Interaction Between Two Leafminer Parasitoids, Halticoptera arduine (Hymenoptera: Pteromalidae) and Diglyphus isaea (Hymenoptera: Eulophidae), in the Management of Liriomyza huidobrensis (Diptera: Agromyzidae)

Interaction Between Two Leafminer Parasitoids, Halticoptera arduine (Hymenoptera: Pteromalidae)... Abstract Liriomyza spp., leafminer flies (Mik; Diptera: Agromyzidae), are economically important quarantine pests that puncture and mine leaves and fruits of various horticultural crops worldwide, affecting yield and trade. Halticoptera arduine (Walker; Hymenoptera: Pteromalidae), a key parasitoid from the pests’ areas of origin in South America, was introduced as a potential alternative management strategy. Prior to H. arduine release, its potential interactions with the dominant local ectoparasitoid, Diglyphus isaea (Walker; Hymenoptera: Eulophidae), were assessed. Halticoptera arduine and D. isaea were released in single, sequential and simultaneous combinations on Liriomyza huidobrensis (Blanchard; Diptera: Agromyzidae) to evaluate possible effect on the parasitism rate, reproduction and host mortality. The combination of both parasitoids did not significantly affect the specific parasitism rates of either of them, an indication that H. arduine and D. isaea can coexist. Parasitism rates of the exotic H. arduine were significantly superior to the indigenous D. isaea in all release combinations except when both species were released simultaneously. While 50 individuals of D. isaea resulted only in 21.23 ± 2.1% parasitism, 50 parasitoids composed of 25 H. arduine and 25 D. isaea caused 53.27 ± 4.99%. Both parasitoids further induced significant nonreproductive host mortalities. Both parasitoids’ F1 progenies sex ratios were female-biased in all parasitoid release combinations except in single release of D. isaea with a balanced sex ratio. The improvement in D. isaea’s sex ratio induced by the presence of H. arduine suggests a synergetic effect on D. isaea’s reproductive performance. The introduction of H. arduine in horticulture production systems may therefore improve natural control of Liriomyza leafminers in East Africa. classical biological control, parasitism, nonreproductive mortality, coexistence, sex ratio Horticulture is a leading sector in Kenya’s economy, contributing annual revenue of $2 billion (KNBS 2016). In 2015, horticulture contributed 27.3% to Kenya’s Gross domestic product (GDP) (KIPPRA 2016, KNBS 2016), with export of horticultural products generating $1 billion in foreign exchange for the country (KNBS 2016). More than six million people are employed in the production, processing and marketing of horticultural products in Kenya (NHP 2012, KHC 2015). A major constraint to the growth of this sector is the occurrence of pests and diseases. Among the important pests is Liriomyza leafminer flies (LMF) (Diptera: Agromyzidae), originating from the neotropics. They have become a hindrance to the production and trade in ornamentals, fruits and vegetables in the East African region. LMF were first reported in Kenya in the early 1970s (Spencer 1973), with L. huidobrensis (Blanchard), L. sativae (Blanchard; Diptera: Agromyzidae) and L. trifolii (Burgess; Diptera: Agromyzidae)) now widely distributed in the country. The three species represent above 95% of total Liriomyza species across East Africa, and known to infest a variety of food crops of commercial value including snow pea (Pisum sativum (L.(Fabales: Fabaceae)), Faba bean (Vicia faba (L.; Fabales: Fabaceae)), French bean (Phaseolus vulgaris (L.; Fabales: Fabaceae)), runner bean (Phaseolus coccineus (L.; Fabales: Fabaceae)), tomato (Solanum lycopersicum (L.; Solanales: Solanaceae)), Irish potatoes (Solanum tuberosum (L.; Solanales: Solanaceae)), and a variety of cut flowers including Chrysanthemum spp. (L.; Asterales: Asteraceae), Eryngium spp.(L.; Apiales: Apiaceae), Gypsophila spp.(L.; Caryophyllales: Caryophyllaceae), and Carthamus spp.( L.; Asterales: Asteraceae) (Chabi-Olaye et al. 2008, Mujica and Kroschel 2013, KEPHIS 2014). Adult female leafminers make punctures on leaves using the ovipositor on which both females and males feed from leaf exudates. The punctures are also used to insert eggs below the leaf surface and may also act as vectors for diseases such as Alternaria alternata ((Fr.) Keissl; Pleosporales: Pleosporaceae) (Parrella et al. 1984, Deadman et al. 2002, Bjorksten et al. 2005). Larval mining is generally the most destructive feeding behavior that reduces the photosynthetic capacity of plants and may lead to leaf fall in severe infestation (EPPO 2013). Liriomyza species have become main contributors to yield losses of greenhouse and field crops (Chabi-Olaye et al. 2008, Mujica and Kroschel 2013, KEPHIS 2014, Foba et al. 2015b). LMF are categorized as of quarantine significance to the main trading partners in the European Union (EU) (EU 2000, IPPC 2005, EPPO 2013). For example, in 2016, 23% of interceptions of Kenyan horticultural products destined for the EU were as a result of the presence of Liriomyza species (EUROPHYT 2016). In an effort to manage LMFs, farmers in Kenya have mostly relied on the use of synthetic insecticides (Gitonga et al. 2010). This overreliance has resulted in the development of resistance by LMF to several groups of insecticides (Tran and Takagi 2005, Liu et al. 2009, Guantai et al. 2015). Moreover, the accumulation of pesticide residues in horticultural products has become a food safety issue while the nonselective use of insecticides adversely affects natural enemies associated with LMF (Mujica and Kroschel 2005, Guantai et al. 2015). Parasitoids and other natural enemies are important in regulating LMF populations in their native and invaded areas (Shepard et al. 1998, Murphy and LaSalle 1999, Rauf et al. 2000, Mujica and Kroschel 2011). Worldwide, more than 300 species of parasitoids are associated with agromyzids, and of these, more than 80 species are known to attack Liriomyza spp. (Noyes 2004), although the majority, have been reported from South America (Liu et al. 2009). For instance, along the Peruvian coast, LMFs’ native region, a complex of 63 parasitoid species is associated with Liriomyza spp. (Mujica and Kroschel 2011). A rich complex of the endoparasitoids Halticoptera arduine (Walker) (Hymenoptera: Pteromalidae), Chrysocharis flacilla (Walker) (Hymenoptera: Eulophidae), Phaedrotoma scabriventris (Nixon; Hymenoptera: Braconidae), Ganaspidium sp. (Weld; Hymenoptera: Eucoilidae), and the two ectoparasitoids Diglyphus websteri (Crawford; Hymenoptera: Eulophidae) and D. begini (Ashmead; Hymenoptera: Eulophidae) are the most important parasitoid species, representing more than 94.1% of total parasitoid species and causing LMF mortality of up to 55% in Peru, Argentina, and Chile (Serantes de González 1974, Salvo and Valladares 1995, Cisneros and Mujica 1998, Neder de Román 2000, Mujica and Kroschel 2011). Under the field conditions of the Peruvian coast, H. arduine was the most abundant and efficient parasitoid species, occurring on 25 host plants infested by a wide range of agromyzid LMF species, including the three key Liriomyza species identified in Kenya (Mujica and Kroschel 2011). The parasitoid is adapted to a wide range of ecologies from the coastal region of Peru and Chile (less than 500 m above sea level [m.a.s.l.]) to high altitudes of up to 4,050 m.a.s.l. in Argentina, Chile, and Peru (Sanchez and Redolfi 1985, Neder de Román 2000, Mujica and Kroschel 2011). The diversity of indigenous parasitoids associated with Liriomyza spp. in East Africa in horticultural field crops is low. In a field survey conducted in 2008 in Kenya, the two parasitoids Opius dissitus (Muesebeck; Hymenoptera: Braconidae) and D. isaea (Walker) were identified as the most important local LMF parasitoids, yet only causing parasitism rates of approximately 6%. Diglyphus isaea represents approximately 35% of all LMF parasitoids found in Kenya, closely followed by O. dissitus (Chabi-Olaye et al. 2008, Foba et al. 2015c). In the effort to improve biological control of Liriomyza leafminers in East Africa by boosting the parasitism rates, H. arduine was imported from Peru into Kenya by the International Centre of Insect Physiology and Ecology (icipe), in collaboration with the International Potato Centre (CIP), Peru. To avoid potential ecological disruptions to the local parasitoid populations as a consequence of the introduction of the exotic biological control agent, an assessment of its impact is necessary (Boettner et al. 2000, Louda et al. 2003). Interacting parasitoids may compete for resources, thereby affecting their performance (Godfray 1994, Hardy et al. 2013). The objective of this study was to evaluate the interactions between the exotic H. arduine and the indigenous D. isaea using L. huidobrensis. The results obtained in these studies will be a key criterion for consideration of H. arduine potential release as a biological control agent against LMF in East Africa. Liriomyza huidobrensis is the most abundant and widely distributed Liriomyza leafminer species found in Kenya (Chabi-Olaye et al. 2013, Foba et al. 2015b). Materials and Methods Plant Materials An open pollinated variety of Kenyan faba bean, Vicia faba (L.; Fabales: Fabaceae) was used for the rearing of L. huidobrensis and its two parasitoid species. Five seeds were planted in plastic pots (5.5 cm diameter × 7.3 cm high) and filled with planting substrate (mixture of soil and manure 5:1 in a ratio). Potted plants were maintained in a screenhouse (2.8 m length × 1.8 m width × 2.2 m height) at icipe’s Duduville campus in Nairobi, Kenya at 25 ± 2°C for 2 wk. Two-week-old plants were used for adult L. huidobrensis exposure, on which the parasitoid interaction experiments were conducted using procedures adapted from Akutse et al. (2015) and Foba et al. (2015a). Insect Colonies Leafminer Colony. The initial colony was started from field collections in Nyeri County (0°21′S, 36°57′E, 2,200 m.a.s.l.) of Central Kenya in 2007 and maintained on faba bean plants at icipe’s rearing unit. Two-day-old adult LMF were released for 24 hr in Perspex cages (60 cm length × 60 width × 60 cm height) for egg laying on potted faba plants. The colony was cultured at 25 ± 2°C, 60 ± 5% relative humidity (RH) and a photoperiod of 12L: 12D. Infested plants were removed and held in wooden cages (45 cm wide × 45 cm long × 60 cm high) for 5 to 6 d for the development of second and third similar age cohort instar larvae of L. huidobrensis. Plants were cropped at the base of the stem and incubated on mesh trays to capture dropping pupae. Pupae were collected and incubated in Petri dishes for adult emergence. Adult LMF were fed on 10% sugar solution for 2 d after emergence for preoviposition period before experimental use as described in Chabi-Olaye et al. (2013), Akutse et al. (2015), and Foba et al. (2015a). Parasitoid Colonies. The initial culture of H. arduine was imported from CIP in 2012, where they were maintained on L. huidobrensis. In the icipe quarantine facility, the parasitoid was also maintained on L. huidobrensis reared on faba beans at 21°C ± 1 and 55% ± 5 RH for 10 generations before experimental use. The initial culture of D. isaea was recovered from LMF-infested Pisum sativum (L.; Fabales: Fabaceae) plants collected from farmer fields in Naromoru (0°18′ S, 036°84′ E, 1975 m.a.s.l) of Nyeri County, Kenya, in 2013. Cultures of D. isaea were then reared on L. huidobrensis at icipe’s insect rearing facilities (25°C ± 2 and 60% ± 5 RH) for five generations before experimental use as described by Akutse et al. (2015). Adult parasitoids of both species were fed on 10% honey solution after emergence for 2 d to allow mating and egg maturation before experimental use and in mass production. Each insect species colony was reared in separate rooms to avoid contamination of colonies. Experimental Procedure Bioassay experiments on parasitoid interactions were conducted in laboratories at icipe’s Duduville campus. Ten pots of 2-wk-old faba bean plants from the screenhouse were placed in aerated Perspex cages (30 cm × 30 cm × 45 cm). Two hundred adults of 2-d-old L. huidobrensis in the ratio of 1:2 (males: females) were exposed for 24 hr to the plants for egg-laying before being removed. Infested plants were held for 5 to 6 d (25 ± 2°C, 60 ± 5% RH and photoperiod (12L: 12D)) for the development of second to third LMF larval instars, thereby generating a similar age cohort. Pots were then covered with aluminum foil to minimize the loss of host larvae in potted soil. Two-day-old adult parasitoids in the ratio of 1:2 (male: females) were subsequently introduced in each of the respective treatments described in Table 1 for 24 hr before their removal by aspiration. LMF larvae were held on the plants for 2 to 3 d for pupa development under the same laboratory conditions as described above. To confirm the solitary nature of H. arduine, pupae were collected using a fine camel-hair brush, transferred singly into transparent gelatin capsules (2.2 cm height and 0.7 cm diameter) and then incubated for 30 d to allow emergence of adult LMF and parasitoids. Because D. isaea is an ectoparasitoid, which parasitizes its host by injecting venom into the larvae before depositing the eggs on or close to the host larvae, parasitized larvae were not able to drop for pupation. Thus, plant foliage was cropped and maintained in separate aerated lunch boxes (19 cm long × 13 cm wide × 8 cm high) for 20 d to allow adult emergence as described by Akutse et al. (2015). The leaves were later examined under the microscope to correct for D. isaea parasitism and host mortality. Pupae without exit holes and where insects failed to exit were dissected under the microscope following the methodology described by Heinz and Parrella (1990) to correct for parasitism rates and host mortality. The experiment was arranged in a randomized complete block design (RCBD) with six blocks and one replicate per block. Table 1. Release strategies, sequences, and densities of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released ♂: males, ♀: females. View Large Table 1. Release strategies, sequences, and densities of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released ♂: males, ♀: females. View Large Assessment of Parasitoid Interactions Interactions between H. arduine and D. isaea in parasitizing L. huidobrensis larvae were studied following the procedures described by Wang and Messing (2002), Bader et al. (2006), Akutse et al. (2015), and Foba et al. (2015a). Six parasitoid combinations comprising sole release of 50 H. arduine (T1), sole release of 50 D. isaea (T2), simultaneous release of 50 H. arduine and 50 D. isaea (T3), simultaneous release of 25 H. arduine and 25 D. isaea (T4), sequential release of 50 H. arduine before 50 D. isaea (T5), sequential release of 50 D. isaea before 50 H. arduine (T6), and a control without parasitoids (T7) were established (Table 1). The average number of larvae per treatment and per replicate was n = 190. The number of adult parasitoids collected for each treatment was pooled per replicate and a specific mean and total parasitism rates generated. To assess the effect of parasitoid combinations on both parasitoids’ performance, specific and total parasitism rates were computed and comparisons made among treatments as well as within treatments for specific parasitism rates. Total parasitism rates in simultaneous release of 50 individuals of each of the two parasitoid species (T5) were compared with sequential releases of 50 individuals of each species (T3 and T4). Each specific parasitism rate in the simultaneous release treatment (T5) was compared with their respective single releases (T1 and T2) for both parasitoid species. Total parasitism rates in sequential release strategies (T3 and T4) were compared among themselves. Similarly, each specific parasitism rate in the sequential releases was compared with the specific parasitism rates in the single (T1 and T2) and simultaneous (T5) releases of 50 individuals of each species to determine the effect of release sequence. Comparisons were also made between total parasitism rates in simultaneous release of 25 individuals of each species (T6) with the two single releases of 50 individuals of each species (T1 and T2) to evaluate the performance of the combined parasitoid species with each parasitoid species’ single release at the same parasitoid density. To assess effects of parasitoid release combinations on sex ratio, F1 progenies from each treatment were compared among and within treatments. Assessment of parasitoids’ nonreproductive host mortality was done using procedures described by Wang and Messing (2002) and Foba et al. (2015b). The pupal mortality rate was expressed as the numbers of un-emerged host pupae divided by total pupae multiplied by 100 in each treatment. Data Analyses Specific parasitism rate for each parasitoid species and the total parasitism rate for both species were calculated using the below equations: SpHa=(CHaCHa+CLh)× 100 SpDi=(CDiCDi+CLh)× 100 TPHaDi=(CHa+CDiCHa+CDi+CLh)× 100 Where SpHa = the specific parasitism of H. arduine; CHa = corrected number of H. arduine; CLh = the corrected number of L. huidobrensis; SpDi = the specific parasitism of D. isaea; CDi = the corrected number of D. isaea; TPHaDi = the total parasitism of H. arduine and D. isaea. Percentage data on specific, total parasitism rates and sex ratios were arcsine transformed and subjected to one-way analysis of variance. The nonreproductive (parasitoid induced) mortality was evaluated using Abbott’s formula (Abbott 1925), while the level of observed mortalities was assessed by comparing each treatment with the control using the chi-square test (P < 0.05). Mean differences in parasitism rates across treatments were separated using Tukey–Kramer honest significant difference test at P ≤ 0.05 while differences in parasitoid species sex ratio within treatments were separated using chi-square test (P < 0.05). The statistical programs used for these analyses were JPM(SAS 2013) and R version 3.1 (R Core Development Team 2013). Results Effect of H. arduine and D. isaea combinations on parasitism rates A total of 7,293 LMF pupae were kept individually in gelatin capsules each of which yielded a single parasitoid or LMF adult. The solitary nature was observed for both H. arduine and D. isaea, even when both parasitoid species were jointly released (T3, T4, T5, and T6). In the single 50-parasitoid species releases (T1 and T2), the specific parasitism rate for H. arduine (42.40 ± 3.27%) was significantly two times higher than that for D. isaea (21.23 ± 2.10%) (χ2 = 197.71, df = 1, P < 0.0001). The presence of H. arduine did not affect the specific parasitism rate of D. isaea, and neither did D. isaea affect the specific parasitism rate of H. arduine (T3 and T4) (Table 2). From simultaneously introducing both parasitoid species at a density of 50 individuals/species (T5), their specific parasitism rates did not differ significantly from the same density in their sequential introduction (T3 and T4) for both parasitoid species. Similarly, simultaneous release of both parasitoid species at 50 individuals/species (T5) did not result in significantly different specific parasitism from T1 and T2. Table 2. Mean (± SE) of total and specific parasitism rates of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on Liriomyza huidobrensis following various release combinations under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Ha- H. arduine, Di- D. isaea. *A comparison between T1 and T2. T1-50 H. arduine only, T2- 50 D. isaea only, T3-50 H. arduine first followed by 50 D. isaea, T4-50 D. isaea first followed by 50 H. arduine, T5 - 50 H. arduine plus 50 D. isaea simultaneously, T6- 25 H. arduine plus 25 D. isaea simultaneously. Within rows (columns), means followed by same lower (upper) case letter are not significantly different at P ≤ 0.05 according to Tukey–Kramer (chi-square) test. View Large Table 2. Mean (± SE) of total and specific parasitism rates of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on Liriomyza huidobrensis following various release combinations under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Ha- H. arduine, Di- D. isaea. *A comparison between T1 and T2. T1-50 H. arduine only, T2- 50 D. isaea only, T3-50 H. arduine first followed by 50 D. isaea, T4-50 D. isaea first followed by 50 H. arduine, T5 - 50 H. arduine plus 50 D. isaea simultaneously, T6- 25 H. arduine plus 25 D. isaea simultaneously. Within rows (columns), means followed by same lower (upper) case letter are not significantly different at P ≤ 0.05 according to Tukey–Kramer (chi-square) test. View Large The sequence of introducing the parasitoids (T3 and T4) had no significant effect on total parasitism when compared with the single release of 50 H. arduine (T1) and the simultaneous release of 100 total individuals of both parasitoid species (T5). However, the total parasitism in T5 (55.46%) was significantly higher compared with that resulting from the 50 D. isaea releases (T2) (21.23%). There was no significant effect in host parasitization of the simultaneous release of 25 individuals of each parasitoid species (T6) (53.27%) compared with 50 individuals of each species (T5). However, the specific parasitism by 25 H. arduine in a simultaneous release strategy (T6) was 1.9 times greater than that of 50 D. isaea (T2) (21.23%) under the same host conditions, and 2.3 and 1.6 times higher than the specific parasitism by 50 D. isaea when used in T5 and T3, respectively. Furthermore, total parasitism rate by simultaneous use of 25 each of H. arduine and D. isaea (T6) was significantly 2.5 times higher than use of 50 D. isaea (T2) (F5,35 = 4.88, P < 0.01) (Table 2). Except for T3, where specific parasitism rates of both parasitoid species were similar (χ2 = 1.87, df = 1, P = 0.1714), those of H. arduine were always significantly superior to those of D. isaea in all treatments where both parasitoids were jointly released, by 1.7–2.6 times (T4, T5, and T6) (Table 2). Effect of H. arduine and D. isaea Combinations on L. huidobrensis Nonreproductive Mortality Liriomyza huidobrensis exhibited mortality in the presence of both H. arduine and D. isaea in single and combined release strategies that were significantly higher compared with the control where no parasitoids were released. However, the nonreproductive moralities of L. huidobrensis resulting from both parasitoid species did not differ significantly (F5,33 = 1.33, P = 0.2823) among the release combinations (Table 3). Table 3. Effect of combinations of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis mortality rates (mean ± SE) under laboratory conditions (25 ± 2oC, 60 ± 5% RH and 12L: 12D photoperiod) Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 *Host mortality induced by parasitoid through host stinging and/or feeding, besides direct parasitization. Means followed by the same letters within columns are not significantly different at P ≤ 0.05 (Tukey–Kramer test). **Comparison of observed mortality in each treatment (presence of parasitoid species) versus the control (absence of parasitoids) View Large Table 3. Effect of combinations of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis mortality rates (mean ± SE) under laboratory conditions (25 ± 2oC, 60 ± 5% RH and 12L: 12D photoperiod) Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 *Host mortality induced by parasitoid through host stinging and/or feeding, besides direct parasitization. Means followed by the same letters within columns are not significantly different at P ≤ 0.05 (Tukey–Kramer test). **Comparison of observed mortality in each treatment (presence of parasitoid species) versus the control (absence of parasitoids) View Large Effect of H. arduine and D. isaea Combinations on Parasitoid F1 Sex Ratios Halticoptera arduine F1 progenies in all parasitoid release strategies were significantly female-biased (Table 4), but the proportion of males and females did not differ significantly among the treatments (F4,29 = 0.73, P = 0.5806). Sole releases of D. isaea resulted in a balanced sex ratio in the F1, but was significantly female-biased in combinations with H. arduine either sequentially (T3 and T4) or simultaneously (T5 and T6) (Table 4). As with H. arduine, the proportion of males and females of D. isaea did not differ among the treatments (F4,29 = 0.48, P = 0.75) (Table 4). Table 4. Effect of various combinations of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on parasitoid F1 progeny sex ratios (mean ± SE) under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Key; ♀= females, ♂= males, T1= 50 Ha, T2= 50 Di, T3= 50 Ha first, 50 Di second, T4= 50 Di first, 50 Ha second, T5= 50 Ha +50 Di, T6= 25 Ha + 25 Di. Within rows, means followed by the same lower case letters are not significantly different at P < 0.05 (Tukey’s test). Within columns, and for each parasitoid species, means followed by the same upper case letters are not significantly different at P< 0.05 (chi-square test). View Large Table 4. Effect of various combinations of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on parasitoid F1 progeny sex ratios (mean ± SE) under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Key; ♀= females, ♂= males, T1= 50 Ha, T2= 50 Di, T3= 50 Ha first, 50 Di second, T4= 50 Di first, 50 Ha second, T5= 50 Ha +50 Di, T6= 25 Ha + 25 Di. Within rows, means followed by the same lower case letters are not significantly different at P < 0.05 (Tukey’s test). Within columns, and for each parasitoid species, means followed by the same upper case letters are not significantly different at P< 0.05 (chi-square test). View Large Discussion For both parasitoid species, irrespective of the treatment, only one parasitoid specimen was recovered from each host pupa. This finding confirms the solitary nature of both H. arduine and D. isaea, corroborating earlier reports on H. arduine by Arellano and Redolfi (1989) and preliminary studies by K. Fiaboe (unpublished data) while studying its performance on three Liriomyza leafminer hosts. Akutse et al. (2015) reported on the solitary nature of D. isaea while carrying out interaction studies between D. isaea and P. scabriventris. Thus, our results indicate the possibility for host resource sharing of the two parasitoid species, which could result in higher levels of LMF control (Foba et al. 2015a). Interactions between parasitoids in the exploitation of a common resource can influence the performance and ability to control the target pest (Briggs 1993, Grover 1997, Bogran et al. 2002). The introduction of a new parasitoid species that shares the same resources as indigenous parasitoids can pose a risk of interspecific competition, possibly leading to ecological disruption and reduced performance of one or both species (Briggs et al. 1993, De Moraes et al. 1999, Pianka 2000, Louda et al. 2003, Shi et al. 2004, Tian et al. 2008, Jones et al. 2009, Harvey et al. 2013). However, coexistence is common between different parasitoid species if the parasitoids attack different host life stages. According to Harvey et al. (2013), coexistence between two or more species sharing the same host and stage may be due to the degree of specificity, searching efficiency, egg load and the ability to discriminate between hosts parasitized by each other in ways that dilute competition. Stiling and Cornelissen (2005) showed that the introduction of two or more biocontrol agents increased pest mortality by 12.97% and decreased pest abundance by 27.17% compared with single releases. In our study, the exotic parasitoid H. arduine had no detrimental effect on the specific parasitism performance of the local parasitoid D. isaea, whether released first, simultaneously or second. Similarly, the local parasitoid’s presence did not affect the specific parasitism of the exotic species independently of combination sequence, indicating coexistence of both species. Possibly, the fact that H. arduine is an endo- and D. isaea an ecto-parasitoid might have reduced the risk of direct competition of the immature stages of both species. The coexistence could also be related to host discrimination abilities in one or both parasitoids (Bakker et al. 1985). Further studies are warranted to assess the potential host discrimination abilities in both parasitoids as well as the potential mechanisms involved. Beyond the coexistence observed, the introduction of H. arduine considerably boosted the total parasitism. Releases of 50 individual parasitoids composed of 25 H. arduine and 25 D. isaea resulted in 2.5 times more parasitism than when 50 individuals of the indigenous parasitoid species were used. At the same host density, releases of 25 or 50 H. arduine resulted in the same level of parasitism, warranting further studies on parasitoid-host density functional and numerical responses. This could guide optimizing the number of parasitoids required for releases in the case of inundative release strategies, such as in greenhouse environments. Moreover, H. arduine proved to be superior in LMF control than D. isaea, with between 1.4 and 2.6 times higher specific parasitism rates. Such relatively low reproductive performance of D. isaea compared with an endoparasitoid have also been reported by Akutse et al. (2015) while studying the interactions between P. scabriventris and D. isaea using L. huidobrensis as host. Yildirim et al. (2011) and Boot et al. (1992) also reported and modeled low D. isaea parasitism rates on L. bryoniae (Kaltenbach; Diptera: Agromyzidae)) and L. sativae, respectively, in field studies. Similarly, Mujica and Kroschel (2011) found that H. arduine coexisted with a complex of 60 parasitoid species in the field and was more important in controlling the majority of LMF including several Diglyphus species. Along the Peruvian coast, H. arduine was found parasitizing different leafminer species with up to 66.7% parasitism rates under field conditions (Mujica and Kroschel 2011). In parasitoids, nonreproductive host mortality is considered an additional and important mortality factor in pest suppression (Honda et al. 2006). Female parasitoids induce this additional mortality through paralyses of the host by stinging, often followed by host feeding (Jervis and Kidd 1986, Liu et al. 2013, Akutse et al. 2015). In our study, both H. arduine and D. isaea caused insignificant nonreproductive mortality of L. huidobrensis in single and combined release strategies. This insignificant nonreproductive mortality by H. arduine is consistent with earlier results observed during our host performance studies on three LMF species (F. Komi, unpublished data). However, the significant mortalities of L. huidobrensis in the presence of D. isaea are in line with previous reports by Minkenberg (1989), Liu et al. (2013), and Akutse et al. (2015). In addition, the observed mortalities of L. huidobrensis in the presence of H. arduine were also notable, though not significant compared with the control. Further studies on the effect of parasitoid density on host nonreproductive mortalities are warranted. In our study, reproduction of the exotic H. arduine always resulted in a female-biased sex ratio, irrespective of the presence of the other parasitoid species. However, for the indigenous D. isaea, the sex ratio of its F1 became female-biased only in the presence of H. arduine, indicating a synergistic potential when combining both parasitoid species. Since only female parasitoids cause host mortality (Pascua and Pascua 2004, Chow and Heinz 2005, Abe and Kamimura 2012, Foba et al. 2015a), the female-biased parasitoid populations suggest better reproductive and parasitism performances. Conclusion Halticoptera arduine host parasitization is superior to the indigenous D. isaea with synergistic improvement on D. isaea reproduction potential when both parasitoid species were released simultaneously. Thus, if successfully introduced into the East African horticultural production systems, H. arduine might not only coexist with D. isaea, but also raise the total parasitism rates to achieve economically important pest control levels. Low pest pressure may cause small holder farmers to shift away from heavy broad-spectrum pesticides use. Upon receiving the official release permit from the Kenyan authorities, we will therefore assess the performance of the parasitoids under East African field conditions. Acknowledgments We are grateful to Jürgen Kroschel and Norman Mujica of the International Potato Center (CIP) in Peru for providing H. arduine specimens to start the colony in Kenya. 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Res . 92 : 423 – 429 . Google Scholar CrossRef Search ADS PubMed Yildirim , E. M. , H. S. Civelek , O. Dursun , and A. Eskin . 2011 . The parasitism rate of Diglyphus isaea (Hymenoptera: Eulophidae) on Liriomyza sativae Blanchard (Diptera: Agromyzidae) in Mugla Province . J. Appl. Biol. Sci . 6 : 21 – 23 . © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Interaction Between Two Leafminer Parasitoids, Halticoptera arduine (Hymenoptera: Pteromalidae) and Diglyphus isaea (Hymenoptera: Eulophidae), in the Management of Liriomyza huidobrensis (Diptera: Agromyzidae)

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Abstract

Abstract Liriomyza spp., leafminer flies (Mik; Diptera: Agromyzidae), are economically important quarantine pests that puncture and mine leaves and fruits of various horticultural crops worldwide, affecting yield and trade. Halticoptera arduine (Walker; Hymenoptera: Pteromalidae), a key parasitoid from the pests’ areas of origin in South America, was introduced as a potential alternative management strategy. Prior to H. arduine release, its potential interactions with the dominant local ectoparasitoid, Diglyphus isaea (Walker; Hymenoptera: Eulophidae), were assessed. Halticoptera arduine and D. isaea were released in single, sequential and simultaneous combinations on Liriomyza huidobrensis (Blanchard; Diptera: Agromyzidae) to evaluate possible effect on the parasitism rate, reproduction and host mortality. The combination of both parasitoids did not significantly affect the specific parasitism rates of either of them, an indication that H. arduine and D. isaea can coexist. Parasitism rates of the exotic H. arduine were significantly superior to the indigenous D. isaea in all release combinations except when both species were released simultaneously. While 50 individuals of D. isaea resulted only in 21.23 ± 2.1% parasitism, 50 parasitoids composed of 25 H. arduine and 25 D. isaea caused 53.27 ± 4.99%. Both parasitoids further induced significant nonreproductive host mortalities. Both parasitoids’ F1 progenies sex ratios were female-biased in all parasitoid release combinations except in single release of D. isaea with a balanced sex ratio. The improvement in D. isaea’s sex ratio induced by the presence of H. arduine suggests a synergetic effect on D. isaea’s reproductive performance. The introduction of H. arduine in horticulture production systems may therefore improve natural control of Liriomyza leafminers in East Africa. classical biological control, parasitism, nonreproductive mortality, coexistence, sex ratio Horticulture is a leading sector in Kenya’s economy, contributing annual revenue of $2 billion (KNBS 2016). In 2015, horticulture contributed 27.3% to Kenya’s Gross domestic product (GDP) (KIPPRA 2016, KNBS 2016), with export of horticultural products generating $1 billion in foreign exchange for the country (KNBS 2016). More than six million people are employed in the production, processing and marketing of horticultural products in Kenya (NHP 2012, KHC 2015). A major constraint to the growth of this sector is the occurrence of pests and diseases. Among the important pests is Liriomyza leafminer flies (LMF) (Diptera: Agromyzidae), originating from the neotropics. They have become a hindrance to the production and trade in ornamentals, fruits and vegetables in the East African region. LMF were first reported in Kenya in the early 1970s (Spencer 1973), with L. huidobrensis (Blanchard), L. sativae (Blanchard; Diptera: Agromyzidae) and L. trifolii (Burgess; Diptera: Agromyzidae)) now widely distributed in the country. The three species represent above 95% of total Liriomyza species across East Africa, and known to infest a variety of food crops of commercial value including snow pea (Pisum sativum (L.(Fabales: Fabaceae)), Faba bean (Vicia faba (L.; Fabales: Fabaceae)), French bean (Phaseolus vulgaris (L.; Fabales: Fabaceae)), runner bean (Phaseolus coccineus (L.; Fabales: Fabaceae)), tomato (Solanum lycopersicum (L.; Solanales: Solanaceae)), Irish potatoes (Solanum tuberosum (L.; Solanales: Solanaceae)), and a variety of cut flowers including Chrysanthemum spp. (L.; Asterales: Asteraceae), Eryngium spp.(L.; Apiales: Apiaceae), Gypsophila spp.(L.; Caryophyllales: Caryophyllaceae), and Carthamus spp.( L.; Asterales: Asteraceae) (Chabi-Olaye et al. 2008, Mujica and Kroschel 2013, KEPHIS 2014). Adult female leafminers make punctures on leaves using the ovipositor on which both females and males feed from leaf exudates. The punctures are also used to insert eggs below the leaf surface and may also act as vectors for diseases such as Alternaria alternata ((Fr.) Keissl; Pleosporales: Pleosporaceae) (Parrella et al. 1984, Deadman et al. 2002, Bjorksten et al. 2005). Larval mining is generally the most destructive feeding behavior that reduces the photosynthetic capacity of plants and may lead to leaf fall in severe infestation (EPPO 2013). Liriomyza species have become main contributors to yield losses of greenhouse and field crops (Chabi-Olaye et al. 2008, Mujica and Kroschel 2013, KEPHIS 2014, Foba et al. 2015b). LMF are categorized as of quarantine significance to the main trading partners in the European Union (EU) (EU 2000, IPPC 2005, EPPO 2013). For example, in 2016, 23% of interceptions of Kenyan horticultural products destined for the EU were as a result of the presence of Liriomyza species (EUROPHYT 2016). In an effort to manage LMFs, farmers in Kenya have mostly relied on the use of synthetic insecticides (Gitonga et al. 2010). This overreliance has resulted in the development of resistance by LMF to several groups of insecticides (Tran and Takagi 2005, Liu et al. 2009, Guantai et al. 2015). Moreover, the accumulation of pesticide residues in horticultural products has become a food safety issue while the nonselective use of insecticides adversely affects natural enemies associated with LMF (Mujica and Kroschel 2005, Guantai et al. 2015). Parasitoids and other natural enemies are important in regulating LMF populations in their native and invaded areas (Shepard et al. 1998, Murphy and LaSalle 1999, Rauf et al. 2000, Mujica and Kroschel 2011). Worldwide, more than 300 species of parasitoids are associated with agromyzids, and of these, more than 80 species are known to attack Liriomyza spp. (Noyes 2004), although the majority, have been reported from South America (Liu et al. 2009). For instance, along the Peruvian coast, LMFs’ native region, a complex of 63 parasitoid species is associated with Liriomyza spp. (Mujica and Kroschel 2011). A rich complex of the endoparasitoids Halticoptera arduine (Walker) (Hymenoptera: Pteromalidae), Chrysocharis flacilla (Walker) (Hymenoptera: Eulophidae), Phaedrotoma scabriventris (Nixon; Hymenoptera: Braconidae), Ganaspidium sp. (Weld; Hymenoptera: Eucoilidae), and the two ectoparasitoids Diglyphus websteri (Crawford; Hymenoptera: Eulophidae) and D. begini (Ashmead; Hymenoptera: Eulophidae) are the most important parasitoid species, representing more than 94.1% of total parasitoid species and causing LMF mortality of up to 55% in Peru, Argentina, and Chile (Serantes de González 1974, Salvo and Valladares 1995, Cisneros and Mujica 1998, Neder de Román 2000, Mujica and Kroschel 2011). Under the field conditions of the Peruvian coast, H. arduine was the most abundant and efficient parasitoid species, occurring on 25 host plants infested by a wide range of agromyzid LMF species, including the three key Liriomyza species identified in Kenya (Mujica and Kroschel 2011). The parasitoid is adapted to a wide range of ecologies from the coastal region of Peru and Chile (less than 500 m above sea level [m.a.s.l.]) to high altitudes of up to 4,050 m.a.s.l. in Argentina, Chile, and Peru (Sanchez and Redolfi 1985, Neder de Román 2000, Mujica and Kroschel 2011). The diversity of indigenous parasitoids associated with Liriomyza spp. in East Africa in horticultural field crops is low. In a field survey conducted in 2008 in Kenya, the two parasitoids Opius dissitus (Muesebeck; Hymenoptera: Braconidae) and D. isaea (Walker) were identified as the most important local LMF parasitoids, yet only causing parasitism rates of approximately 6%. Diglyphus isaea represents approximately 35% of all LMF parasitoids found in Kenya, closely followed by O. dissitus (Chabi-Olaye et al. 2008, Foba et al. 2015c). In the effort to improve biological control of Liriomyza leafminers in East Africa by boosting the parasitism rates, H. arduine was imported from Peru into Kenya by the International Centre of Insect Physiology and Ecology (icipe), in collaboration with the International Potato Centre (CIP), Peru. To avoid potential ecological disruptions to the local parasitoid populations as a consequence of the introduction of the exotic biological control agent, an assessment of its impact is necessary (Boettner et al. 2000, Louda et al. 2003). Interacting parasitoids may compete for resources, thereby affecting their performance (Godfray 1994, Hardy et al. 2013). The objective of this study was to evaluate the interactions between the exotic H. arduine and the indigenous D. isaea using L. huidobrensis. The results obtained in these studies will be a key criterion for consideration of H. arduine potential release as a biological control agent against LMF in East Africa. Liriomyza huidobrensis is the most abundant and widely distributed Liriomyza leafminer species found in Kenya (Chabi-Olaye et al. 2013, Foba et al. 2015b). Materials and Methods Plant Materials An open pollinated variety of Kenyan faba bean, Vicia faba (L.; Fabales: Fabaceae) was used for the rearing of L. huidobrensis and its two parasitoid species. Five seeds were planted in plastic pots (5.5 cm diameter × 7.3 cm high) and filled with planting substrate (mixture of soil and manure 5:1 in a ratio). Potted plants were maintained in a screenhouse (2.8 m length × 1.8 m width × 2.2 m height) at icipe’s Duduville campus in Nairobi, Kenya at 25 ± 2°C for 2 wk. Two-week-old plants were used for adult L. huidobrensis exposure, on which the parasitoid interaction experiments were conducted using procedures adapted from Akutse et al. (2015) and Foba et al. (2015a). Insect Colonies Leafminer Colony. The initial colony was started from field collections in Nyeri County (0°21′S, 36°57′E, 2,200 m.a.s.l.) of Central Kenya in 2007 and maintained on faba bean plants at icipe’s rearing unit. Two-day-old adult LMF were released for 24 hr in Perspex cages (60 cm length × 60 width × 60 cm height) for egg laying on potted faba plants. The colony was cultured at 25 ± 2°C, 60 ± 5% relative humidity (RH) and a photoperiod of 12L: 12D. Infested plants were removed and held in wooden cages (45 cm wide × 45 cm long × 60 cm high) for 5 to 6 d for the development of second and third similar age cohort instar larvae of L. huidobrensis. Plants were cropped at the base of the stem and incubated on mesh trays to capture dropping pupae. Pupae were collected and incubated in Petri dishes for adult emergence. Adult LMF were fed on 10% sugar solution for 2 d after emergence for preoviposition period before experimental use as described in Chabi-Olaye et al. (2013), Akutse et al. (2015), and Foba et al. (2015a). Parasitoid Colonies. The initial culture of H. arduine was imported from CIP in 2012, where they were maintained on L. huidobrensis. In the icipe quarantine facility, the parasitoid was also maintained on L. huidobrensis reared on faba beans at 21°C ± 1 and 55% ± 5 RH for 10 generations before experimental use. The initial culture of D. isaea was recovered from LMF-infested Pisum sativum (L.; Fabales: Fabaceae) plants collected from farmer fields in Naromoru (0°18′ S, 036°84′ E, 1975 m.a.s.l) of Nyeri County, Kenya, in 2013. Cultures of D. isaea were then reared on L. huidobrensis at icipe’s insect rearing facilities (25°C ± 2 and 60% ± 5 RH) for five generations before experimental use as described by Akutse et al. (2015). Adult parasitoids of both species were fed on 10% honey solution after emergence for 2 d to allow mating and egg maturation before experimental use and in mass production. Each insect species colony was reared in separate rooms to avoid contamination of colonies. Experimental Procedure Bioassay experiments on parasitoid interactions were conducted in laboratories at icipe’s Duduville campus. Ten pots of 2-wk-old faba bean plants from the screenhouse were placed in aerated Perspex cages (30 cm × 30 cm × 45 cm). Two hundred adults of 2-d-old L. huidobrensis in the ratio of 1:2 (males: females) were exposed for 24 hr to the plants for egg-laying before being removed. Infested plants were held for 5 to 6 d (25 ± 2°C, 60 ± 5% RH and photoperiod (12L: 12D)) for the development of second to third LMF larval instars, thereby generating a similar age cohort. Pots were then covered with aluminum foil to minimize the loss of host larvae in potted soil. Two-day-old adult parasitoids in the ratio of 1:2 (male: females) were subsequently introduced in each of the respective treatments described in Table 1 for 24 hr before their removal by aspiration. LMF larvae were held on the plants for 2 to 3 d for pupa development under the same laboratory conditions as described above. To confirm the solitary nature of H. arduine, pupae were collected using a fine camel-hair brush, transferred singly into transparent gelatin capsules (2.2 cm height and 0.7 cm diameter) and then incubated for 30 d to allow emergence of adult LMF and parasitoids. Because D. isaea is an ectoparasitoid, which parasitizes its host by injecting venom into the larvae before depositing the eggs on or close to the host larvae, parasitized larvae were not able to drop for pupation. Thus, plant foliage was cropped and maintained in separate aerated lunch boxes (19 cm long × 13 cm wide × 8 cm high) for 20 d to allow adult emergence as described by Akutse et al. (2015). The leaves were later examined under the microscope to correct for D. isaea parasitism and host mortality. Pupae without exit holes and where insects failed to exit were dissected under the microscope following the methodology described by Heinz and Parrella (1990) to correct for parasitism rates and host mortality. The experiment was arranged in a randomized complete block design (RCBD) with six blocks and one replicate per block. Table 1. Release strategies, sequences, and densities of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released ♂: males, ♀: females. View Large Table 1. Release strategies, sequences, and densities of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released Treatments (T) Parasitoid species release combinations Sole releases  T1-H. arduine alone 50 adults of H. arduine (1:2 for ♂and ♀)  T2-D. isaea alone 50 adults of D. isaea (1:2 for ♂and ♀) Sequential releases  T3-H. arduine first, D. isaea second 50 adults of H. arduine, followed by 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T4-D. isaea first, H. arduine second 50 adults of D. isaea, followed by 50 adults of H. arduine (1:2 for ♂and ♀ of each species) Simultaneous releases  T5-H. arduine and D. isaea 50 adults of H. arduine + 50 adults of D. isaea (1:2 for ♂and ♀ of each species)  T6-H. arduine and D. isaea 25 adults of H. arduine + 25 adults of D. isaea (1:2 for ♂and ♀ of each species) Control  T7-L. huidobrensis reared alone No parasitoids released ♂: males, ♀: females. View Large Assessment of Parasitoid Interactions Interactions between H. arduine and D. isaea in parasitizing L. huidobrensis larvae were studied following the procedures described by Wang and Messing (2002), Bader et al. (2006), Akutse et al. (2015), and Foba et al. (2015a). Six parasitoid combinations comprising sole release of 50 H. arduine (T1), sole release of 50 D. isaea (T2), simultaneous release of 50 H. arduine and 50 D. isaea (T3), simultaneous release of 25 H. arduine and 25 D. isaea (T4), sequential release of 50 H. arduine before 50 D. isaea (T5), sequential release of 50 D. isaea before 50 H. arduine (T6), and a control without parasitoids (T7) were established (Table 1). The average number of larvae per treatment and per replicate was n = 190. The number of adult parasitoids collected for each treatment was pooled per replicate and a specific mean and total parasitism rates generated. To assess the effect of parasitoid combinations on both parasitoids’ performance, specific and total parasitism rates were computed and comparisons made among treatments as well as within treatments for specific parasitism rates. Total parasitism rates in simultaneous release of 50 individuals of each of the two parasitoid species (T5) were compared with sequential releases of 50 individuals of each species (T3 and T4). Each specific parasitism rate in the simultaneous release treatment (T5) was compared with their respective single releases (T1 and T2) for both parasitoid species. Total parasitism rates in sequential release strategies (T3 and T4) were compared among themselves. Similarly, each specific parasitism rate in the sequential releases was compared with the specific parasitism rates in the single (T1 and T2) and simultaneous (T5) releases of 50 individuals of each species to determine the effect of release sequence. Comparisons were also made between total parasitism rates in simultaneous release of 25 individuals of each species (T6) with the two single releases of 50 individuals of each species (T1 and T2) to evaluate the performance of the combined parasitoid species with each parasitoid species’ single release at the same parasitoid density. To assess effects of parasitoid release combinations on sex ratio, F1 progenies from each treatment were compared among and within treatments. Assessment of parasitoids’ nonreproductive host mortality was done using procedures described by Wang and Messing (2002) and Foba et al. (2015b). The pupal mortality rate was expressed as the numbers of un-emerged host pupae divided by total pupae multiplied by 100 in each treatment. Data Analyses Specific parasitism rate for each parasitoid species and the total parasitism rate for both species were calculated using the below equations: SpHa=(CHaCHa+CLh)× 100 SpDi=(CDiCDi+CLh)× 100 TPHaDi=(CHa+CDiCHa+CDi+CLh)× 100 Where SpHa = the specific parasitism of H. arduine; CHa = corrected number of H. arduine; CLh = the corrected number of L. huidobrensis; SpDi = the specific parasitism of D. isaea; CDi = the corrected number of D. isaea; TPHaDi = the total parasitism of H. arduine and D. isaea. Percentage data on specific, total parasitism rates and sex ratios were arcsine transformed and subjected to one-way analysis of variance. The nonreproductive (parasitoid induced) mortality was evaluated using Abbott’s formula (Abbott 1925), while the level of observed mortalities was assessed by comparing each treatment with the control using the chi-square test (P < 0.05). Mean differences in parasitism rates across treatments were separated using Tukey–Kramer honest significant difference test at P ≤ 0.05 while differences in parasitoid species sex ratio within treatments were separated using chi-square test (P < 0.05). The statistical programs used for these analyses were JPM(SAS 2013) and R version 3.1 (R Core Development Team 2013). Results Effect of H. arduine and D. isaea combinations on parasitism rates A total of 7,293 LMF pupae were kept individually in gelatin capsules each of which yielded a single parasitoid or LMF adult. The solitary nature was observed for both H. arduine and D. isaea, even when both parasitoid species were jointly released (T3, T4, T5, and T6). In the single 50-parasitoid species releases (T1 and T2), the specific parasitism rate for H. arduine (42.40 ± 3.27%) was significantly two times higher than that for D. isaea (21.23 ± 2.10%) (χ2 = 197.71, df = 1, P < 0.0001). The presence of H. arduine did not affect the specific parasitism rate of D. isaea, and neither did D. isaea affect the specific parasitism rate of H. arduine (T3 and T4) (Table 2). From simultaneously introducing both parasitoid species at a density of 50 individuals/species (T5), their specific parasitism rates did not differ significantly from the same density in their sequential introduction (T3 and T4) for both parasitoid species. Similarly, simultaneous release of both parasitoid species at 50 individuals/species (T5) did not result in significantly different specific parasitism from T1 and T2. Table 2. Mean (± SE) of total and specific parasitism rates of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on Liriomyza huidobrensis following various release combinations under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Ha- H. arduine, Di- D. isaea. *A comparison between T1 and T2. T1-50 H. arduine only, T2- 50 D. isaea only, T3-50 H. arduine first followed by 50 D. isaea, T4-50 D. isaea first followed by 50 H. arduine, T5 - 50 H. arduine plus 50 D. isaea simultaneously, T6- 25 H. arduine plus 25 D. isaea simultaneously. Within rows (columns), means followed by same lower (upper) case letter are not significantly different at P ≤ 0.05 according to Tukey–Kramer (chi-square) test. View Large Table 2. Mean (± SE) of total and specific parasitism rates of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on Liriomyza huidobrensis following various release combinations under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Treatments (T) regime T1 T2 T3 T4 T5 T6 Ha specific parasitism (%) 42.40 ± 3.27aA 29.72 ± 3.14aA 30.84 ± 3.70aA 40.14 ± 2.50aA 34.60 ± 5.27aA Di specific parasitism (%) 21.23 ± 2.10aB 21.78 ± 2.67aA 18.68 ± 3.316aB 15.32 ± 1.99aB 18.67 ± 2.36aB χ2 values 197.71* 1.87 90.18 347.63 137.98 P-values 0.0001 0.1714 0.0001 0.0001 0.0001 Total parasitism (%) 42.40 ± 3.27a 21.23 ± 2.10b 51.50 ± 3.82a 49.52 ± 5.17a 55.46 ± 2.60a 53.27 ± 4.99a Ha- H. arduine, Di- D. isaea. *A comparison between T1 and T2. T1-50 H. arduine only, T2- 50 D. isaea only, T3-50 H. arduine first followed by 50 D. isaea, T4-50 D. isaea first followed by 50 H. arduine, T5 - 50 H. arduine plus 50 D. isaea simultaneously, T6- 25 H. arduine plus 25 D. isaea simultaneously. Within rows (columns), means followed by same lower (upper) case letter are not significantly different at P ≤ 0.05 according to Tukey–Kramer (chi-square) test. View Large The sequence of introducing the parasitoids (T3 and T4) had no significant effect on total parasitism when compared with the single release of 50 H. arduine (T1) and the simultaneous release of 100 total individuals of both parasitoid species (T5). However, the total parasitism in T5 (55.46%) was significantly higher compared with that resulting from the 50 D. isaea releases (T2) (21.23%). There was no significant effect in host parasitization of the simultaneous release of 25 individuals of each parasitoid species (T6) (53.27%) compared with 50 individuals of each species (T5). However, the specific parasitism by 25 H. arduine in a simultaneous release strategy (T6) was 1.9 times greater than that of 50 D. isaea (T2) (21.23%) under the same host conditions, and 2.3 and 1.6 times higher than the specific parasitism by 50 D. isaea when used in T5 and T3, respectively. Furthermore, total parasitism rate by simultaneous use of 25 each of H. arduine and D. isaea (T6) was significantly 2.5 times higher than use of 50 D. isaea (T2) (F5,35 = 4.88, P < 0.01) (Table 2). Except for T3, where specific parasitism rates of both parasitoid species were similar (χ2 = 1.87, df = 1, P = 0.1714), those of H. arduine were always significantly superior to those of D. isaea in all treatments where both parasitoids were jointly released, by 1.7–2.6 times (T4, T5, and T6) (Table 2). Effect of H. arduine and D. isaea Combinations on L. huidobrensis Nonreproductive Mortality Liriomyza huidobrensis exhibited mortality in the presence of both H. arduine and D. isaea in single and combined release strategies that were significantly higher compared with the control where no parasitoids were released. However, the nonreproductive moralities of L. huidobrensis resulting from both parasitoid species did not differ significantly (F5,33 = 1.33, P = 0.2823) among the release combinations (Table 3). Table 3. Effect of combinations of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis mortality rates (mean ± SE) under laboratory conditions (25 ± 2oC, 60 ± 5% RH and 12L: 12D photoperiod) Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 *Host mortality induced by parasitoid through host stinging and/or feeding, besides direct parasitization. Means followed by the same letters within columns are not significantly different at P ≤ 0.05 (Tukey–Kramer test). **Comparison of observed mortality in each treatment (presence of parasitoid species) versus the control (absence of parasitoids) View Large Table 3. Effect of combinations of Halticoptera arduine and Diglyphus isaea on Liriomyza huidobrensis mortality rates (mean ± SE) under laboratory conditions (25 ± 2oC, 60 ± 5% RH and 12L: 12D photoperiod) Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 Treatment (T) regime L. huidobrensis nonreproductive mortality* Significance level of treatment mortality versus control** χ2 values P values Single releases  T1-H. arduine 15.79 ± 0.58a 10.72 0.001  T2-D. isaea 14.78 ± 1.76a 9.23 0.0024 Sequential releases  T3-H. arduine first, D. isaea second 12.12 ± 3.81a 29.30 0.0001  T4-D. isaea first, H. arduine second 14.98 ± 3.74a 23.00 0.0001 Simultaneous releases  T5-H. arduine and D. isaea 13.71 ± 2.03a 5.61 0.0178  T6-H. arduine and D. isaea 22.59 ± 4.85a 92.35 0.0001 *Host mortality induced by parasitoid through host stinging and/or feeding, besides direct parasitization. Means followed by the same letters within columns are not significantly different at P ≤ 0.05 (Tukey–Kramer test). **Comparison of observed mortality in each treatment (presence of parasitoid species) versus the control (absence of parasitoids) View Large Effect of H. arduine and D. isaea Combinations on Parasitoid F1 Sex Ratios Halticoptera arduine F1 progenies in all parasitoid release strategies were significantly female-biased (Table 4), but the proportion of males and females did not differ significantly among the treatments (F4,29 = 0.73, P = 0.5806). Sole releases of D. isaea resulted in a balanced sex ratio in the F1, but was significantly female-biased in combinations with H. arduine either sequentially (T3 and T4) or simultaneously (T5 and T6) (Table 4). As with H. arduine, the proportion of males and females of D. isaea did not differ among the treatments (F4,29 = 0.48, P = 0.75) (Table 4). Table 4. Effect of various combinations of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on parasitoid F1 progeny sex ratios (mean ± SE) under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Key; ♀= females, ♂= males, T1= 50 Ha, T2= 50 Di, T3= 50 Ha first, 50 Di second, T4= 50 Di first, 50 Ha second, T5= 50 Ha +50 Di, T6= 25 Ha + 25 Di. Within rows, means followed by the same lower case letters are not significantly different at P < 0.05 (Tukey’s test). Within columns, and for each parasitoid species, means followed by the same upper case letters are not significantly different at P< 0.05 (chi-square test). View Large Table 4. Effect of various combinations of Halticoptera arduine (Ha) and Diglyphus isaea (Di) on parasitoid F1 progeny sex ratios (mean ± SE) under laboratory conditions (25 ± 2°C, 60 ± 5% RH and 12L: 12D photoperiod) Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Parasitoid species Sex ratio T1 T2 T3 T4 T5 T6 H. arduine %♀ 65.02 ± 1.03aA 67.32 ± 1.41aA 66.21 ± 1.00aA 64.65 ± 1.37aA 61.94 ± 2.00aA %♂ 34.98 ± 1.03aB 32.68 ± 1.41aB 33.79 ± 1.00aB 35.34 ± 1.37aB 38.08 ± 2.00aB χ2 20.50 27.76 24.00 19.60 13.01 P <0.0001 <0.0001 <0.0001 <0.0001 0.0003 D. isaea %♀ 56.45 ± 3.71aA 58.27 ± 2.16aA 66.43 ± 1.98aA 58.07 ± 4.45aA 59.83 ± 3.55aA %♂ 43.55 ± 3.71aA 41.73 ± 2.16aB 33.57 ± 1.98aB 41.93 ± 4.45aB 40.16 ± 3.55aB χ2 3.75 6.22 25.21 6.88 9.21 P 0.0529 0.0127 <0.0001 0.0087 0.0024 Key; ♀= females, ♂= males, T1= 50 Ha, T2= 50 Di, T3= 50 Ha first, 50 Di second, T4= 50 Di first, 50 Ha second, T5= 50 Ha +50 Di, T6= 25 Ha + 25 Di. Within rows, means followed by the same lower case letters are not significantly different at P < 0.05 (Tukey’s test). Within columns, and for each parasitoid species, means followed by the same upper case letters are not significantly different at P< 0.05 (chi-square test). View Large Discussion For both parasitoid species, irrespective of the treatment, only one parasitoid specimen was recovered from each host pupa. This finding confirms the solitary nature of both H. arduine and D. isaea, corroborating earlier reports on H. arduine by Arellano and Redolfi (1989) and preliminary studies by K. Fiaboe (unpublished data) while studying its performance on three Liriomyza leafminer hosts. Akutse et al. (2015) reported on the solitary nature of D. isaea while carrying out interaction studies between D. isaea and P. scabriventris. Thus, our results indicate the possibility for host resource sharing of the two parasitoid species, which could result in higher levels of LMF control (Foba et al. 2015a). Interactions between parasitoids in the exploitation of a common resource can influence the performance and ability to control the target pest (Briggs 1993, Grover 1997, Bogran et al. 2002). The introduction of a new parasitoid species that shares the same resources as indigenous parasitoids can pose a risk of interspecific competition, possibly leading to ecological disruption and reduced performance of one or both species (Briggs et al. 1993, De Moraes et al. 1999, Pianka 2000, Louda et al. 2003, Shi et al. 2004, Tian et al. 2008, Jones et al. 2009, Harvey et al. 2013). However, coexistence is common between different parasitoid species if the parasitoids attack different host life stages. According to Harvey et al. (2013), coexistence between two or more species sharing the same host and stage may be due to the degree of specificity, searching efficiency, egg load and the ability to discriminate between hosts parasitized by each other in ways that dilute competition. Stiling and Cornelissen (2005) showed that the introduction of two or more biocontrol agents increased pest mortality by 12.97% and decreased pest abundance by 27.17% compared with single releases. In our study, the exotic parasitoid H. arduine had no detrimental effect on the specific parasitism performance of the local parasitoid D. isaea, whether released first, simultaneously or second. Similarly, the local parasitoid’s presence did not affect the specific parasitism of the exotic species independently of combination sequence, indicating coexistence of both species. Possibly, the fact that H. arduine is an endo- and D. isaea an ecto-parasitoid might have reduced the risk of direct competition of the immature stages of both species. The coexistence could also be related to host discrimination abilities in one or both parasitoids (Bakker et al. 1985). Further studies are warranted to assess the potential host discrimination abilities in both parasitoids as well as the potential mechanisms involved. Beyond the coexistence observed, the introduction of H. arduine considerably boosted the total parasitism. Releases of 50 individual parasitoids composed of 25 H. arduine and 25 D. isaea resulted in 2.5 times more parasitism than when 50 individuals of the indigenous parasitoid species were used. At the same host density, releases of 25 or 50 H. arduine resulted in the same level of parasitism, warranting further studies on parasitoid-host density functional and numerical responses. This could guide optimizing the number of parasitoids required for releases in the case of inundative release strategies, such as in greenhouse environments. Moreover, H. arduine proved to be superior in LMF control than D. isaea, with between 1.4 and 2.6 times higher specific parasitism rates. Such relatively low reproductive performance of D. isaea compared with an endoparasitoid have also been reported by Akutse et al. (2015) while studying the interactions between P. scabriventris and D. isaea using L. huidobrensis as host. Yildirim et al. (2011) and Boot et al. (1992) also reported and modeled low D. isaea parasitism rates on L. bryoniae (Kaltenbach; Diptera: Agromyzidae)) and L. sativae, respectively, in field studies. Similarly, Mujica and Kroschel (2011) found that H. arduine coexisted with a complex of 60 parasitoid species in the field and was more important in controlling the majority of LMF including several Diglyphus species. Along the Peruvian coast, H. arduine was found parasitizing different leafminer species with up to 66.7% parasitism rates under field conditions (Mujica and Kroschel 2011). In parasitoids, nonreproductive host mortality is considered an additional and important mortality factor in pest suppression (Honda et al. 2006). Female parasitoids induce this additional mortality through paralyses of the host by stinging, often followed by host feeding (Jervis and Kidd 1986, Liu et al. 2013, Akutse et al. 2015). In our study, both H. arduine and D. isaea caused insignificant nonreproductive mortality of L. huidobrensis in single and combined release strategies. This insignificant nonreproductive mortality by H. arduine is consistent with earlier results observed during our host performance studies on three LMF species (F. Komi, unpublished data). However, the significant mortalities of L. huidobrensis in the presence of D. isaea are in line with previous reports by Minkenberg (1989), Liu et al. (2013), and Akutse et al. (2015). In addition, the observed mortalities of L. huidobrensis in the presence of H. arduine were also notable, though not significant compared with the control. Further studies on the effect of parasitoid density on host nonreproductive mortalities are warranted. In our study, reproduction of the exotic H. arduine always resulted in a female-biased sex ratio, irrespective of the presence of the other parasitoid species. However, for the indigenous D. isaea, the sex ratio of its F1 became female-biased only in the presence of H. arduine, indicating a synergistic potential when combining both parasitoid species. Since only female parasitoids cause host mortality (Pascua and Pascua 2004, Chow and Heinz 2005, Abe and Kamimura 2012, Foba et al. 2015a), the female-biased parasitoid populations suggest better reproductive and parasitism performances. Conclusion Halticoptera arduine host parasitization is superior to the indigenous D. isaea with synergistic improvement on D. isaea reproduction potential when both parasitoid species were released simultaneously. Thus, if successfully introduced into the East African horticultural production systems, H. arduine might not only coexist with D. isaea, but also raise the total parasitism rates to achieve economically important pest control levels. Low pest pressure may cause small holder farmers to shift away from heavy broad-spectrum pesticides use. Upon receiving the official release permit from the Kenyan authorities, we will therefore assess the performance of the parasitoids under East African field conditions. Acknowledgments We are grateful to Jürgen Kroschel and Norman Mujica of the International Potato Center (CIP) in Peru for providing H. arduine specimens to start the colony in Kenya. The first author was supported through the Dissertation and Research Internship Program (DRIP) of icipe. The present study was conducted with financial support from the German Federal Ministry for Economic Cooperation and Development (BMZ) (Grant number: 09.7860.1-001.00; Contract number: 81121261). We are also grateful to UK Aid of the UK Government, the Swedish International Development Cooperation Agency (Sida), the Swiss Agency for Development and Cooperation (SDC), and the Kenyan Government for their core support to icipe that facilitated the present work. References Cited Abbott , W. S . 1925 . A method of computing the effectiveness of insecticides . J. Econ. Entomol . 18 : 265 – 267 . Google Scholar CrossRef Search ADS Abe , J. , and Y. Kamimura . 2012 . Do female parasitoid wasps recognize and adjust sex ratios to build cooperative relationships ? J. Evol. Biol . 25 : 1427 – 1437 . Google Scholar CrossRef Search ADS PubMed Akutse , K. S. , J. Van den Berg , N. K. 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Environmental EntomologyOxford University Press

Published: Apr 11, 2018

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