Trichogramma (Hymenoptera: Trichogrammatidae) are minute polyphagous wasps and endoparasitoids of lepidopteran eggs. The objective of this study was to identify Trichogramma species emerging from the eggs of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) attacking corn, Zea mays L. (Poaceae) in Bunga Raya, Siak, Riau, Sumatra, Indonesia, and individuals from an unidentified species obtained from a commercial laboratory in Jatisari, Karawang, West Java, Java, Indonesia. The emergence rate of Trichogramma yousufi sp. nov. Khan & Ikram (Hymenoptera: Trichogrammatidae) on an infestation containing eggs of both pest species, beet armyworm, Spodoptera exigua (Hübner), and the tobacco cutworm, Spodoptera litura (F.) (both Lepidoptera: Noctuidae), on Acacia crassicarpa A. Cunn ex Benth. (Fabaceae) also was evaluated in a nursery in Pangkalan Kerinci, Riau. Two Trichogramma species, namely Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) and a new species, T. yousufi sp. nov., were recovered from the eggs of O. furnacalis in Bunga Raya. Trichogramma poliae Nagaraja (Hymenoptera: Trichogrammatidae) was identified as the species reared in the laboratory in Jatisari. Trichogramma yousufi sp. nov. was compared and separated from its closer species, Trichogramma latipennis Haliday (Hymenoptera: Trichogrammatidae). Trichogramma species, recovered from corn crops and 1 that also was reared in the laboratory, successfully parasitized the eggs of S. exigua and S. litura under controlled conditions. The emergence rate of T. yousufi sp. nov. was recorded at 24.79 to 40.82%, with the highest percentage for more adults of this parasitoid released per m2 in the nursery, indicating its potential to be employed as a biological control agent of Spodoptera in natural conditions.
The beet armyworm, Spodoptera exigua (Hübner) and the tobacco cutworm, Spodoptera litura (F.) (both Lepidoptera: Noctuidae) are important polyphagous pests with a cosmopolitan distribution (Li et al. 2015). They are recorded as severe defoliating pests of Acacia crassicarpa A. Cunn. ex Benth. (Fabaceae) plantings that are growing in commercial nurseries in Riau, Sumatra, Indonesia (Sulistyono et al. 2020). Spodoptera exigua and S. litura possibly migrate to A. crassicarpa from onion, Allium cepa L. (Amaryllidaceae), crops because of the extensive area in which this vegetable is cultivated, which is recorded annually as being infested with these pests in Riau (Sulistyono et al. 2020).
The minute polyphagous wasps, Trichogramma (Hymenoptera: Trichogrammatidae), are endoparasitoids of lepidopteran eggs (Leite et al. 2015, 2017; Pontes et al. 2019). Trichogramma could be recovered locally from crops such as corn, Zea mays L., and sugarcane, Saccharum officinarum L. (both Poaceae), and used in a program to control S. exigua and S. litura on A. crassicarpa plantings in nurseries in Sumatra. Trichogramma species are not well understood in Indonesia (Yunus 2018). Studies on taxonomy characters and the biology of Trichogramma species on the eggs of alternative and natural hosts in Indonesia are important information for the management of lepidopterans in several crops (Jiang et al. 2019; Khan et al. 2019).
The objectives of this study were undertaken to identify the Trichogramma species recovered from the eggs of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), attacking corn in Bunga Raya, Siak, Riau, Sumatra, Indonesia, and from an established colony in a laboratory in Jatisari, Karawang, West Java, Java, Indonesia. Additionally, to evaluate some biological parameters of these parasitoids on eggs of the rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) (an alternative host), S. exigua, and S. litura (natural hosts) in the laboratory. Finally, we wanted to discover the parasitism rate of Trichogramma yousufi sp. nov. Khan & Ikram (Hymenoptera: Trichogrammatidae) on eggs of a mixed population of S. exigua and S. litura recovered after a release of this parasitoid in an A. crassicarpa nursery in Pangkalan Kerinci, Riau.
Materials and Methods
COLLECTION OF TRICHOGRAMMA SPECIES
Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) and T. yousufi sp. nov. were reared on eggs of O. furnacalis collected from a corn field during an exploration at Bunga Raya, Indonesia (0.7500°N, 102.0500°E, 15 masl). Trichogramma poliae Nagaraja (Hymenoptera: Trichogrammatidae) was obtained from Belai Besar Peramalan Organisme Pengganggu Tumbuhan, a commercial laboratory in Jatisari, Indonesia (6.3000°S, 107.0000°E, 100 masl), where it had been reared for about 5 yr on eggs of C. cephalonica and delivered to farmers to manage lepidopteran pests on sugar cane crops.
LABORATORY REARING
Eggs of O. furnacalis, parasitized by T. chilonis and T. yousufi sp. nov., and of C. cephalonica by T. poliae were taken to the Entomology Laboratory of PT. Riau Andalan Pulp and Paper in Pangkalan Kerinci, Riau, Indonesia, where they were kept at 26 ± 2 °C, 75 ± 5% RH, and a 12:12 h (L:D) photoperiod. Corcyra cephalonica (Cruz 1980) and O. furnacalis (Seo et al. 2014) were identified based on the analysis of the external body morphology of adults. Colonies of these parasitoids were established on C. cephalonica eggs in the Entomology Laboratory.
MAINTENANCE OF TRICHOGRAMMA COLONIES
Corcyra cephalonica eggs were obtained from oviposition cages in the laboratory. These cages each consisted of a cylindrical PVC tube (12 cm diam × 30 cm high) with its top opening covered by organza fabric. Newly deposited eggs were separated from impurities using a cotton ball and were glued with diluted Arabic gum (90:10% Arabic gum:distilled water) onto sheets of paperboard (10 cm long × 2 cm wide), totaling around 5,000 C. cephalonica eggs per paperboard sheet. Paperboard sheets then were placed on a plastic tray and exposed to ultraviolet light for 1 h in a chamber (40 cm long × 40 cm high × 40 cm wide) equipped with 2 lamps (Gaxindo®, GX-N093, T5-8W, Jakarta, Indonesia; purple light, lamp length = 29 cm, lamp diam = 1.5 cm, lamp wavelength = 28.5 cm, height from lamp to paperboard sheets = 30 cm) to make the C. cephalonica embryo unviable. After the inviabilization process, paperboard sheets were exposed individually to a colony of T. chilonis, T. poliae, or T. yousufi sp. nov. adults in a glass jar (12 cm long × 3 cm wide) for 72 h (3,000–3,500 adults per jar). Drops of honey were placed on the inner surface of the jars daily as food for the Trichogramma adults. Steps used to rear the larvae, pupae, and adults of C. cephalonica followed Amadou et al. (2019) with slight modifications.
BIOLOGICAL PARAMETERS OF TRICHOGRAMMA PARASITIZING SPODOPTERA EGGS IN THE LABORATORY
Spodoptera exigua and S. litura eggs and larvae were collected manually from an A. crassicarpa commercial nursery in Pangkalan Kerinci, and taken to the Entomology Laboratory where the larvae were reared on a solid, cube-shaped artificial diet, following fabrication steps described by Tavares et al. (2019). Spodoptera exigua and S. litura newly deposited eggs, laid on paper sheets used as oviposition subtrate in cages as previously described, were exposed to ultraviolet light for 1 h in a chamber to make the Spodoptera embryo unviable as previously described. Spodoptera exigua or S. litura paperboard sheets were prepared by cutting the egg masses from the paper sheets leaving 1 mass per sheet, followed by gluing the egg masses onto sheets of paperboards. Each sheet of paperboard was exposed to a colony of T. chilonis, T. poliae, or T. yousufi sp. nov. adults in a jar for 72 h as previously described (each sheet of paperboard had 6 egg masses exposed to 3,000–3,500 adults per jar). One egg mass had an average of 60 and 75 eggs for S. exigua and S. litura, respectively.
After subjection to parasitism, 1 sheet of paperboard was placed into a test tube (2 cm diam × 12 cm high), where it was kept until adult Trichogramma or larval Spodoptera hatched. Individuals of S. exigua and S. litura, from the same laboratory colony, were recently identified through morphology and confirmed via molecular analysis (Sulistyono et al. 2020). The design was completely randomized with 9 treatments (each Trichogramma species parasitizing 1 of Lepidoptera), and with 25 replicates each with a sheet of paperboard. The following parameters were evaluated: incubation period, life cycle, adult longevity (d), female-biased sex ratio (number of females/number of insects), parasitism, and emergence (%). Blackened eggs, 4 d after subjection to parasitism, were classified as parasitized. Males and females of Trichogrammatidae were identified by dimorphism of their antennae, feathery in males, and nailed in females (Santos et al. 2020). The data were submitted to Analysis of Variance, and averages were compared by the Tukey range test (Tukey 1949) at 5% probability.
PARASITISM RATE OF SPODOPTERA BY TRICHOGRAMMA YOUSUFI SP. NOV. IN THE NURSERY
Trichogramma yousufi sp. nov. sheets of paperboard, consisting of fresh eggs of C. cephalonica subjected to parasitism for 72 h, were taken to a commercial nursery of A. crassicarpa and were infested by about 95% S. exigua and 5% S. litura in Pangkalan Kerinci. The percentage of Spodoptera species was previously evaluated by collecting the moths using light traps. There were 3 treatments each with 5 replicates: 1, 2, or 5 paperboard sheets stapled per 1, 2, or 3 ground sand beds, respectively. Paperboard sheets were stapled on the abaxial surface of a leaf from the middle plant. Each bed (24 m long × 1 m wide) was planted with 1,280 plantings. The experimental area was visited every d after the trial was established, and all new Spodoptera egg masses were eliminated. Five d after the paperboard sheets were stapled, 100 Spodoptera egg masses were randomly selected and recovered per treatment by cutting them from the entire leaf using scissors; then they were placed in 500 mL plastic containers (Easiyo®; Pekanbaru, Riau, Indonesia) and taken to the Entomology Laboratory where they were kept under incubation in glass jars (1 mass per jar) until adult Trichogramma or larval Spodoptera hatched. Trichogramma emergence rate was evaluated using the following formula: emergence rate = number of egg masses with adult Trichogramma hatched/number of recovered egg masses × 100. The data were submitted to Analysis of Variance, and averages were compared by the Duncan new multiple range test (Duncan 1955) at 5% probability.
TRICHOGRAMMA IDENTIFICATION, DESCRIPTION, PHOTOGRAPHY, AND DEPOSIT
Trichogramma species were identified and described after the external and internal morphology analysis of the male. Photographs were taken under a stereo zoom binocular microscope (ARK-zoom Star-VI, AmScope®; Irvine, California, USA) using a Leitz Labor Lux S (Leica®, Wetzlar, Germany) camera. Body measurements were made with oculometer of different magnification powers (10×, 20×, 40×) in the Leitz Labor Lux S. Trichogramma yousufi sp. nov. is deposited under the registration NFIC Accession No.: 22321, Depository: National Forest Insect Collection, Forest Protection Division, Forest Entomology Discipline, Forest Research Institute, Dehradun, Uttarakhand, India 248006.
Results
PARASITISM IN THE LABORATORY
This is the first report of T. chilonis and T. yousufi sp. nov. parasitizing eggs of O. furnacalis. Parasitism of eggs of C. cephalonica, S. exigua, and S. litura was successful using T. chilonis, T. poliae, and T. yousufi sp. nov. The mean incubation period, life cycle, adult longevity, and female-biased sex ratio of Trichogramma were similar on eggs of Spodoptera and C. cephalonica (P < 5%). The parasitism and emergence rates were greater on C. cephalonica eggs than on those of Spodoptera (P > 5%) (Table 1).
PARASITISM IN THE NURSERY
The emergence rate of T. yousufi sp. nov. parasitizing egg masses of a mixed infestation of S. exigua and S. litura in a commercial nursery of A. crassicarpa (Fig. 1A, B) was greater with the density of 1 paperboard sheet stapled per ground sand bed (P > 5%; Table 2).
Table 1.
Biological parameters (mean) of Trichogramma chilonis, Trichogramma poliae, and Trichogramma yousufi sp. nov. (Hymenoptera: Trichogrammatidae) parasitizing egg masses of Corcyra cephalonica (CC) (Lepidoptera: Pyralidae), Spodoptera exigua (SE), and Spodoptera litura (SL) (both Lepidoptera: Noctuidae) in the laboratory.
DESCRIPTION OF TRICHOGRAMMA YOUSUFI SP. NOV. KHAN & IKRAM (FIG. 2A–F)
MALE. Body length 0.51 mm. Body honey yellow; ocelli and eyes bright red. Antennae pale brown with 32 to 35 thick and pointed hairs on flagellum. Midlobe of mesoscutum and scutellum light brown, forewings hyaline except light infuscation beneath STV; RS1 having 4 setae. Genital capsule longer than combined length of adeagus and apodemes.
HEAD (Fig. 2E). 1.45× wider than long in facial view (192: 132); ocelli arranged in obtuse triangle; mandible with 3 denticles. Antennae (Fig. 2E) with scape, 3.6× as long as broad (77: 21); pedicel 1.5× as long as wide (35: 23) and slightly wider than scape (21: 23), 2-anelli (A1 and A2) present; flagellum having 31 to 35 thick and pointed hairs, 4.7× as long as broad (151: 65); flagellar setae longest of which about 2× as long as maximum width of flagellum (65: 32).
MESOSOMA. Mid lobe of mesoscutum about as long as wide; scutellum about 2× as wide as long (70: 35); propodeum about 2× longer than dorsellum medially. Forewings (Fig. 2C) about 2× as long as wide (428: 228) having RS1 4 seate (Fig. 2D); discal setae arranged in rows; costal cell narrow; marginal fringe about 1/8th of forewing width (30: 228).
METASOMA. Gaster about 2× longer than mesosoma (Fig. 2A); genital capsule slightly shorter than hind tibia (144: 140); aedeagal length slightly shorter than apodemes, both together slightly shorter than genital capsule (132: 140).
FEMALE (Fig. 2B). Body length 0.50 mm. Same as male except antenna having scape 3.7× as long as wide (85: 23); pedicel 1.7× as long as wide; club 2.4× as long as wide (85: 35). Ovipositor slightly longer than hind tibia (159: 156).
HOST. Eggs of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae).
TYPE SPECIMEN EXAMINED. Holotype, ♀ (on slide). Bunga Raya, Siak, Riau, Sumatra, Indonesia; 28-29 Nov 2018. Coll. Wagner de Souza Tavares. Ex. Eggs of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae).
PARATYPES. 2♂♂ & 2♀, same data as holotype.
DISTRIBUTION. Indonesia: Riau.
ETYMOLOGY. The species name is derived from the name of Dr. Mohd. Yousuf, Scientist-G, Forest Research Institute, Uttarakhand (India), who has provided the major contribution in taxonomy of the world Trichogrammatidae.
REMARKS. Trichogramma yousufi sp. nov. is very close to Trichogramma latipennis Haliday, but can be distinguished by the male body length of 0.51 mm. Antennae with scape, 3.6× as long as broad; pedicel 1.5× as long as wide; flagellum 4.7× as long as broad; longest hair of flagellum about 2× as long as maximum width of flagellum; flagellum having 31 to 35 thick, pointed hairs. Forewings (Fig. 2C) about 2× as long as wide (428: 228) having RS1 4 setae; discal setae arranged in rows; costal cell narrow; marginal fringe about 1/8th of forewing width (30: 228). FEMALE. Body length 50 mm. Antenna with club more than 2× as long as wide. Ovipositor hidden, slightly shorter than hind tibia.
Fig. 1.
Adults of Trichogramma yousufi sp. nov. (Hymenoptera: Trichogrammatidae) parasitizing an egg mass of Spodoptera exigua (Lepidoptera: Noctuidae) (A) and a parasitized egg mass (B) in a commercial nursery of Acacia crassicarpa (Fabaceae) in Sumatra, Indonesia.
In T. latipennis, male body length 0.42 mm. Antennae with scape, slightly less than 4× as long as broad; pedicel about 2× as long as wide; flagellum 4.7× as long as broad; longest hair of flagellum about 1.5× as long as maximum width of flagellum; flagellum having 41 blunt hairs. Forewings about 2× as long as wide (428: 228) having RS1 4 setae; discal setae arranged in rows; costal cell narrow; marginal fringe about 1/8th of forewing width (30: 228). FEMALE. Body length 41 mm. Antenna with club less than 2× as long as wide. Ovipositor slightly exserted, about as long as hind tibia.
TRICHOGRAMMA CHILONIS ISHII (FIG. 3A)
DIAGNOSIS. Trichogramma chilonis. Body about 0.45 mm long, honey yellow; male antennae with 35 to 45 long and tapering flagellar hairs, longest hair about 2.5× as long as maximum width of flagellum; forewings with fringe on tornus about 1/6th the wing width; genitalia having DEG triangular with prominent lateral lobes; chelate structure clearly below the level of gonoforceps; median ventral process broad at base; aedeagus longer than apodemes; both together slightly shorter than hind tibia; female with ovipositor as long as hind tibia.
TRICHOGRAMMA POLIAE NAGARAJA (FIG. 3B)
DIAGNOSIS. Trichogramma poliae. Body slightly more than 0.5 mm long, light brownish yellow; male antennae having flagellum with 30 to 35 long tapering hairs, longest hair about 3× the maximum width of flagellum; forewings with marginal fringe on tornus about 1/6th the wing width; male genitalia having DEG with prominent lateral lobes; CS below the level of GF; MVP large and broad at base; apodemes shorter than aedeagus, both together slightly shorter than hind tibia. Female with ovipositor about as long as hind tibia.
Table 2.
Emergence rate (mean) of Trichogramma yousufi sp. nov. (Hymenoptera: Trichogrammatidae) parasitizing egg masses of a mixed population of Spodoptera exigua and Spodoptera litura (both Lepidoptera: Noctuidae) in a commercial nursery of Acacia crassicarpa (Fabaceae) in Sumatra, Indonesia.
Discussion
The similar incubation period of Trichogramma shows the eggs of Spodoptera and C. cephalonica being able to provide adequate nutrients to the larva of this parasitoid. Eggs of C. cephalonica (573.5 µm long × 346.1 µm wide, with a volume of 0.036 mm3) and Spodoptera (454.9 µm long and 390.2 µm wide, with a volume of 0.036 mm3), similar in size and volume (Cônsoli et al. 1999), are suitable to allow Trichogramma to produce large and vigorous adults, resulting in a standard life cycle period and survival rate (Firake & Khan 2014; Edwin et al. 2016). The higher number of females produced with eggs of both host species is beneficial in laboratory rearing for the release of this parasitoid (Brotodjojo & Walter 2006; Oliveira et al. 2017). The lower parasitism and emergence rate of Trichogramma on Spodoptera is possibly due to the ability of the eggs of this pest to encapsulate the parasitoid larva (Dorémus et al. 2013) and promote physical protection against parasitoids (Sá & Parra 1994). Spodoptera eggs are laid in 2 to 3 layers and are protected with hairs deposited by moths, with the parasitoid ovipositor encountering difficulties reaching those in deeper layers (Cônsoli et al. 1999).
The greater emergence rate of T. yousufi sp. nov. with a density of 1:1 (paperboard sheet:ground sand bed) was expected because a higher number of adults of this parasitoid was released per m2. Higher densities of Trichogramma released per m2 are more successful in parasitism (Jin et al. 2019; Mohammadpour et al. 2019). The emergence rate of T. yousufi sp. nov. in the current study was greater than that of Trichogramma japonicum Ashmead (Hymenoptera: Trichogrammatidae) on eggs of the rice yellow stem borer, Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), by 2.05% in Asian rice, Orysa sativa L. (Poaceae), cabbage, Brassica oleraceae L. (Brassicaceae), corn, and tomato, Solanum lycopersicum L. (Solanaceae), crops and of Trichogramma chilotrae Nagaraja & Nagarkatti (Hymenoptera: Trichogrammatidae) on those of a mixed infestation of the cabbage cluster, Crocidolomia pavonana (Fabricius) (Lepidoptera: Crambidae), and S. litura by 1.67% on cabbage crops in Solok, West Sumatra, Indonesia (Hidrayani et al. 2013).
Fig. 2.
Trichogramma yousufi sp. nov. (Hymenoptera: Trichogrammatidae) adult male (A), adult female (B), fore wing (C), RS1 (D), head with antennae (E), and male genitalia (F).
Fig. 3.
Genitalic difference between adults of Trichogramma chilonis (A) and Trichogramma poliae (B) (Hymenoptera: Trichogrammatidae).
In conclusion, T. chilonis and the new species, T. yousufi sp. nov., were recovered from O. furnacalis eggs during a field exploration in Bunga Raya, whereas T. poliae was obtained from C. cephalonica eggs in a laboratory in Jatisari. The separation of T. yousufi sp. nov. from its closer species, T. latipennis, has been discussed in greater detail. The taxonomic characters of all 3 Trichogramma species have been given precisely. The Trichogramma species, recovered from corn crops and reared in a laboratory, successfully parasitized S. exigua and S. litura eggs under environmentally controlled conditions, with the parasitism rate of T. yousufi sp. nov. ranging from 24.79 to 40.82% on the eggs of S. exigua and S. litura attacking A. crassicarpa plants in a commercial nursery in Pangkalan Kerinci. It therefore appears from this study that T. yousufi sp. nov. may be employed as a biological control agent to manage Spodoptera in Acacia nurseries.
Acknowledgments
We would like to thank the Kerinci Central Nursery 1 management for their logistical support. Adam Batten, a Senior Editor at Get It Right in Jakarta, Indonesia, revised and corrected the English language used in this manuscript. This study received financial support from the following Indonesian companies: Asia Pacific Resources International Holdings Ltd. and PT. Riau Andalan Pulp and Paper.
