Developmental Biology of Trichospilus pupivorus Ferrière (Hymenoptera: Eulophidae), a Pupal Parasitoid of the Coconut Black-headed Caterpillar, Opisina arenosella Walker (Lepidoptera: Xyloryctidae)

Background: The coconut black-headed caterpillar, Opisina arenosella Walker (Lepidoptera: Xyloryctidae) has been a serious defoliator of coconut plants in many coconut producing countries including Vietnam. Trichospilus pupivorus Ferrière (Hymenoptera: Eulophidae) is pupal endo-parasitoid of O . arenosella with high percent parasitism and abundance in the coconut fields and appears to be a good biological control agent against the coconut black-headed caterpillar in Vietnam. The objective of this study was to investigate the developmental biology of T . pupivorus on O . arenosella . Methods: The experiment was carried out at the Faculty of Agronomy, Nong Lam University, Viet Nam during 2020 and 2021. The developmental biology of T . pupivorus on O . arenosella was studied in the laboratory at a constant temperature of 28  C and a photoperiod of 12L: 8D. Result: Total developmental time from egg to adult emergence was 14.0 days. The females laid a mean of 124.2 eggs during an average lifespan of 9.4 days. The number of progenies emergence from a single pupa of O . arenosella was 103.8. The offspring sex ratio was female-biased (8.0%). The results would contribute to the knowledge of the biology of this parasitoid to optimize a mass rearing system for a biological control program against O . arenosella .


INTRODUCTION
The coconut black-headed caterpillar, Opisina arenosella Walker (Lepidoptera: Xyloryctidae), an indigenous pest and outbreak insect pest of coconut in Sri Lanka and India (Perera et al., 2010;Chalapathi et al., 2016), has been one of the most serious defoliator of coconut plants in many coconut producing countries.Its serious damage has been reported in some Southeast Asian countries including Thailand (Kumara et al., 2015), Malaysia (Nor Ahya and Tajul Ariffin, 2018), Vietnam (Le et al., 2020).Recently, outbreak of the defoliator has been found in coconut across Vietnam and it controlled by a wide range of conventional insecticides, which was ineffective (Le et al., 2020).It is necessary to consider a biological control program based on the use of parasitoids against this pest.
Information on basic biology of a parasitoid species (e.g.developmental time, longevity, fecundity, sex ratio) is fundamentally necessary to evaluate its effectiveness as a biological control agent.However, biological studies of T. pupivorus developing on O. arenosella has been limited.The objectives of the present studies are to determine development time for immature developmental stages and longevity, fecundity

MATERIALS AND METHODS
The experiment was carried out at the Laboratoty of Entomology, Faculty of Agronomy, Nong Lam University, Viet Nam during 2020 and 2021.The coconut blackheaded caterpillar, O. arenosella andthe wasp parasitoid, T. pupivorus used for the present study were collected from infested coconut plants at Tan Thanh Binh commune, Mo Cay Bac district, Ben Tre province, Southern Vietnam (101676N 1063572E).Opisina arenosella was reared on coconut leaves inside 30  30  25 cm cages.The colony of O. arenosella was maintained on coconut leaves for two to three generations prior to use in experiments.The parasitoids were offered with three-days-old pupae of O. arenosella in plastic bottles (500 ml).Parasitoid ad ults were p rovid ed with a ho ney solu tio n (30 %) imm ed iately after emerg en ce.T he c olo nies w ere maintained under the condition of 28±1 C, 60-70% humidity and 12L: 12D photoperiod.
Sixty 3-days-old pupae of O. arenosella and a piece of tissue paper (2 cm  2 cm) saturated with a honey solution (10%) were placed in a plastic bottle (160 ml) covered with a fine nylon mesh.About 20 couples of T. pupivorus were introduced into the box.After the exposure for 24 h,parasitized pupae were transferred into other plastic boxes.The boxes with parasitized pupae were maintained in the environmental chambers set at 28±1C, 60-70% humidity and 12L: 12D photoperiod until emergence of the parasitoids.Every day, 2 parasitized pupae were dissected using a stereo microscope (Olympus SZX10, Japan) to determine the developmental times of parasitoid eggs, larvae and pupae.
Three 3-days-old pupae of O. arenosella were placed in a 5 ml glass vials covered with a fine nylon mesh.One pair of newly emerged wasps was released into the vial.Honey solution (30%) was streaked on a piece of Sealon film (Fuji Photo Film Co., Ltd.).The streaked honey film was attached to the top of the vial and replaced daily to provide wasps with fresh food.These vials were kept in the environmental chambers set at 28±1 C, 60-70% humidity and 12L : 12D photoperiod.After the exposure for 24 h, the pupae were removed and were then dissected under a microscope to check for paralyzed eggs.Pupae of O. arenosella were exchanged daily until the females died.The number of parasitoid eggs was recorded as fec un dity c apacity and lo ng evity of females was determined.A total of 10 females were used for test.
Ten parasitized pupae were individually placed in 5 ml glass vials maintained at the same experimental condition until wasp emergence.All offspring wasps were sexed.The sex ratio is expressed as the proportion of males among the offspring (Godfray, 1994).

RESULTS AND DISCUSSION
Developmental time for immature stages of T. pupivorus is summarized in Table 1.Total developmental time from egg to adult emergence was 14.0 days.The duration of egg and larva periods were 1.3 and 6.7 days, respectively.The pupal development lasted an average of 6.0 days.Although T. pupivorusis a potential biological control agent to control O. arenosella (Nor Ahya et al., 2019, Le at al., 2020), limited data on the developmental biology of T. pupivorus on O. arenosella are available.The present study indicated that total developmental time from egg to adult emergence of T. pupivorus was ranged from 13-15 days.Our results are in agreement with the results of Gosh and Abburahiman (1985) who reported that developmental times for immature stages of T. pupivorus on O. arenosella was 13-15 days.
The females produced a mean of 124.2 eggs during an average lifespan of 9.4 days (Table 2).Longevity of the female (9.4 days) was longer than that of the male (2.8 days).W hile the pre-oviposition period lasted for 2.5 days after emergence, the females stopped laying eggs (postoviposition) about an average of 4.1 day before death.The fecundity of T. pupivorus on O. arenosella was higher than the results of Nor Ahya et al. (2020) who reported that fecundity of T. pupivorus rearedon O. arenosella with 30% honey at 30C was 105.4 progenies.However, female longevity (9.4 days) was longer than that was recorded by Gosh and Abburahiman (1985) (5.0 days) and Nor Ahya et al.
The number of progenies emergence from a single pupa of O. arenosella was 103.8 in average with a range of 28-161 (Table 3).Gosh and Abburahiman (1985) also reported that the number of eggs laid on a single O. arenosella was 22-162 days.The offspring sex ratio (8.0%) was female-biased (Table 3).There was no other information on the offspring sex ratio o f T .p upiv oru s to be a comparison.However female-biased sex ratio of T .pupivorus is similar with other eulophid parasitoids (Tran and Takagi, 2006).Because laboratory rearing O. arenosella on coconut leaves is laborious, a maintain of O. arenosella on large scale on natural host (e.g.coconut leaves) is not possible to supply insect hosts to a mass-rearing of the parasitoid T. pupivorus.Murthy et al. (2002) indicated that an artificial diet reared O. arenosella can be used for mass rearingthe parasitoidB.nephantidis without depending on the natural host, plant host and factitious hosts.Moreover, previous studies reported that Galleria mellonella L. (Lepidoptera: Galleridae) was a potential alternative host for the rearing of Goniozus nephantidis Muesebeck (Hymenoptera: Bethylidae), a larval parasitoid of O. arenosella (Mohan and Shameer, 2003;Venkatesan et al., 2007) Therefore, those alternative rearing systems can be used for a mass rearing of T. pupivorus.W hile previous studies have indicated that biology of an insect parasitoid depends on abiotic and biotic factors including temperature, relative humidity and host available (Tran et al., 2007;Tran et al., 2012), suitability of T. pupivorus reared with artificial diet and alternative hosts as well as temperature dependent development of T. pupivorus are needed further studies.

CONCLUSION
It is concluded that T. pupivorus could completely develop on the pupae of O. arenosella.The total developmental time from egg to adult emergence of T. pupivoruswas 14.0 days.The females produced a mean of 124.2 eggs during an average lifespan of 9.4 days.The number of progenies emergence from a single pupa of O. arenosella was 103.8.The offspring sex ratio was female-biased (8.0%).The results would contribute to the knowledge of the biology of T. pupivorusto optimize the mass-rearing system for a biological program against O. arenosella.
Developmental Biology of Trichospilus pupivorus Ferrière (Hymenoptera: Eulophidae), a Pupal Parasitoid of the Coconut Black... of T. pupivorus reared on O. arenosella.The results would contribute to the knowledge of the biology of this parasitoid to optimize biological control program against O. arenosella. 2

Table 3 :
Progenies emergence and offspring sex ratio of T. purivorus