricultural stored - product in Dong Thap, and 8 species were considered as natural enemy species in stored - product in Viet Nam. Among these species, A. calandrae was found at over 80% in 5/7 types of stored - product. 2. The life cycle of A. calandrae grew on L. serricorne is 17.3 days (at 30°C, 75% RH). An individual of A. calandrae laid 71.1 eggs and parasitized 63.2 young larvae on average. 55 individuals (37.0 females) were produced from a female. A femele laid from 1.0 to 12.0 eggs/day, and from 1.0 to 5.0 eggs/host larva. 82.5% females laid 1 egg on a host. 23 3. A. calandrae has a behavior to lay eggs on hosts who hide in agricultural seeds. There is no egg laid on freely moving larvae outside the seeds. Females prefer to lay eggs on 3rd larvae instar and 4th larvae instar, while no egg was laid on 1st l