Effect of Larval Diets on House Fly (Diptera: Muscidae) Production and Host Suitability for a Pupal House Fly Parasitoid (Spalangia gemina) (Hymenoptera: Pteromalidae).

A larval medium for house fly (Musca domestica L.) suitable for house fly parasitoid production was developed from locally available and inexpensive food ingredients. Biological parameters and life table parameters were estimated for house flies treated with five different diets. It was found that percentage survival of 1st-3rd instar larvae of house fly, percentage of pupation, percentage of hatching adults, growth index, and life table parameters (net reproductive rate [R0] the cohort generation time [T], intrinsic rate of increase [r], and finite rate of increase [λ]) were significantly highest on larvae produced on diet 5 (composed of rice barn and chicken feed). Diet composition and performance of house fly larvae in larval medium are discussed, based on the results. The results of Spalangia gemina Boucek (Hymenoptera, Pteromalidae) parasitization on house fly pupae revealed that the number of total parasitized pupa and number of parasitoids hatched from pupa reared with diet 5 (rice bran and chicken feed) were greater than with diet 1 (rice bran, powdered milk, dry yeast, fish meal, soybean meal, and chicken feed). Additionally, S. gemina offspring from diet 5 treatment had a higher proportion of females. Our results indicated that pupal size resulting from larval diet was an important factor for parasitization.

Effects, interactions, and localization of Rickettsia and Wolbachia in the house fly parasitoid, Spalangia endius.

Many insect species harbor facultative microbial symbionts that affect their biology in diverse ways. Here, we studied the effects, interactions, and localization of two bacterial symbionts-Wolbachia and Rickettsia-in the parasitoid Spalangia endius. We crossed between four S. endius colonies-Wolbachia only (W), Rickettsia only (R), both (WR), and none (aposymbiotic, APS) (16 possible crosses) and found that Wolbachia induces incomplete cytoplasmic incompatibility (CI), both when the males are W or WR. Rickettsia did not cause reproductive manipulations and did not rescue the Wolbachia-induced CI. However, when R females were crossed with W or WR males, significantly less offspring were produced compared with that of control crosses. In non-CI crosses, the presence of Wolbachia in males caused a significant reduction in offspring numbers. Females' developmental time was significantly prolonged in the R colony, with adults starting to emerge one day later than the other colonies. Other fitness parameters did not differ significantly between the colonies. Using fluorescence in situ hybridization microscopy in females, we found that Wolbachia is localized alongside Rickettsia inside oocytes, follicle cells, and nurse cells in the ovaries. However, Rickettsia is distributed also in muscle cells all over the body, in ganglia, and even in the brain.

Heat Adaptation of the House Fly (Diptera: Muscidae) and Its Associated Parasitoids in Israel.

Insects are ectothermic organisms; hence, all aspects of their biology are strongly influenced by ambient temperatures. Different insect species respond differently with phenotypic plasticity and/or genetic adaptation to changing temperatures. Here, we tested the thermal adaptation of the house fly and three of its parasitoids species by comparing life-history parameters in populations from a hot climate region (Jordan Valley) and from a moderate-climate region (Galilee). No significant differences were found between the two house fly populations, both under hot and moderate experimental conditions. Life-history parameters of the parasitoids (Muscidifurax raptor Girault & Sanders, Spalangia endius Walker, and Spalangia cameroni Perkins [Hymenoptera: Pteromalidae]) varied markedly between origins, species, sexes, and experimental conditions. Of the three species tested, only M. raptor collected in the Jordan Valley proved better adapted to experimental heat conditions, compared to its counterpart population that was collected in the Galilee. Additionally, we tested the effect of elevating temperatures on a house fly lab population for 17 consecutive generations and found no evidence for heat adaptation. We discuss our results in the context of house fly control and global warming.

Experimental and field investigation of non-biting flies as potential mechanical vectors of Echinococcus granulosus eggs.

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1-3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.

A SIMPLE AND EFFECTIVE MULTIPLEX PCR TECHNIQUE FOR DETECTING HUMAN PATHOGENIC TAENIA EGGS IN HOUSEFLIES.

Taenia solium, T. saginata, and T. asiatica are cestode pathogens causing taeniasis in humans. Houseflies can transfer Taenia eggs to food. However, houseflies are thought to carry only small numbers of Taenia eggs, sometimes fewer than 10. Although several PCR-based methods have been developed to detect Taenia DNA, these require more than 10 eggs for adequate detection. We developed a multiplex PCR method with high specificity for the discrimination among the eggs of the three Taenia species, T. solium, T. saginata, and T. asiatica, using 18S ribosomal DNA (rDNA) as a genetic marker. This technique was found to be highly sensitive, capable of identifying the Taenia species from only one egg. This multiplex PCR technique using 18S rDNA specific primers should be suitable to diagnose Taenia eggs.

Effects of Size and Age of the Host Musca domestica (Diptera: Muscidae) on Production of the Parasitoid Wasp Spalangia endius (Hymenoptera: Pteromalidae).

One method of control of house flies, Musca domestica L. (Diptera: Muscidae), and other filth flies is by repeated release of large numbers of pupal parasitoids such as Spalangia endius Walker. Rearing these parasitoids may be facilitated by understanding how host factors affect their production. Previous studies have examined the effects of host size and host age on parasitoid production, but have not examined the interaction between host size and host age or the effects with older females, which may be less capable of drilling tough hosts. Females were given hosts of a single size-age category (small young, small old, large young, or large old) for 2 wk. The effect of host size and of host age on parasitoid production depended on female age. On their first day of oviposition, females produced more offspring from large than from small hosts, but host age had no significant effect. The cumulative number of parasitoids produced in the first week was not significantly affected by host size or host age. However, the cumulative number of parasitoids produced over 2 wk was affected by both host size and host age, with the greatest number of parasitoids produced from small young hosts. Thus, not only are smaller hosts cheaper to produce, but these results suggest that their use may have no effect or a positive effect on the number of parasitoids that can be produced when females are ovipositing for a week or two.

Temperature- and Age-Dependent Survival, Development, and Oviposition Rates of the Pupal Parasitoid Spalangia cameroni (Hymenoptera: Pteromalidae).

The combined effect of temperature and age on development, survival, attack rate, and oviposition of the parasitoid Spalangia cameroni (Perkins) (Hymenoptera: Pteromalidae) exploiting house fly pupae was investigated by conducting life-table experiments at 15, 20, 25, 30 and 35°C. Temperature had a pronounced effect on survival and development of the immature stages. Survival was highest at 25°C, where 88.5% of the parasitized host pupae resulted in adult parasitoids, and lowest at 35°C when only 3.78% emerged. Females constituted between 50% (at 20°C) and 100% (at 35°C) of the surviving immatures. Males developed faster than females, with the shortest developmental times at 30°C (18.18 d for males and 19.41 d for females). Longevity of adult females decreased with temperature from 80 d at 15°C to 18 d at 35°C. Total attack rate of female parasitoids was highest at 20°C (106 hosts per female), and life-time reproduction highest at 20°C and 25°C (about 60 offspring per female). Sex ratio was female biased (65% females). A generic model was used to estimate and predict the temperature effect on the intrinsic rate of increase (rm), the net reproduction rate (R0), and the generation time (G). The model predicted that rm peaks at 33.5°C (rm = 0.182 d(-1)), that maximum R0 is reached at 27.2°C (R0 = 50.2), and that the shortest generation time occurs at 34.5°C (G = 21.1 d). Doubling time was 4.19 d at 33°C. In the temperature range between 20°C and 30°C, S. cameroni has the potential to be an efficient control agent against nuisance flies.

Brevibacillus laterosporus inside the insect body: Beneficial resident or pathogenic outsider?

Brevibacillus laterosporus is an entomopathogenic bacterium showing varying degrees of virulence against diverse insect pests. Conversely, it is regarded as a beneficial component of the intestinal flora in different animals and in some insect species including the honeybee. B. laterosporus was detected through a species-specific PCR assay in the body of different insects, including Apis mellifera and Bombus terrestris. A strain isolated from a honeybee worker was pathogenic to the house fly Musca domestica, thus supporting the development of either mutualistic or pathogenic interactions of this bacterium with diverse insect species, as the result of a coevolutionary process.

House and Stable Fly Seasonal Abundance, Larval Development Substrates, and Natural Parasitism on Small Equine Farms in Florida.

House flies, Musca domestica Linnaeus, and stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), are common pests on horse farms. The successful use of pupal parasitoids for management of these pests requires knowledge of seasonal fluctuations and biology of the flies as well as natural parasitism levels. However, these dynamics have not been investigated on small equine farms. A 1-year field study began in July 2010, in north central Florida, to determine adult fly population levels and breeding areas on four small equine farms. Weekly surveillance showed that pest flies were present year-round, though there were differences in adult population levels among farms and seasons. Fly development was not confirmed on two of the four small farms, suggesting that subtle differences in husbandry may adversely affect the development of immature flies. In six substrates previously identified as the most common among the farms, stable fly puparia were found overwhelmingly in hay mixed with equine manure and house fly puparia were found in fresh pine shavings mixed with equine manure. Natural parasitism was minimal as expected, but greatest numbers of natural parasitoids collected were of the genus Spalangia. Differences in adult and immature fly numbers recovered emphasizes the need for farm owners to confirm on-site fly development prior to purchase and release of biological control agents. Additionally, due to the low natural parasitism levels and domination of parasitism by Spalangia cameroni, augmentative releases using this species may be the most effective.

Comparison of the Olfactory Preferences of Four of Filth Fly Pupal Parasitoid Species (Hymenoptera: Pteromalidae) for Hosts in Equine and Bovine Manure.

House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans (L.)) (Diptera: Muscidae) are common pests in equine and cattle facilities. Pupal parasitoids, primarily in the genera Spalangia and Muscidifurax (Hymenoptera: Pteromalidae), can be purchased for biological control of these flies. However, little is known about the host-habitat preferences associated with host-seeking by these parasitoids. The preferences of two Spalangia and two Muscidifurax species to odors associated with house fly hosts in equine and bovine manure were investigated in the laboratory using a Y-tube olfactometer. Odor stimuli from manure without developing flies, third-instar house flies in manure, and fly host puparia in manure were evaluated. In choice tests, S. cameroni and S. endius were strongly attracted to odor associated with equine manure against clean air. Although S. cameroni was attracted to all bovine manure-containing treatments against clean air, S. endius was only attracted to the bovine manure with third-instar flies. There were no significant differences between the Spalangia species in odor responses. Neither Muscidifurax species were attracted to equine manure treatments and were only attracted to the bovine manure with puparia over clean air. In manure comparison studies, bovine treatments with developing flies were more attractive than the equivalent equine treatments to both Muscidifurax species The data suggest that coexistence between the competing pteromalid parasitoids might be promoted by different host-seeking behaviors. Additionally, manure preferences may indicate parasitoid suitability for releases on different livestock and equine facilities.

Drilling-in and Chewing-out of Hosts by the Parasitoid Wasp Spalangia endius (Hymenoptera: Pteromalidae) When Parasitizing Musca domestica (Diptera: Muscidae).

Many organisms are protected from natural enemies by a tough exterior. Such protection is particularly important for immobile stages, such as pupae. The pupa of some insects is protected by a puparium, which is a shell formed from the exoskeleton of the last larval instar. However, the puparium of certain fly species is drilled through by adult females of the wasp Spalangia endius Walker. The female wasp then deposits an egg on the fly pupa within the puparium. After the wasp offspring finishes feeding on the fly pupa, it chews through the puparium to complete emergence. Despite the apparent toughness of the puparium, there was no detectable wear on the ovipositor of S. endius females even when females had been encountering fly pupae (Musca domestica L.) for weeks, and regardless of whether the pupae were large or old or both. Energy dispersive spectroscopy did not reveal any metal ions in the ovipositor's cuticle to account for this resistance against wear. Offspring of S. endius that chewed their way out of pupae also showed no detectable wear on their mandibles. Tests with a penetrometer showed that the force required to penetrate the center of a puparium was greater for larger and for older pupae; and an index of overall thickness was greater for large old pupae than for small old pupae. The lack of an effect of pupal size or age on wear may result from wasps choosing locations on the puparium that are easier to get through.

Livestock bedding effects on two species of parasitoid wasps of filth flies.

Choice of livestock bedding has been shown to affect density of filth fly maggots. Here, laboratory experiments indicate that bedding type can also affect natural enemies of the flies, specifically the parasitoid wasps Spalangia endius Walker and Urolepis rufipes (Ashmead) (Hymenoptera: Pteromalidae) parasitizing a natural host, the house fly Musca domestica L. (Diptera: Muscidae). For both parasitoid species, when females parasitized hosts under bedding, cedar shavings resulted in fewer parasitoids compared with pine shavings, but pine shavings did not differ from wood pellets and corn cob pellets. In the absence of exposure to hosts, longevity of adult females was reduced in cedar shavings compared with pine shavings and pellets. In contrast to the effects on parasitization and on adult survival, shavings treatment had no significant effect on the number of parasitoids or flies that emerged when hosts were not exposed to shavings until after parasitization.

Competition between the filth fly parasitoids Muscidifurax raptor and M. raptorellus (Hymenoptera: Pteromalidae).

Competition bioassays were conducted with the filth fly pupal parasitoids Muscidurax raptor (Girault & Sanders) and M. raptorellus (Kogan & Legner) (Hymenoptera: Pteromalidae) using house fly Musca domestica L. (Diptera: Muscidae) hosts at different host densities. Muscidifurax raptor had a significant impact on M. raptorellus when hosts were limiting in sequential parasitism tests. Fewer than six M. raptorellus adult progeny emerged from groups of 50 fly pupae that were parasitized by M. raptor at the same time or when M. raptor parasitism preceded M. raptorellus by 48 h, respectively, compared with 42-55 M. raptorellus progeny produced when this species was tested alone. Production of M. raptor was significantly lower when parasitism by this species was preceded by M. raptorellus (25) than when M. raptor was tested alone (43). When the two species parasitized hosts at the same time in different proportions at low host:parasitoid densities (5:1), M. raptorellus produced 13 progeny per parent female when it was the sole species present and fewer than two when M. raptor was present. No negative impact of M. raptorellus on M. raptor was observed. Neither species had a substantial effect on the success of the other at higher host:parasitoid densities.

Habronema muscae (Nematoda: Habronematidae) larvae: developmental stages, migration route and morphological changes in Musca domestica (Diptera: Muscidae).

The present paper describes the morphological modifications occurring during the larval development of Habronema muscae (Nematoda: Habronematidae) in Musca domestica (Diptera: Muscidae), along with the reactions caused by parasitism and the migration route of the nematodes inside the flies. Houseflies were reared on faeces of a H. muscae-infected horse, then dissected and processed by histology. The experimental part of the study was performed in 1996 in the Parasitological Experimental Station W.O. Neitz, Federal Rural University of Rio de Janeiro, Seropédica, Rio de Janeiro State, Brazil. Three different larval stages of H. muscae were recovered, measured and described. The encapsulation of larval nematodes was found in the third larval instar (L3) of M. domestica and cryptocephalic pupa. The mature capsules were observed in dipteran L3, pupae and mainly adults. In 1day-old or more M. domestica adults an active rupturing of capsules by H. muscae L3 and the migration to the head through the circulatory system and insect hemocoel were observed. Infective H. muscae L3s remained exclusively in the head of adult 5days-old or more M. domestica.

Studies on the potential and public health importance of non-biting synanthropic flies in the mechanical transmission of human enterohelminths.

BACKGROUND: This study was aimed at examining the potential of non-biting synanthropic filth flies to acquire naturally eggs of human intestinal helminths from unsanitary sites, and its attendant public health importance. METHODS: Body surface washings and gut contents of flies caught foraging while infected human faeces lay exposed at a garbage dump in Iperu, Ogun State, Nigeria and within 24 hours subsequently after removal of faeces from the dump were examined parasitologically by the formol-ether concentration technique. The viability of helminth eggs isolated from flies was determined by incubation under laboratory conditions. RESULTS: A total of 303 flies were examined: Musca domestica (107; 35.3%), Chrysomya megacephala (125; 41.3%) and Musca sorbens (71; 23.4%). Eggs of Ascaris lumbricoides and Trichuris trichiura isolated from exposed human faeces were recovered from the body surfaces and or gut contents of flies caught before (141; 77.5%) and after removal of exposed faeces (44; 36.4%). Eggs of Taenia sp. were isolated only from the gut contents of three C. megacephala flies caught after removal of exposed faeces. Significantly more (p<0.05) eggs were recovered from fly gut contents than body surfaces and from flies caught before than after removal of exposed faeces. 93.1% (552; from exposed faeces) and 57.4% (408; from flies) of A. lumbricoides and T. trichiura eggs were viable. CONCLUSIONS: Synanthropic flies may, because they carry viable eggs acquired naturally from unsanitary sites, be involved in the epidemiology of human intestinal helminthiases.

A xenodiagnostic method using Musca domestica for the diagnosis of gastric habronemosis and examining the anthelmintic efficacy of moxidectin.

Equine habronematidosis has a global distribution and is caused by three spirurid species, Habronema muscae, Habronema microstoma and Draschia megastoma. A case of cutaneous habronematidosis in a stallion in a stable in Dubai, UAE gave occasion to investigate the parasite situation on the farm. Patent H. muscae infections were diagnosed in 18 out of 49 horses in a stable in Dubai, UAE with a xenodiagnostic test using houseflies as indicator host. All horses in the stable were treated with a single dose of moxidectin administered orally as 2% gel in a dosage of 0.4 mg/kg body weight and the efficacy of this targeted treatment was studied. Habronema infection was terminated in all horses. A fly survey conducted at the farm prior and after treatment revealed two muscid species: Musca domestica and Stomoxys calcitrans. Only M. domestica caught at the farm showed a natural infection with Habronema larvae prior and shortly after anthelmintic treatment. Later, examination of flies caught at the farm in monthly intervals up to the end of observation (8 months after treatment) gave negative results. The absence of infection in the intermediate host was an indication of the eradication of stomach worms. The described xenodiagnostic test is a useful tool to diagnose H. muscae infections and can be used to evaluate the efficacy of nematocides in equines.

Venom of the ectoparasitoid, Nasonia vitripennis, influences gene expression in Musca domestica hemocytes.

Insect hosts have evolved potent innate immunity against invasion by parasitoid wasps. Host/parasitoids live in co-evolutionary relationships. Nasonia vitripennis females inject venom into their dipteran hosts just prior to laying eggs on the host's outer integument. The parasitoid larvae are ectoparasitoids because they feed on their hosts within the puparium, but do not enter the host body. We investigated the influence of N. vitripennis venom on the gene expression profile of hemocytes of their hosts, pupae of the housefly, Musca domestica. We prepared venom by isolating venom glands and treated experimental host pupae with venom. We used suppression subtractive hybridization (SSH) to determine the influence of venom on hemocyte gene expression. At 1 h post treatment, we recorded decreases in transcript levels of 133 EST clones derived from forward a subtractive library of host hemocytes and upregulation in transcript levels of 111 EST clones from the reverse library. These genes are related to immune and stress response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, and transcription/translation regulation. We verified the reliability of our data with reverse transcription quantitative real-time PCR analysis of randomly selected genes, and with assays of enzyme activities. These analyses showed that the expression level of all selected genes were downregulated after venom treatment. Outcomes of our experiments support the hypothesis that N. vitripennis venom influences the gene expression in host hemocytes. We conclude that the actions of venom on host gene expression influence host biology in ways that benefit the development and emergence of the next generation of parasitoids.

[Effects of different treatment methods of housefly pupae for the reproduction of Pachycrepoideus vindemmiae Rondani].

It is an important way to massively rear parasitoid wasps by using appropriate methods to treat the wasps' hosts and preserve them for a long duration. Pachycrepoideus vindemmiae Rondani is a pupal parasitoid of several dipteral pests, being of significance for the biological control of the pests. In this paper, housefly pupae were frozen at -20 degrees C, cold storage-preserved at 6 degrees C, and CO2-asphyxiated for 1-, 3-, and 30 days, respectively, and some pupae were heat-killed and cold storage-preserved for 30 days, aimed to approach the effects of these treatment methods on the reproduction of P. vindemmiae on the pupae. The results showed that P. vindemmiae could reproduce on the pupae treated with the above-mentioned methods, and the tibia length of the offspring had less difference with that on the fresh pupae. However, the reproduction of P. vindemmiae on the pupae treated with the above-mentioned methods except frozen decreased with the increasing preserving duration of the pupae. At the prerequisite of preserving for 30 days, frozen pupae had the highest P. vindemmiae offspring reproduction, suggesting that P. vindemmiae could be massively reared when the housefly pupae were treated by frozen and cold storage-preserved.

Individual and combined releases of Muscidifurax raptor and M. raptorellus (Hymenoptera: Pteromalidae) as a biological control tactic targeting house flies in dairy calf facilities.

The impact of commercially reared house fly parasitoids released into nine dairy calf coverall facilities were evaluated over 3 yr. Individual and equally mixed ratios of the pteromalid parasitoids Muscidifurax raptor Girault and Saunders and M. raptorellus Kogan and Legner were released at a rate of 500 parasitoids per calf per week for 8 wk. Prerelease, release, and postrelease parasitism was monitored using nearly 100,000 sentinel house fly, Musca domestica L., pupae during the 3 yr study. In general, very few adult parasitoids were recovered during the prerelease period and on the no-release farms during any period. However, considerable numbers of M. raptor and M. raptorellus were recovered from sentinel pupae on respective release farms. As expected, the greatest successful parasitism occurred during release periods, with a drop during postrelease periods. High successful parasitism and uneclosed pupae on M. raptorellus release farms suggests that this parasitoid was aggressive in attacking hosts with progeny production at approximately four wasps per pupa. Solitary releases of M. raptor provided sentinel mortality between 31 and 38%, whereas sentinel mortality on M. raptorellus-release farms was double, at 59-80%. Using mixed releases of the two species, overall fly mortality was slightly lower than that observed on M. raptorellus-only farms. This study documents the advantage of releasing M. raptorellus rather than M. raptor on New York dairy calf facilities, as supported by higher parasitism rates and lower costs (35-75%) for purchase of these gregarious wasps, as 75-80% fewer parasitized pupae are needed to achieve similar adult parasitoid levels.

Host suitability of house fly, Musca domestica (Diptera: Muscidae), pupae killed by high or low temperature treatment for a parastoid, Spalangia endius (Hymenoptera: Pteromalidae).

The objective of this study was to establish a high quality progeny production system for the house fly parasitoid, Spalangia endius (Hymenoptera: Pteromalidae), by stockpiling hosts. We performed two host killing methods before host storage: (i) heat-killed by 30 min exposure to 50°C or (ii) freeze-killed by 10 min exposure to -80°C. The average number of parasitoids that emerged from nonstored house fly pupae after heat- or freeze-killing was not significantly different from live pupae. When house fly pupae stored at -20°C after heat-killing were supplied to S. endius, progeny production was significantly less than live pupae. Moreover, productivity became very low when house fly pupae refrigerated at 3°C after heat- or freeze-killing were supplied to S. endius. On the other hand, when house fly pupae stored at -80°C for 1 year after heat-killing were supplied to S. endius, the average number of parasitoids that emerged was not significantly different from live pupae. The average number of parasitoids that emerged from freeze-killed hosts kept for more than 8 weeks at -80°C was significantly fewer than live pupae. Thus, this study clarified that a higher-quality host can be maintained not only by simply storing at -80°C but also by adding heat treatment before storage.