DOI: 10.1111/eea.12587 MINI REVIEW Bethylids attacking stored-product pests: an overview Marco Amante1,* , Matthias Sch€oller2, Pompeo Suma1 & Agatino Russo1 1 Dipartimento di Agricoltura, Alimentazione e Ambiente, University of Catania, via Santa Sofia, 100 95123 Catania, Italy, and 2Faculty of Life Sciences, Humboldt-Universit€at zu Berlin, Lentzeallee 55/57, 14195 Berlin, Germany Accepted: 5 April 2017 Key words: Bethylidae, biological control, integrated pest management, natural enemies, parasitoid, ectoparasitoid, IPM, Hymenoptera, idiobionts, oligophagous Abstract Bethylidae is a family belonging to the insect order Hymenoptera and contains about 2 200 described species. Bethylids typically parasitize larvae of Lepidoptera and Coleoptera, including species that are serious pests of stored products. Here, we review the main characteristics of each of the bethylids reported as biological control agent of these pests. The biological characteristics and peculiarities are reported for each species, and the potential for their practical application is discussed. Introduction Much of the research on the control of pests of stored grain has recently focused on biological control, which is an important component of integrated pest management (IPM) (Flinn et al., 1994). This strategy is highly attractive because many pests have become resistant to the main insecticides, so new organic molecules or innovative systems to control pests need to be found (Muggleton, 1987; Herron, 1990; Muggleton et al., 1991; Collins et al., 1993; Lord, 2001; Goubault et al., 2007b; Adler et al., 2012; Eliopoulos et al., 2016). Politicians and organizations such as the Food and Agriculture Organization (FAO) of the United Nations (UN), the European and Mediterranean Plant Protection Organization (EPPO), and the International Organisation for Biological and Integrated Control (IOBC) have begun in recent decades to encourage ecofriendly solutions. Examples are provided by the Code of Conduct for the Import and Release of Biological Control Agents, endorsed by FAO on 28 November 1995, The Rotterdam Convention developed by FAO and the United Nations Environment Programme (UNEP), which was enforced on 24 February 2004, and the worldwide phaseout and ban of the fumigant methyl bromide (Fields & White, 2002). Parasitoids and some biopesticides are among the most adopted tools for the biological control of pests of stored products (Sch€ oller et al., 1997; Lord, 2006, 2008; Sch€ oller, *Correspondence: Marco Amante, Dipartimento di Agricoltura, Alimentazione e Ambiente, University of Catania, via Santa Sofia, 100 95123 Catania, Italy. E-mail: mamante@unict.it 2015; Amante et al., 2017a). The diversity and efficacy of available microbial agents under the storage conditions of durables for the management of stored-product pests, however, remain limited (Copping & Menn, 2000;  arkova et al., 2003; Konecka et al., 2015). The vast Zd majority of parasitoid species used as biocontrol agents against these pests belong to the order Hymenoptera. Biological control has been studied for many decades, and the use of parasitoids is becoming more frequent, especially in central Europe, but gaps in our knowledge remain, and the use of parasitoids is not always adopted or successful. Many parasitoids have been studied extensively, but others are less well known, including those belonging to Bethylidae (Hymenoptera). The majority of wasps reported as promising biological control agents are in the families Pteromalidae and Bethylidae (Hagstrum & Flinn, 1992; Kapranas et al., 2016a,b), so more information on natural enemies belonging to Bethylidae could clearly enhance the future of IPM. Bethylidae is a primitive family of Aculeata with a worldwide distribution and containing about 2 200 described species (Hawkins & Gordh, 1986). Bethylids are idiobiont ectoparasitoids and typically oligophagous. Morphological, behavioural, and biological aspects of this family have been studied, but here we review general life histories, including competitive and agonistic behaviour, because these aspects have a basic importance for the outcome of mass rearing and for the application of biological pest control treatments. Bethylidae life history – basic biology The biology of some bethylid species that attack storedproduct pests has been studied in detail (Powell, 1938; © 2017 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 163: 251–264, 2017 251 252 Amante et al. Rilett, 1949; Finlayson, 1950a; Hardy & Mayhew, 1998; Hardy et al., 1998, 2013; Perez-Lachaud & Hardy, 1999; Mayhew & Heitmans, 2000; Cheng et al., 2004; Goubault et al., 2007b). Bethylid males characteristically emerge before females (protandry) and enter the cocoon of females to mate, which results in a high degree of sib-mating (inbreeding) (Hardy & Mayhew, 1998). This inbreeding does not appear to be genetically detrimental, but females may subsequently become sperm-limited and may re-mate various times with unrelated males (Clausen, 1940, 1962; Rilett, 1949; Finlayson, 1950a; Itoh, 1980; Ahmed & Islam, 1988; Howard & Flinn, 1990; Cook, 1993; Ahmed & Khatun, 1996a; Cheng et al., 2003, 2004). The practical advantage of protandry is strictly associated with the period before oviposition. Mayhew & Heitmans (2000) and Amante et al. (2017a) indicated that unmated females either lay unfertilized eggs (producing only male offspring) or wait for a mating opportunity (after which they produce both male and female offspring). This difference has a temporal cost; for example, Amante et al. (2017a) reported a significant difference in the times until the first eggs were laid between mated (mean  SE = 3.68  0.28 days) and unmated (5.36  0.51 days) Holepyris sylvanidis (Brethes) females. The male plays an active role in the courtship before each mating, stimulating the sexual receptivity of the female. The beginning of the mating can thus be influenced by the courtship behaviour. Males and females mate multiple times, and the female is thought to need more matings to replenish sperm. In fact, some authors have reported that this aspect is very common in bethylid females; evidence of sperm depletion is lacking. Female bethylids begin to search for larval hosts after emergence, and when one is found, she immediately stings it and hides the larva in a safe place where she will lay her eggs. Bethylids are solitary or gregarious parasitoids able to highly control sex allocation. The clutch size in the gregarious species is primarily influenced by host size. Some studies have indicated a sex ratio biased towards females when sib-mating is common, but to our knowledge, no empirical data demonstrate how sib-mating can influence biological control programs based on bethylids (Frank, 1985; Herre, 1985; Flinn, 1991; Morgan & Cook, 1994; Flinn & Hagstrum, 1995; Ahmed et al., 1997; Mayhew & Godfray, 1997; Mayhew, 1998; Mayhew & Heitmans, 2000; Cheng et al., 2003, 2004). Parasitization Paralysis is the first step prior to egg laying, and the hatching larva will develop as an ectoparasitoid (Powell, 1938; Clausen, 1940, 1962; Finlayson, 1950a; Hardy et al., 1992; Al-Kirshi, 1998; Howard et al., 1998). The female attacks the host to paralyse it by initially crawling over the host larva and curving her abdomen beneath the thorax, then inserting her ovipositor into the intersegmental membrane of the host and injecting the venom. The larva rolls itself during the attack as a defensive behaviour against the wasp, which is trying to sting and inject the venom (Hu et al., 2012). The female bethylid transports the paralysed prey, dragging it along the surface of the infested substrate, to a crevice or cavity where she oviposits (Powell, 1938; Finlayson, 1950a; Ahmed & Islam, 1988; Cheng et al., 2004). An aspect that is of interest from a biological as well as applied perspective is that the number of hosts attacked and paralysed by a female often exceeds those that receive eggs. A typical example was reported for Prorops nasuta Waterston, in which females fed on small hosts but oviposited on large larvae of Hypothenemus hampei (Ferrari) (Clausen, 1940, 1962; Amante et al., 2017a,b). Oviposition and larval development Bethylids are either gregarious or solitary wasps, although some can act as semi-gregarious parasitoids. Gregarious species lay a clutch of eggs disposed transversely on one host, and the size of the clutch is influenced by the size of the host. Solitary species lay one egg with the end directed caudally (Clausen, 1940, 1962; Godfray, 1987; Hardy et al., 1992). Host larvae with a setose cuticle may be prepared by the female for oviposition by removing the setae at the site of oviposition (Al-Kirshi, 1998). Eggs can hatch after varying periods depending on environmental conditions; for example, eggs laid by Cephalonomia gallicola (Ashmead) required 13 days to hatch at 22.6 °C, but eggs laid by Parascleroderma berlandi Maneval required 1–4 days, with an extreme of 7 days. The emerging larva begins to feed immediately after completely freeing itself from the eggshell. Clausen (1940, 1962) reported that the larva makes only one feeding puncture during its life. The duration of the larval stage generally varies, with a mean of 5 days. The larval stage in Goniozus and Cephalonomia is completed in 2–3 days, but 10 days has been reported for Bethylus cephalotes (F€ orster). The larvae are able to move a few centimetres from the host after feeding to spin a cocoon. Intraspecific competition Bethylids exhibit behaviour that can influence the choice of method for mass rearing and potentially the efficacy of applications of biological pest control. This behaviour often leads to competitive and agonistic conflicts, whose interactions and consequences vary based on the sociality Bethylids attacking stored-product pests 253 of the species, physiological state, past experiences and environmental conditions. Adults of both sexes can be injured or sometimes killed as a consequence of competitive and agonist behaviour. Identifying the most common agonistic behaviour and its interactions for each species can thus be important for the population ecology of the bethylids of stored-product pests and can be exploited to enhance biocontrol applications. How this behaviour is involved in competitive and agonistic interactions has mostly been reported for gregarious bethylids, and little information is available for solitary species (Goertzen & Doutt, 1975; Hardy & Blackburn, 1991; Mayhew, 1997, 1998; Mayhew & Hardy, 1998; Takasu & Overholt, 1998; Stokkebo & Hardy, 2000; Perez-Lachaud et al., 2002, 2004; Batchelor et al., 2005, 2006; Hardy & Goubault, 2007; Venkatesan et al., 2009a,b). Examples of these behaviours include female–female (for offspring production sites or food sources) or male– male (for mating opportunities) interactions, involving biting and stinging. These behaviours are exhibited when conspecific (and sometimes allospecific) individuals are defending or acquiring resources. Competitive and agonistic behaviours are also exhibited when guarding broods and caring for the larvae, so clutch-size strategies may be influenced (Lawrence, 1981; Petersen & Hardy, 1996; Humphries et al., 2006; Goubault et al., 2007a; Matthews & Deyrup, 2007; Hardy et al., 2013). Other competitive behaviours are exhibited after the eggs have been laid. For example, female bethylids have been observed to adopt the ovicid tactics of eating the eggs of conspecifics or killing the offspring of other females when a parasitized host is found. Superparasitism is a further example of competitive behaviour when an egg is laid on a host that has been previously parasitized by a conspecific female, which leads to fighting among the newly emerged larvae, suppressing one or more contenders for resources (Clausen, 1940, 1962; Hardy et al., 1992, 2013; Godfray, 1994; Ahmed et al., 1997; Mayhew, 1997). These conflicts are closely linked to the postdefensive ovipositional behaviours; in fact, ovipositing females guard their offspring from harmful actions by conspecific females, especially for gregarious species (Clausen, 1940, 1962; Goertzen & Doutt, 1975; Hardy & Blackburn, 1991; Takasu & Overholt, 1998; Hardy et al., 1999, 2013). This behaviour consists of remaining with the host and the developing broods for days after oviposition, with the goal of increasing reproductive success (Batchelor et al., 2005; Goubault et al., 2006, 2007b, 2008; Venkatesan et al., 2009b; Hu et al., 2012; Lize et al., 2012). Sex allocation is a further behaviour that is of interest for the application of biological control and the programs for rearing Bethylidae. Strategies of sex allocation determine how females adjust the sex ratios of the broods and have been mostly investigated for gregarious bethylids (Ode & Hardy, 2008). The ways in which female parasitoids adjust the sex ratio can significantly influence population dynamics and have also been used to understand the reproductive strategies of other parasitoids of stored-product pests (Hardy & Mayhew, 1998; Hardy et al., 1998; Tang et al., 2014; Kapranas et al., 2016a). Host size (female eggs are laid on larger hosts), resource availability, clutch size, risks of immature-male mortality, probability of mating between siblings, environmental constraints, and level of competition can all influence sex allocation (Hamilton, 1967; van den Assem, 1971; Werren, 1980, 1984; Charnov et al., 1981; Charnov, 1982; King, 1987, 2002; Brault, 1991; Mayhew & Godfray, 1997; West et al., 2000; Lebreton et al., 2009). Bethylidae for biological control of stored-product pests Interest in methods of biological control using parasitoids for stored products has grown considerably in recent decades and has been mostly focused on Pteromalidae, Braconidae, and Ichneumonidae parasitoids (Sch€ oller et al., 2006; Suma et al., 2014). Applied entomologists are now focusing their attention on Bethylidae, because some species are promising candidates for programs of biological pest control. The main bethylids in stored-grain ecosystems are species of Cephalonomia, Holepyris and Laelius (Table 1; Howard et al., 1998). Some researchers have thus conducted applied studies on how the biological characteristics of bethylids and their possible interactions with other organisms can be useful in biological treatments, although several gaps in knowledge remain (Bridwell, 1919, 1920; Itoh, 1980; Klein & Beckage, 1990; Flinn, 1991; Klein et al., 1991; Flinn & Hagstrum, 1995; Mayhew & Heitmans, 2000; Lord, 2001, 2006; Eliopoulos et al., 2002, 2016; Cheng  arkova et al., 2003; Lim et al., 2007; et al., 2003; Zd Reichmuth et al., 2007; Collatz & Steidle, 2008; Lorenz et al., 2010; Amante et al., 2017a,b). Considering bethylids as biocontrol agents is important because their hosts are widely reported as pests of crops and stored products and because bethylids are biologically different from parasitoids belonging to other families (Perez-Lachaud & Hardy, 1999). Studies of parasitoid biology are essential for determining whether a parasitoid is suitable for a biological pest control program (Evans, 1964). We report a review of the main biological and beneficial characteristics of Bethylidae as biological control agents. 254 Amante et al. Table 1 Bethylid species attacking stored-product pests Host (s) Bethylid Order Family Species Reference Holepyris glabratus Lepidoptera Pyralidae Corcyra cephalonica Stainton Plodia interpunctella (H€ ubner) Cadra (Ephestia) cautella (Walker) Erechthias flavistriata (Walsingham) Araecerus fasciculatus (DeGeer) Gibbium psylloides (Czempinski) Niptus hololeucus (Faldermann) Ptinus fur (L.) Ptinus tectus Boieldieu Lasioderma serricorne (Fabricius) Stegobium paniceum (L.) Oryzaephilus mercator (Fauvel) Oryzaephilus surinamensis L. Bridwell (1919, 1920); Pemberton (1932); Clausen (1940); Li (1976) Cephalonomia gallicola Coleoptera Phycitidae Tineidae Anthribidae Ptinidae Anobiidae Cephalonomia tarsalis Silvanidae Cephalonomia waterstoni Holepyris sylvanidis Cucujidae Cucujidae Tenebrionidae Silvanidae Laelius pedatus Dermestidae Plastanoxus westwoodi Cucujidae Cryptolestes ferrugineus (Stephens) Cryptolestes minutus (Olivier) (= C. pusillus) C. ferrugineus Tribolium castaneum (Herbst) Tribolium confusum J. du Val O. mercator O. surinamensis Anthrenus flavipes LeConte Anthrenus sarnicus Mroczkowski Anthrenus verbasci L. Trogoderma angustum Solier Trogoderma glabrum Herbst Trogoderma granarium Everts Cryptolestes pusillus (Sch€ onherr) Cryptolestes turcicus (Grouvelle) Genus Cephalonomia Cephalonomia gallicola This species is an arrhenotokous, cosmopolitan, synovigenic, gregarious ectoparasitoid of larvae and pupae of Coleoptera infesting stored products. It is characterized by its wing polymorphism with the females always apterous and the males either apterous or macropterous. The female sometimes opens the integument of the host before laying eggs. This behaviour offers an easy way for the newly hatched larvae to feed on the host, especially during their early stages (Kearns, 1934a). The larval stage lasts for 6–9 days at 20–26 °C. The larva begins to spin a cocoon as soon as the host is completely devoured. In this sense, C. gallicola differs from other Bethylidae reported as Kearns (1934a); Itoh (1980); Yamasaki (1982); Kuwahara (1984); Lim et al. (2007) Lord (2001, 2006); Cheng et al.  arkova et al. (2003); (2003); Zd Collatz & Steidle (2008) Flinn (1991); Flinn & Hagstrum (1995); Reichmuth et al. (2007) Mertins (1980); Ahmed & Islam (1988); Morgan & Cook (1994); Ahmed et al. (1997); Mayhew (1997, 1998); Eliopoulos et al. (2002); Reichmuth et al. (2007); Lorenz et al. (2010); Adler et al. (2012); F€ urstenau et al. (2016); Amante et al. (2017a) Klein & Beckage (1990); Klein et al. (1991); Al-Kirshi (1998); Mayhew & Heitmans (2000); Reichmuth et al. (2007) Sch€ oller (1998, 2012); Rahman et al. (2008) biological control agents of stored-product pests, because the parasitoid larva can pupate inside the empty cocoon of its host, Lasioderma serricorne (Fabricius) (Kearns, 1934a). The pupal stage can last 7–18 days, depending on the sex and adult form. The pupal stage lasts for 11–18 days for females, 9–15 days for winged males, and 7–12 days for wingless males at 20–26 °C. Total developmental time consequently varies with temperature, requiring 34 days at 22.6 °C and 64 days at 18.4 °C (Kearns, 1934a,b). Itoh (1980) evaluated the duration of the developmental period under natural conditions, reporting 60 days in spring and 20–30 days in summer. This species has four or five generations per year in the absence of artificial heating (Kearns, 1934a,b; Tanioka, 1982; Yamasaki, 1982; Lim et al., 2007; Lee et al., 2014). Bethylids attacking stored-product pests 255 Cephalonomia gallicola is a candidate for the biological control of stored-product pests, and some studies have evaluated its characteristics for this purpose (Table 1). This wasp is not attracted by light and so can be released in the presence of light traps and can enter cracks and crevices, penetrating substrates deeply when looking for hosts. It has a large number of hosts, for example, L. serricorne and Stegobium paniceum (L.), Araecerus fasciculatus (Degeer), Niptus hololeucus (Faldermann), Ptinus fur (L.), and Ptinus tectus Boieldieu (Table 1). Itoh (1980) reported that C. gallicola was able to parasitize larvae of the ptinid Gibbium psylloides (Czempinski) under laboratory conditions. The same author collected the wasp in stored oil cakes infested by S. paniceum and concluded from his biological studies in Japan that its reproductive capacity was highest from July to September. Cephalonomia gallicola shows potential as a biocontrol agent but can sting humans, for example, on bare arms, legs, trunks and necks (Kearns, 1934a). The symptoms are redness around the stung area, swelling, pain and pruritic erythematous papules (Matsuura, 1981; Yamasaki, 1982; Kuwahara, 1984; Lee et al., 2014). This aspect has been evaluated only from a medical perspective, and further studies are absolutely needed to fully understand the conditions in which the female stings and whether stinging is a genuine obstacle during pest management programs based on the use of this natural enemy. Cephalonomia tarsalis Cephalonomia tarsalis (Ashmead), common in stored-product facilities, is a predator and an ectoparasitoid of the sawtoothed grain beetle, Oryzaephilus surinamensis L., and the merchant grain beetle, Oryzaephilus mercator (Fauvel) (Powell, 1938; Lord, 2001). Cheng et al. (2003) reported that a mated female could lay either a single egg or a pair of eggs on a host. Of the eggs laid by a mated wasp 80% are female, and when two eggs are laid on a single host, one becomes male and the other becomes female. The first egg is typically laid on the prothorax of the host, whereas the second is laid on the mesothorax (Powell, 1938; Cheng et al., 2003). Mating status can influence the number of eggs laid: virgin females lay an average of 50 eggs, whereas non-virgins lay an average of 85 eggs (Cheng et al., 2003). Eggs hatch within 24 h after oviposition and males emerge 1–2 days before the females (Powell, 1938). The larval period of C. tarsalis begins when contractions and expansions of the digestive tract, due to the ingestion of food, are visible. The young larva will feed as a semi-ectoparasitoid, completely consuming its host in 4 days (Powell, 1938; Cheng et al., 2003). The number of eggs laid on the host can influence the feeding time. The host larva is consumed in two-thirds of the time required by a single larva when two eggs are deposited. The larva spins a cocoon in about 6 h after it stops feeding. The cocoon plays an important protective role against parasitic mites (Powell, 1938). The pupal phase of C. tarsalis is the longest pre-imaginal stage, the abdomen typically begins to darken, and the entire body becomes dark grey to black after 1 day. Ecdysis to the adult then occurs. The adult needs about 4–5 days to emerge from the cocoon due to weakness and is sluggish during the first hours of life. Lukas & Stejskal (2004) indicated that the developmental time for all stages was shortest at 27 and 30 °C but did not differ significantly between these two temperatures. Egg development was shortest at 30 °C, lasting for 1.1 days. Larval development required 5.2 days at 30 °C, but in contrast, the shortest pupal development (5.2 days) was recorded at 27 °C. Lifespan differs considerably between the two sexes, and mating does not influence it. Males live about 6 days, and do not feed, whereas females live an average of 35 days (Powell, 1938; Cheng et al., 2003, 2004). Cephalonomia tarsalis females find their hosts by two main sensory modalities. The first is sight – the wasp has well-developed eyes, which can help when some light is present – the second is by following chemical trails deriving from the host’s food and faeces (Howard et al., 1998; Eliopoulos et al., 2016). The latter was first reported for Cephalonomia waterstoni Gahan, which is able to locate Cryptolestes ferrugineus (Stephens) following the trails laid by the beetle. Howard et al. (1998) hypothesized that the host’s cuticular hydrocarbons provide the first signal to the wasp, even though C. tarsalis needs to perceive movement from the larvae before attacking. This wasp also recognizes its host by the stimuli deriving from the trails and surface of O. surinamensis larvae. Host faecal odour, which is host-specific, is also used, and the female can discriminate areas with host activities when it detects the odour (Collatz & Steidle, 2008; Eliopoulos et al., 2016). Cephalonomia tarsalis is able to parasitize both the larvae and pupae of the sawtoothed grain beetle and the merchant grain beetle, O. mercator, so is a useful agent for their biological control (Cheng et al., 2003; Lukas & Stejskal, 2004; Lukas, 2005; Collatz et al., 2009). Gordh & M oczar (1990) listed C. tarsalis as a parasitoid of various species of Sitophilus and Tribolium castaneum (Herbst), and Howard et al. (1998) reported that this natural enemy was an obligate parasitoid of the sawtoothed grain beetle. The literature offers some examples of combining the use of C. tarsalis with other biological control agents (Table 1). One study combined C. tarsalis and the entomopathogenic fungus Beauveria bassiana (Balsamo) but found that the eggs laid by the wasp did not survive and that the parasitoid was also susceptible to the fungus 256 Amante et al.  arkova et al. (2003) evaluated the com(Lord, 2001). Zd patibility of Cheyletus eruditus (Schrank) and C. tarsalis as combined agents against O. surinamensis. Beetle populations were controlled better when the two natural enemies were applied in combination than when each was applied alone. Lord (2006) evaluated the interactions between C. tarsalis females and the parasitic protozoan Mattesia oryzaephili Ormieres and found that they could interact either complementarily or synergistically, suppressing the pest beetles in both cases. The wasp could easily disseminate the pathogen while moving around in the environment of the stored product, enhancing its action, although M. oryzaephili is transmitted by oviposition. Mattesia oryzaephili shortens the lifespan of C. tarsalis, but the lifespan remains sufficient to produce a benefit when the parasitoid and neogregarine are combined. The combination did not influence the behaviour of this biocontrol agent. Eliopoulos et al. (2016) demonstrated mutual interference in C. tarsalis, especially at higher densities of parasitoids. Eliopoulos et al. (2016) thus considered augumentative and inundative releases of C. tarsalis as a potential means of suppressing pest populations, but an efficient system for mass rearing the parasitoids is needed. Cephalonomia waterstoni This is an arrhenotokous ectoparasitoid, reported as a parasite of the rusty grain beetle, C. ferrugineus. Cephalonomia waterstoni is able to find hosts by recognizing residual kairomonal cues on infested substrates, similar to other parasitoids (Howard & Flinn, 1990). Immediately after the female wasp encounters a larva, she begins host recognition by touching the host with her antennae. This process is completed quickly, and the parasitoid then attacks the prey suddenly. The female has powerful mandibles, the host larva cannot escape when she bites it (Rilett, 1949). The host larva is often larger than the C. waterstoni adult and tries to avoid the paralysis by moving continuously, but the host always succumbs to the attack and is consequently paralysed. The wasp must sometimes fight for several minutes against the defensive reaction, and many attempts may be required before it successfully stings the host. The sting is inserted in a randomly chosen part of the host body, and the injection is rapid, causing paralysis after a few seconds. The envenomation is so prolonged that the host larva has no opportunity to recover (Rilett, 1949). The female:male ratio of C. waterstoni in laboratory cultures is 2:1, and at 25 °C, the complete lifespan is about 3 weeks for females and much shorter for males. The presence of this parasitoid was indicated in specimens collected from stored grain, in which the rusty grain beetle was also registered as a pest (Sinha et al., 1979; Flinn, 1991). Rilett (1949) reported a rapid increase in a laboratory population of C. waterstoni in wheat infested by C. ferrugineus, and the concurrent presence of the parasitoid caused a marked delay in the increase of the beetle population. Hagstrum (1987) and Reichmuth et al. (2007) reported the ability of C. waterstoni to maintain the population of rusty grain beetles below the economic threshold. Further suggestions for the suitability of C. waterstoni in programmes of biological pest control were reported by Flinn & Hagstrum (1995). The authors emphasized the ability of this parasitoid to find its host, which usually develops between grains or in coarse substrate. One of the most important characteristics of C. waterstoni is its capacity to penetrate the substrate. This behaviour likely enhances the potential of C. waterstoni as a biocontrol agent. Finlayson (1950a) first reported this behaviour, and Flinn et al. (1994) subsequently reported the ability of C. waterstoni to spread from one bin to another under field conditions, even though the bins were tightly sealed. Reichmuth et al. (2007) confirmed this ability, indicating that the wasp could enter through narrow cracks and crevices when searching for hosts. Cephalonomia waterstoni is able to attack various species of Cryptolestes but prefers C. ferrugineus when a choice is provided (Finlayson, 1950b; Flinn, 1991). About half to two-thirds of the Cryptolestes minutus (Olivier) [= Cryptolestes pusillus (Sch€ onherr)] and Cryptolestes turcicus (Grouvelle) in the same experiment were not attacked, or if attacked, were not seriously damaged. Finlayson (1950b) supplied larvae of O. surinamensis and O. mercator to C. waterstoni, but no eggs were laid even though some larvae were stung and paralysed. Cephalonomia waterstoni females, like many parasitoids, generally either feed or lay eggs when in contact with a host. They are able to find hosts in any region of the grain mass. This natural enemy feeds on first, second and third instars, but eggs have only been found on fourth instars. The female feeds from her prey by piercing the cuticle and then sucking the haemolymph. The male does not feed on the body fluids and remains passive when encountering a host larva. Flinn & Hagstrum (1995) demonstrated in simulation models of C. waterstoni how the use of the appropriate time of release could control C. ferrugineus better. According to the authors, the ‘optimal timing of parasitoid release’ was strictly correlated with the possibility of the parasitoid finding the first fourth-instar beetles produced. In other words, the control would be most effective when parasitoids are released in time to suppress the newly formed fourth instars. Flinn & Hagstrum (1995) estimated that the immigration rate for C. ferrugineus in the field was ca. 10 beetles per 27 ton per day. If 200 C. ferrugineus Bethylids attacking stored-product pests 257 could immigrate into an 81-ton (3 000 bu, wheat bushel) bin during the first week, then releasing 200–400 parasitoids 20 days after the grain is stored should produce a good control. Lord (2006) demonstrated that the wasps can serve as mechanical vectors of M. oryzaephili, enhancing the pathogen’s dissemination. Mattesia oryzaephili is transmitted by the mandibles and ovipositor during the normal activities of the biocontrol agent. Infection with M. oryzaephili reduced the lifespan of C. waterstoni by 21%, but the survival period of the wasp was sufficient to benefit the efficacy of the combined wasp-neogregarine biological control. Other bethylid species, as for C. waterstoni, can cause irritation to humans by stinging (van Emden, 1931; Finlayson, 1950a). Genus Holepyris Holepyris glabratus Holepyris glabratus (Fabricius) is the only natural enemy among the Bethylidae of stored-product pests considered here able to develop on Lepidoptera larvae. This parasitoid is also the least well studied. Bridwell (1920) observed the behaviour of this parasitoid associated with Erechthias flavistriata (Walsingham), Plodia interpunctella (H€ ubner) and Corcyra cephalonica Stainton. Bridwell (1919) found H. glabratus in warehouses in Honolulu (HI, USA) and indicated that Ehrhorn (unpubl.) reared this natural enemy from Cadra (Ephestia) cautella (Walker) (Table 1). Pemberton (1932) stated that this wasp was reared on small Lepidoptera in stored foods on dried and broken algaroba [Prosopis juliflora (Sw.) DC.] seed pods. This natural enemy can cause irritation due to stinging, similar to C. gallicola. Holepyris sylvanidis The host-searching behaviour of H. sylvanidis is influenced by the presence of host faeces, in which two compounds are thought to be responsible for the attraction: (E)-2-nonenal and 1-pentadecene (F€ urstenau et al., 2016). Host recognition begins when a female meets a larva of Tribolium spp., followed by the injection of venom. The H. sylvanidis female brings the paralysed host to a potential hiding place where the egg will be laid. The host larva cannot move freely due to the paralysis but remains alive during egg laying and for the next few days. The paralysis causes continuous but uncoordinated movements of the legs and head (Ahmed et al., 1997). The lifespan of males is strongly influenced by the presence of a food source. The number of offsprings of H. sylvanidis is not influenced by mating, although it can strongly influence the preoviposition period (Amante et al., 2017a). The ability of H. sylvanidis to penetrate cracks and crevices makes it a promising natural enemy against stored-product pests. Pest larvae are often hidden under thin layers of substrate, in aeration ducts, in machines, and in areas that are difficult to clean, but this wasp is able to access these critical environments (Reichmuth et al., 2007; Lorenz et al., 2010). Holepyris sylvanidis females are able to penetrate layers of grist when they search for hidden larvae. Lorenz et al. (2010) reported that the ability to find hosts was hindered by small particles: the parasitoid could easily penetrate 1 and 2 cm of fine grist, was less successful in penetrating a layer of 4 cm, and could not penetrate deeper than 8 cm into coarse grist. The favourite hosts (T. castaneum and Tribolium confusum Jacquelin du Val) often live in milled grain (Sokoloff, 1974), so the ability of H. sylvanidis to penetrate substrates would be an asset for use in biological control. Eliopoulos et al. (2002) reported that H. sylvanidis was the most ‘dominant’ parasitoid collected in storage facilities in Greece but was the second most frequent parasitoid. Eliopoulos et al. (2002) stated that H. sylvanidis was able to suppress C. tarsalis when both occurred in the same substrate. The authors suggested two explanations: H. sylvanidis was able to disperse better and could reach the hosts more quickly, and H. sylvanidis was able to develop on a wider range of hosts, whereas C. tarsalis parasitized O. surinamensis almost exclusively. Holepyris sylvanidis is a natural enemy of Tribolium species and in some cases has attacked other pests of stored environments (Table 1); however, the host range of this species needs further study. Adler et al. (2012) evaluated the efficacy of releases of H. sylvanidis in a five-storey mill after heat treatments. The heat treatments were conducted in April and between May and November. About 300 unsexed H. sylvanidis were released every 2 weeks in batches of 150 on the first and second floors, and the presence of the pests was monitored. The number of flour beetles remained low until November but increased considerably in January, especially in the cellar around the base of the grain elevator. Laelius pedatus Laelius contains 15 described species (Mayhew & Heitmans, 2000). The best known and most widely distributed is the arrenotokous Laelius pedatus (Say), which has been widely reported as a semi-gregarious ectoparasitoid of Dermestidae (Coleoptera) larvae (Mertins, 1980; Mayhew, 1998; Mayhew & Heitmans, 2000). Laelius pedatus has been described by various authors from the following Dermestidae: Anthrenus flavipes (LeConte), Anthrenus sarnicus Mroczkowski, Anthrenus verbasci (L.), Trogoderma 258 Amante et al. glabrum Herbst, Trogoderma angustum (Solier), Trogoderma granarium (Everts) and Trogoderma variabile (Ballion) (Mertins, 1980; Klein & Beckage, 1990; Mayhew & Heitmans, 2000; Reichmuth et al., 2007). The wasp has been reared in laboratory conditions using A. flavipes and T. variabile (Klein & Beckage, 1990; Mayhew, 1997) or A. verbasci and T. angustum (Al-Kirshi, 1998) as hosts (Table 1). Clutch size can be directly influenced by larval weight and/or dimensions (Mertins, 1980; Klein et al., 1991; Mayhew, 1998). The wasp investigates its host with its antennae, but the mechanisms used by L. pedatus to determine the host dimension remain unclear. Females choose a host by its volume, perhaps by using the ‘conditional sex expression’ strategy in which a female can obtain the best benefits from large hosts (Klein et al., 1991). The time of exposure to hosts, age, nutritional state and mating status can also influence the number of eggs. The last egg deposited by the wasp in a clutch is usually a male (Mayhew & Heitmans, 2000). The daily average number of eggs laid by a female wasp is 1.42 when larvae of T. angustum are the hosts. A female wasp laid 62 and 25 eggs at low (<10%) and high (>90%) humidity, respectively, at 28 °C. Oviposition was highest at temperatures between 25 and 28 °C, and no oviposition occurred at 15 °C (Reichmuth et al., 2007). Mertins (1980) reported a mean period between oviposition and egg hatching of 4.0  0.6 days. The first instar makes a hole through the host’s exoskeleton immediately after emergence in order to feed. The larvae grow rapidly, completing development in 3–4 days (Mertins, 1980; Mayhew, 1998). Mertins (1980) reported three instars for L. pedatus. The total developmental time from egg to adult was 34.7 days at 28 °C. The lifespan of L. pedatus ranges from 27 days for females to 9 days for males (Mertins, 1980), but the lifespan of females can be influenced by temperature: 3 weeks at 35 °C, but up to 16 weeks at 20 °C (Reichmuth et al., 2007). Laelius pedatus females display an adaptive behaviour that has not been reported for the other bethylids that attack stored-product pests. They remove the eggs laid by conspecific females before laying a clutch of their own on the same host (Mayhew & Heitmans, 2000). This parasitoid has a long handling time, but no brood guarding has been reported (Mayhew, 1997). The offspring of L. pedatus are subject to attack by various natural enemies throughout development, reducing reproductive efficacy. The main factors causing mortality are predatory mites, which kill eggs, possible bacterial infection (causing eggs to become red), and the eulophid hyperparasitoid Melittobia acasta (Walker), which attacks larvae and pupae (Mayhew & Heitmans, 2000). The female wasp acts opportunistically and will always sting and paralyse a larva when encountered. This natural enemy can thus be adopted as a control agent of storedproduct pests (Klein & Beckage, 1990; Yuntai & Burkholder, 1990; Klein et al., 1991). Laelius pedatus is negatively phototactic, which enhances its ability to penetrate wheat and successfully parasitize host larvae of T. granarium to a depth of 90 cm (Mertins, 1980). Laelius pedatus reduced populations of T. granarium by 75–80% within 6–8 weeks at a parasitoid:host ratio of 1:25 and could parasitize and kill larvae of T. variabile (Yuntai & Burkholder, 1990). Klein et al. (1991) found that L. pedatus laid more eggs on T. variabile compared to those reported by Mertins (1980) on A. verbasci, whereas Al-Kirshi (1998) reported that one L. pedatus female paralysed 74 larvae of A. verbasci and 44 larvae of T. granarium, using only one-third of the paralysed larvae of the first host species for oviposition. Plastanoxus westwoodi This is a parasitoid associated with the flat grain beetle, Cryptolestes pusillus (Sch€ onherr) (Rahman & Islam, 2006), and C. turcicus. It is a biocontrol agent and external parasitoid able to parasitize fourth instars and pupae of C. pusillus, although it can also paralyse and feed on first through third instars (Ahmed & Khatun, 1996a; Rahman & Islam, 2006). Plastanoxus westwoodi (Kieffer) females search for hosts upon which to lay eggs 24 h after mating (Ahmed & Khatun, 1996b). The eggs are laid individually between the second and third or between the third and fourth abdominal segments ventrolaterally. Eggs are rarely laid on the first thoracic segment dorsally. The parasitoid prefers the third and fourth abdominal segments ventrolaterally when laying eggs on pupae. The mean ( SD) incubation period is 1.7  0.13 days. The newly hatched larva inserts its mouthparts in the cuticle of the C. pusillus larva, devouring the entire contents of the host body in 1.5–2.0 days. Plastanoxus westwoodi prefers to oviposit on fourth instars but can complete its cycle on all instars of C. pusillus except the first, probably because this instar is too small to receive eggs. Developmental time from egg to adult is 12–15 days at 27  1 °C and 70  5% r.h. (Rahman et al., 2008), although variation in these parameters can strongly affect the biological performance of P. westwoodi (Ahmed & Khatun, 1996a). In fact, developmental times are shortest at 25 and 35 °C, where the wasp can maximize its reproduction and longevity. The entire developmental period of P. westwoodi can be influenced by the instar on which it feeds. The wasp may obtain proper nourishment from fourth instars due to the high nutrient content; the developmental period of P. westwoodi is shorter for this Bethylids attacking stored-product pests 259 instar than for the pupa and prepupa. Ahmed & Khatun (1996b) reported superparasitism for P. westwoodi, also indicating that two larvae developed on the host, both emerging as adults. Wasp females search continually for suitable hosts (Ahmed & Khatun, 1996b). When a host is encountered, the female immediately paralyses it by attacking its thoracic and abdominal region with her ovipositor. Rahman et al. (2008) reported that P. westwoodi is an efficient and successful biological control agent in maintaining C. pusillus populations under damaging levels (Table 1). Plastanoxus westwoodi quickly and substantially decreased the size of a laboratory population of the flat grain beetle, but unfortunately no information is available on the suppressive effect of the wasp on populations of C. pusillus under natural conditions (Rahman et al., 2008). Plastanoxus westwoodi may play an important role in the biological control of this beetle, due to its ability to parasitize all stages. Discussion The ecological requirements (related to factors such as temperature, humidity and food source) of stored-grain pests have been widely demonstrated to match those of some natural enemies, and the environment in the cereal food industry is generally suitable for biological control programmes. The life cycles of the main pest species are sometimes not synchronized with those of the biological control agents, but an artificial synchronization by augmentative biological control in these confined environments is achievable. Suitable candidates for the implementation of any biological control program should generally have specific attributes. One of the most important is a positive and rapid density responsiveness (e.g., high reproductive capacity by a short generation time, high fecundity or both), so that a parasitoid species can enhance its rate of increase in response to numerous hosts. The ecological requirements of a parasitoid species should also be as similar as possible to those of the target host species. The ability to find hosts/prey at low densities and thus to reduce their numbers is another primary characteristic of a suitable natural enemy. The degree of biological adaptation to the host (i.e. host specificity) is an important factor in the design of any biological control program of stored-product pests. Finally, native natural enemies should be preferred (Sch€ oller, 1998). The bethylids have been less well studied as biological control agents than parasitoids belonging to some other families. Based on the features reviewed herein, bethylids may be suitable for the biological control of some of the most important pests of stored products and can also be combined with other natural enemies. To our knowledge, C. tarsalis is the only bethylid reported here that is currently sold by producers of natural enemies in large quantities, and L. pedatus is sold in minor numbers against museum pests. The peculiarities of these wasps can be summarized as follows. Bethylids actively search for hosts when released at the surface of infested stored products, using chemical stimuli deriving from the host, host faeces and substrates. They are able to quickly penetrate cracks and crevices, deep into the food substrate mass to locate larval hosts. The ability to penetrate substrates is an advantage during a biological control treatment, especially when reaching hidden places (machines, crevices, commodities) is difficult. Female bethylids typically paralyse most of the hosts on which they oviposit and feed destructively on the larvae, compensating for their low fecundity (Mertins, 1980; Klein & Beckage, 1990; Batchelor et al., 2005; Reichmuth et al., 2007). For example, H. sylvanidis needs to feed on first and second instars to obtain nutrients for egg maturation (Amante et al., 2017a). Bethylids adapt well to combined use with other biocontrol agents, which satisfies the legal requirement and definition of IPM, stipulating that pest suppression should be obtained by integrating ecofriendly methods (Lord, 2001, 2006; Directive 2009/128/ EC). Furthermore, bethylids respond positively to volatile compounds released by plants, substrates and hosts (adults, pupae, larvae). Future applied perspectives of bethylids may thus be enhanced by the use of the chemicals known to elicit host-searching behaviour in olfactometric studies (Howard & Flinn, 1990; Collatz & Steidle, 2008; Collatz et al., 2009; F€ urstenau et al., 2016; Amante et al., 2017a,b). Other peculiarities of bethylids involved in biological control should be considered. For example, aggression towards larvae, especially when bethylids feed on them, makes larvae unsuitable for oviposition. The aggression of female bethylids is a positive aspect for pest control, but it decreases the number of sites for oviposition. The sex-allocation strategy can have a negative impact on a biological control program if the sex bias is towards males. Published data indicate that individual females can vary sex allocation under laboratory conditions, but unfortunately nothing is known about potential methods to manage the sexallocation strategies of bethylids during a pest control treatment (Charnov et al., 1981; Brault, 1991; Ode & Hardy, 2008). We have reviewed seven species of bethylid wasps as candidates for the biological control of storedproduct pests, but the family contains at least 2 200 species (Hawkins & Gordh, 1986), so the list will likely be expanded. Many studies have focused on the biological aspects of the wasps rather than directly on their 260 Amante et al. application in biocontrol programmes. Most studies have linked specific bethylids to several possible hosts but have provided insufficient evidence to support these relationships. In our opinion, scrutinizing these relationships is urgently needed, identifying the links between specific hosts and natural enemies. Relevant biological and behavioural attributes should be taken into account when bethylid species are evaluated for biological control in the future. Cephalonomia gallicola is resistant to adverse climatic conditions both outside and inside buildings, is not attracted by light traps, and is able to penetrate cracks and crevices when searching for hosts (Kearns, 1934a). Some doubt remains about the ability of this wasp to cause dermatitis in humans. Cephalonomia tarsalis is reported as the principal natural enemy of O. surinamensis and O. mercator (Powell, 1938). Holepyris sylvanidis is among the less well studied Bethylidae of stored-product pests, even though it is a promising candidate for the control of T. confusum and T. castaneum. Its ability to penetrate various substrates to find hosts was demonstrated by Lorenz et al. (2010). Holepyris glabratus is the only bethylid reported as a biological control agent against Lepidoptera infesting stored products. This natural enemy can cause irritation to humans due to its sting, similar to C. gallicola (Pemberton, 1932). Laelius pedatus is a wasp that has been studied from an evolutionary perspective, and although knowledge of its applied characteristics is scarce, it has interesting features, such as the ability to penetrate a grain mass to a depth of 90 cm, and it can parasitize T. granarium larvae. Plastanoxus westwoodi can parasitize all stages of C. pusillus and is considered a highly host-specific parasitoid of this beetle, but no information is available on its suppressive effect on populations of C. pusillus under natural conditions (Rahman et al., 2008). The information presented in this overview indicates that various bethylid species may be used in IPM. Information on the methods of use is still lacking, except for C. waterstoni and to a lesser extent for H. sylvanidis. Flinn et al. (1994) and Flinn & Hagstrum (1995) have evaluated aspects of control using C. waterstoni, such as the optimal timing of release and its response to host density and temperature. Adler et al. (2012) evaluated the efficacy of release of H. sylvanidis in a five-storey mill after heat treatments. The literature on bethylids that attack stored-product pests also contains little information on practical aspects, such as collection methods and laboratory rearing. A first step will be to study the production of bethylids in mass rearing, defining the optimal parasitoid:host ratio (Wei et al., 2016). Current information is insufficient for making best use of these species as biocontrol agents, especially compared to other parasitoid taxa, such as Trichogramma spp., Braconidae, and Pteromalidae. Further studies are thus needed to provide solutions to the problem of the stored-product pests considered here. 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