Interactions between the egg and larval parasitoids of a gall-forming midge and their impact on the host

1. The gall‐forming midge Rhopalomyia californica was exposed experimentally to parasitism and predation during only the egg stage, during only the larval stage, during neither stage, or during both stages. 2. The combined action of natural enemies that attack during both the egg stage and the larval stage led to the lowest number of midges and total insects (midges + parasitoids) in the next generation, and the highest percentage parasitism. 3. The larval parasitoid killed a large fraction of hosts without producing new parasitoid offspring, while there is some indication that the egg parasitoid on its own tended to produce the most parasitoid offspring. The contrasting implications of host mortality versus parasitoid production for biological control are discussed. 4. Exposure to larval parasitoids resulted in a reduction in the number of egg parasitoid offspring produced, but exposure to the egg parasitoid did not affect the number of larval parasitoid offspring produced significantly.     

Dispersal and foraging behaviour of Platygaster californica: hosts can't run, but they can hide

Abstract.  1. Host–parasitoid models often identify foraging behaviour and dispersal distance as important for system persistence.
2. Laboratory observations and field trials were used to characterise foraging behaviour and dispersal capability of Platygaster californica Ashmead (Platygasteridae), a parasitoid of the gall midge Rhopalomyia californica Felt (Cecidomyiidae).
3. Although foraging parasitoids meticulously searched plants in laboratory observations, none of the laboratory trials resulted in 100% parasitism, and the proportion of parasitism declined as midge egg density increased.
4. The field trials showed that the distribution of parasitism over distance from a central release point was hump-shaped, as predicted by a simple diffusion model. Mean parasitoid dispersal distance was 4.5 m, considerably farther than the 1.7 m mean midge dispersal found in previous work.
5. Although the parasitoid appears to search thoroughly for midge eggs and to disperse farther than the midge, the results of this study show how this host–parasitoid system may persist due to spatially variable incomplete parasitism.     

Description,phenology and biology of Zelostemma chionochloae Buhl sp. nov., a platygastrid parasitoid of Eucalyptodiplosis chionochloae (Diptera: Cecidomyiidae) in New Zealand

Abstract

A platygastrid wasp that feeds on the snow tussock flower midge in the inflorescences of New Zealand snow tussock grasses (Chionochloa spp.) is formally described. Zelostemma chionochloae is a specialist natural enemy of Eucalyptodiplosis chionochloae Kolesik which is the most ubiquitous and sophisticated seed predator of Chionochloa. Z. chionochloae is a koinobiont parasitoid and some larvae enter prolonged diapause inside their host for at least 2 years. Methods for adult rearing are described. The phenology of Z. chionochloae is highly synchronised with its host even after 2 years in diapause. Parasitism levels were found to differ between years and elevations, while sex ratios differed among years. Z chionochloae probably suffers inter‐specific competition with another host‐specific hymenopteran parasitoid (Gastrancistrus sp.) which also parasitises E. chionochloae.     

Parasitism rates and parasitoid complexes of the wheat midges, <Emphasis Type="Italic">Sitodiplosis mosellana</Emphasis>, <Emphasis Type="Italic">Contarinia tritici</Emphasis> and <Emphasis Type="Italic">Haplodiplosis marginata</Emphasis>

Three species of cecidomyiid midges (Diptera: Cecidomyiidae), whose larvae overwinter in the soil, can cause significant yield losses on wheat in Europe: the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), the yellow wheat blossom midge, Contarinia tritici (Kirby), and the saddle gall midge, Haplodiplosis marginata (von Roser). The biological control of wheat midges by their parasitoids can contribute to reduce the midge populations. Soil samples were collected in several fields in Belgium in 2012–2014 in order to characterize the parasitism rates and parasitoid complexes in overwintering larvae. The parasitism rates varied greatly between the sampled fields: 3–100, 0–100 and 2% for S. mosellana, H. marginata and C. tritici, respectively. The parasitism rate was not related to the larval density of wheat midge. The three wheat midges have totally distinct parasitoid complexes in Belgium. Eight species (Hymenoptera: Pteromalidae and Platygastridae) were found as parasitoid of S. mosellana: Macroglenes penetrans (Kirby), Amblypasis tritici (Walker), Euxestonotus error (Fitch), Euxestonutus sp. Fouts, Leptacis sp. Foerster, Platygaster gracilipes (Huggert), Platygaster nisus Walker, and Platygaster tuberosula (Kieffer). According to their abundance, M. penetrans, E. error and P. tuberosula appeared as the main parasitoids of S. mosellana in Belgium. For the two other wheat midges, only one species of the family Platygastridae was found for each midge: Platygaster equestris (Spittler) for H. marginata and Synopeas myles (Walker) for C. tritici.     

The reproductive strategy of the gregarious parasitoid,Pteromalus puparum (Hymenoptera: Pteromalidae)

Summary Pteromalus puparum is a gregarious parasitoid of lepidopterous pupae. To determine in which phase of a host outbreak superparasitism occurs, field investigations were carried out on seasonal prevalence of the host, Papilio xuhtus, and parasitism by P. puparum in a citrus grove in Fukuoka, Japan in 1974. Host pupation occurred from May to November and the parasitoid attacked the host throughout this period. Pupal density increased rapidly after mid-August and the percentage parasitism decreased during this period. A high level of parasitism was attained after one or more parasitoid generations in mid-September. The superparasitism was observed after mid-September when the parasitoid attained extremely high density, and resulted in an increase in the proportion of males, high mortality, and a decrease in the size of the progeny.     

Response to host age of the egg parasitoid Anaphes n.sp. (Hymenoptera: Mymaridae)

The mymarid Anaphes n.sp. was observed to parasitize the eggs of the carrot weevil Listronotus oregonensis (LeConte) up to 138 h old at 23±1°C and 18L:6D. Adult emergence of the parasitoid was observed in 85%‐97.5% of the weevil eggs 42–138 h old and host embryogenesis was interrupted in nearly 100% of these eggs. In host eggs of 18 h old, parasitism was very high but not all parasitoids completed their development and their subsequent emergence was generally retarded. An.sp. could parasitize host eggs 162 h old, but such an attack rarely retarded the normal development of the host. The parasitoid was unable to complete its development in sterile carrot weevil eggs.     

The natural enemy complex of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in different phases of its population dynamics in eastern Austria and Slovakia – a comparative study

Parasitism and pathogen mortality of Lymantria dispar were compared between host populations of different densities in Slovakia and Austria. Over a period of 4 years, L. dispar eggs, larvae, and pupae were collected in a stage‐specific manner at three mixed oak stands in each country and reared in the laboratory to assess parasitoid and pathogen mortality. At sites with low host densities, L. dispar abundance was artificially augmented by exposing egg masses and young larvae. We ascertained marked differences between the natural enemy complexes of L. dispar populations in Slovakia and Austria. Overall, pathogens caused highest mortality in the former and parasitoids in the latter. Moreover, the species composition differed significantly between both countries. High variation was also observed between years and host populations at different densities. The investigations revealed that egg parasitization was low in Slovak L. dispar populations, and no egg parasitoids were found in Austria. Larval and pupal parasitism was low at sites with outbreak populations, but higher at the Austrian than at the Slovak site. The tachinid Parasetigena silvestris was the dominant species at elevated host densities. Nuclear polyhedrosis virus also caused significant mortality in these cases. An increase in parasitism by several species was noticed after the breakdown of L. dispar populations. The ichneumonid Phobocampe spp. caused high levels of parasitism during the post‐culmination years. At sites where host abundance increased in the first year of the study, significant differences between the Austrian and the Slovak locality could be ascertained. Mortality was low in the population at the latter site, and host abundance increased to outbreak levels the year after. The population at the Austrian site suffered high parasitism by the tachinids P. silvestris and Blepharipa pratensis and this might have contributed to the prevention of a further increase in L. dispar abundance in this locality. Braconids showed a strong reaction to the local, artificial increase in host density at study plots with innocuous L. dispar populations. Particularly high parasitism by Glyptapanteles liparidis was observed at the Austrian site, but G. porthetriae and Cotesia melanoscela also accounted for significant mortality in both low‐density populations. The artificial L. dispar populations were usually eradicated by natural enemies before pupation of the hosts.     

An Endoparasitoid Avoids Hyperparasitism by Manipulating Immobile Host Herbivore to Modify Host Plant Morphology

Many parasitic organisms have an ability to manipulate their hosts to increase their own fitness. In parasitoids, behavioral changes of mobile hosts to avoid or protect against predation and hyperparasitism have been intensively studied, but host manipulation by parasitoids associated with endophytic or immobile hosts has seldom been investigated. We examined the interactions between a gall inducer Masakimyia pustulae (Diptera: Cecidomyiidae) and its parasitoids. This gall midge induces dimorphic leaf galls, thick and thin types, on Euonymus japonicus (Celastraceae). Platygaster sp. was the most common primary parasitoid of M. pustulae. In galls attacked by Platygaster sp., whole gall thickness as well as thicknesses of upper and lower gall wall was significantly larger than unparasitized galls, regardless of the gall types, in many localities. In addition, localities and tree individuals significantly affected the thickness of gall. Galls attacked by Platygaster sp. were seldom hyperparasitized in the two gall types. These results strongly suggest that Platygaster sp. manipulates the host plant''s development to avoid hyperparasitism by thickening galls.     

Oxycephalomyia, gen. nov., and life history strategy of O. styraci comb. nov. (Diptera: Cecidomyiidae) on Styrax japonicus (Styracaceae)

A new genus Oxycephalomyia is described to contain the gall midge that was previously known as Asteralobia styraci (Shinji). Oxycephalomyia styraci, comb. nov., produces leaf vein galls on Styrax japonicus (Styracaceae). The adult of O. styraci is redescribed, and its full‐grown larva and pupa are described for the first time. The annual life cycle of the gall midge in northern Kyushu was clarified; the first instars overwinter in the galls on the host plant. However, the galls of O. styraci mature much later in the season than those of other gall midges with a similar life history pattern, and the durations of second and third larval instars are remarkably short. Such a life history pattern is considered to have an adaptive significance in avoiding larval parasitism, particularly by early attackers. The number of host axillary buds as oviposition sites decreased in bearing years and increased in off years, but there was no sign of oviposition site shortage even in bearing years, probably due to the low population density of the gall midge. An unidentified lepidopteran that feeds on galled and ungalled host buds and a Torymus sp. that attacks pupae of O. styraci were recognized as mortality factors of the gall midge.     

Reciprocal influences and costs of parasitism on the development of Corcyra cephalonica and its endoparasitoid Venturia canescens

Many endoparasitoids develop successfully within a range of host instars. Parasitoid survival is highest when parasitism is initiated in earlier host instars, due to age-related changes in internal (physiological) host defences. Most studies examining fitness-related costs associated with differences in host instar have concentrated on the parasitoid, ignoring the effects of parasitism on the development of surviving hosts that have encapsulated parasitoid eggs. A laboratory experiment was undertaken examining fitness-related costs associated with encapsulation of Venturia canescens (Hymenoptera: Ichneumonidae) eggs by fifth (L5) instar larvae of Corcyra cephalonica (Lepidoptera: Pyralidae). Growth and development of both host and parasitoid were monitored in C. cephalonica larvae containing 0, 1, 2, or 4 parasitoid eggs. Adult size and fecundity of C. cephalonica did not vary with the number of eggs per host. However, there was a distinct increase in host mortality with egg number, although most parasitoids emerged from hosts containing a single egg. The most dramatic effect on the host was a highly significant increase in development time from parasitism to adult eclosion, with hosts containing 4 parasitoid eggs taking over 2.5 days longer to complete development than unparasitized larvae. The egg-to-adult development time and size of adult V. canescens did not vary with egg number per host, as demonstrated in a previous experiment using a different host (Plodia interpunctella). The results described here show that there are fitness-related costs to the host associated with resistance to parasitism.     

Do distances among host patches and host density affect the distribution of a specialist parasitoid?

The effect of spatial habitat structure and patchiness may differ among species within a multi-trophic system. Theoretical models predict that species at higher trophic levels are more negatively affected by fragmentation than are their hosts or preys. The absence or presence of the higher trophic level, in turn, can affect the population dynamics of lower levels and even the stability of the trophic system as a whole. The present study examines different effects of spatial habitat structure with two field experiments, using as model system the parasitoid Cotesia popularis which is a specialist larval parasitoid of the herbivore Tyria jacobaeae. One experiment examines the colonisation rate of the parasitoid and the percentage parasitism at distances occurring on a natural scale; the other experiment examines the dispersal rate and the percentage parasitism in relation to the density of the herbivore and its host plant. C. popularis was able to reach artificial host populations at distances up to the largest distance created (at least 80 m from the nearest source population). Also, the percentage parasitism did not differ among the distances. The density experiment showed that the total number of herbivores parasitised was higher in patches with a high density of hosts, regardless of the density of the host plant. The percentage parasitism, however, was not related to the density of the host. The density of the host plant did have a (marginally) significant effect on the percentage parasitism, probably indicating that the parasitoid uses the host plant of the herbivore as a cue to find the herbivore itself. In conclusion, the parasitoid was not affected by the spatial habitat structure on spatial scales that are typical of local patches.     

Density-dependent egg mortality in early stages of gall induction by the fruit gall midge <Emphasis Type="Italic">Asphondylia aucubae</Emphasis> Yukawa and Ohsaki

Density-dependent mortality has been considered a symptom of intraspecific competition. We examined the occurrence of such mortality in the early stages of gall induction by the gall midge Asphondylia aucubae Yukawa et Ohsaki (Diptera: Cecidomyiidae). Female midges deposit eggs into young fruit of the dioecious shrub Aucuba japonica Thunberg to induce gall formation. Each host fruit received 0–67 eggs (mean 18.5 eggs) from multiple females, whereas established galls each contained one to ten larvae. Midges suffered intense mortality (65–90%) at the egg stage. Egg mortality occurred even in fruit in which no larvae had hatched, suggesting that this mortality cannot be wholly attributed to larval interference. Egg mortality was affected by fruit size, i.e., resource capacity. Midges distributed more eggs in larger fruit. Egg mortality increased as the per-fruit density increased relative to fruit size. In contrast, the mortality of hatchlings was density-independent. Our results suggest that A. aucubae intensely compete for gall-induction substrates, which are spatiotemporally rare resources.     

Vine mealybug sex pheromone increases citrus mealybug parasitism by Anagyrus sp. near pseudococci (Girault)

The present study was aimed at elucidating the role of lavandulyl senecioate (LS), the sex pheromone of Planococcus ficus, in host selection of the parasitoid Anagyrus sp. near pseudococci. Field trials were carried out in Portugal, Italy and Israel. The effect of LS on the parasitism rate of the wasp was determined by exposing sentinel mealybugs combined with pheromone dispensers impregnated with LS, in comparison with other baits: lavandulyl isovalerate (LI); planococcyl acetate (PAc); and unbaited control traps. In addition, in order to study the host location behavior of A. sp. near pseudococci, pheromone dispensers were placed at three different distances: inside the trap, 30 or 60 cm away from the trap. The number of parasitoid females inside the traps, the number of parasitized mealybugs, and the number of days required for the first parasitoid emergence were recorded. The response of A. sp. near pseudococci females to different doses of LS (25–1350 μg) was also evaluated using sticky plate traps. The rate of mealybug parasitism by A. sp. near pseudococci was significantly increased by LS in the three parasitoid populations. PAc and LI had no significant effect on the wasp parasitism rate in most of the trials. However, the Italian population of the parasitoid responded to PAc, showing apparently a different behavioral pattern. The number of parasitoid females trapped did not significantly differ between tested doses of LS. The use of LS as an arrestant in host location by the A. sp. near pseudococci female is discussed.     

Interactions between the Parasitoid Rhopus nigroclavatus (Ashmead) (Hymenoptera: Encyrtidae) and its Mealybug Hosts Tridiscus sporoboli (Cockerell) and Trionymus sp. (Homoptera: Pseudococcidae)

This research investigated age-class-specific parasitism rates of the buffalograss mealybugs Tridiscus sporoboli (Cockerell) and Trionymus sp. by Rhopus nigroclavatus (Ashmead) (Hymenoptera; Encyrtidae), size class preference of this parasitoid, and mealybug–parasitoid interactions through choice and no-choice studies. In the no-choice studies, the mean rates of parasitism by R. nigroclavatus were 45, 20, 0, and 0%, respectively, for mealybugs adult female, third and fourth instars, first and second instars, and eggs. Choice studies indicated that rate of parasitism increased with host size. The mean rates of parasitism on mealybugs in the choice studies were 100% for adult females, 24% for third and fourth instars, 0% for first and second instars, and 0% for eggs. A second set of choice studies investigating mealybug/parasitoid behavior revealed that R. nigroclavatus oviposits in all post-egg mealybug age classes, but first and second instars were less often parasitized than older mealybugs.     

Seasonal variation in abundance and diversity of eavesdropping frog‐biting midges (Diptera,Corethrellidae) in a neotropical rainforest

1. In the tropics, precipitation patterns result in seasonal fluctuations in the abundance and distribution of plant and animal species. Tropical predators and parasites are therefore faced with seasonal changes in prey and host availability. 2. This study investigates the seasonal interaction among a specialised ectoparasite, eavesdropping frog‐biting midges (Corethrella spp.), and their anuran hosts, examining how the abundance and diversity of the frog‐biting midge community fluctuate between the rainy (host abundant) and dry (host sparse) seasons. 3. Midges were captured in both the rainy and dry seasons using acoustic playbacks of calls from a common frog species that breeds during the rainy season, the túngara frog (Engystomops, Physalaemus, pustulosus). During the dry season túngara frog choruses are absent. To explore seasonal shifts in host preference or changes in the midge community due to host specificity, midges were also captured using playbacks of calls from a frog that breeds during the dry season, the pug‐nosed tree frog (Smilisca sila). 4. While the overall abundance of midges decreased in the dry season, only slight differences in the relative abundance between midge species were found. These results suggest that midge populations can shift between hosts as they become available across seasons, allowing adult populations of frog‐biting midges to persist year‐round. To overcome the challenge of detecting and localising different host species, it is proposed that frog‐biting midges have evolved a generalised acoustic template, allowing them to respond to a broad range of available hosts, regardless of seasonal host composition.     

Demographic dynamics of Platygaster demades in response to host density

Platygaster demades Walker (Hymenoptera: Platygastridae) is the only hymenopteran parasitoid of apple leaf-curling midge, Dasineura mali Kieffer (Diptera: Cecidomyiidae), in New Zealand. Prior to the present study the mechanisms behind the parasitoid–host density interactions were poorly understood. In this study we carried out both laboratory and field experiments to determine the response of P. demades to D. mali density. In the laboratory, when only a single parasitoid was allowed to forage D. mali eggs of a given density, P. demades displayed a Type II functional response leading to an inverse density-dependent parasitism. However, P. demades showed a Type III functional response in the field where females were able to freely search and disperse between apple shoots infested with D. mali eggs of different densities. As a result, the Type III response reflects a more realistic nature of P. demades in response to D. mali density. Our results also indicate that the numeric response and mutual interference in P. demades significantly contributed to the stability of the parasitoid–host system. The density-dependent parasitism in a host range of 50–300 D. mali eggs per apple shoot suggests that P. demades is highly efficient in controlling D. mali populations of the first, third and fourth generations and when necessary, argumentation measures may be taken before the onset of the second generation.     

Effects of storage at low-temperature of various heteropteran host eggs on the egg parasitoid, <Emphasis Type="Italic">Trissolcus semistriatus</Emphasis>

Various heteropteran host eggs stored under two low-temperatures were tested in the laboratory for their usability in the production of sunn pest egg parasitoid, Trissolcus semistriatus. Parasitism, adult emergence rate and development time were assesed on stored and fresh eggs of Eurygaster integriceps, Dolycoris baccarum, Graphosoma lineatum and Eurydema ornatum. Masses of fresh host eggs in microcentrifuge tubes were maintained at +6 °C and −20 °C. Every 30 days, 50 eggs of host species were exposed to parasitism by T. semistriatus. The host eggs stored at 6 °C remained viable to parasitism by T. semistriatus up to 2 months, while those of stored at −20 °C were parasitized at high rates up to 4 months, alhough succesful parasitism rates decrease with time. However, it was indicated that both fresh or stored E. ornatum eggs were not preferred to parasitism by the parasitoid. A longer development time from egg to adult was observed in stored eggs under two tested storage techniques when compared with fresh eggs.     

Assessing augmentative releases of parasitoids using the «Recruitment method», with reference toEdovum puttleri,a parasitoid of the colorado potato beetle [Coleoptera: chrysomelidae]

Release of approximately 17,700 experienced adult femaleEdovum puttleri Grissell against 1^st generation Colorado potato beetle eggs in 1987 in a 0.4 ha potato field in S. Deerfield, Massachusetts resulted in only 3.6% parasitism as assessed by direct measurement of host and parasitoid recruitment. Levels of non-viability indicated an additional 2.8% of hosts killed by parasitoid hostfeeding, for an overall impact of 6.4%. Release in 1988 of 126,300 parasitoid against 1^st generation hosts in a 0.4 ha potato (Solanum tuberosum L.) field at the same site produced only slightly higher levels of parasitism (10.6%) and host feeding (2.0%). Release in 1987 of 32,800 wasps against 2^nd generation eggs resulted in only 0.7% parasitism and 0.3% host feeding due to the toxicity of fenvalerate (Pydrin^R) residues from a single application applied for control of the potato leafhopper,Empoasca fabae (Harris). Release in 1988 of 47,400 wasps against the 2^nd host generation in the absence of any pesticide applications resulted in 34.4% parasitism and 16.1% host feeding, for a total impact of 50.5%. Difference in parasitization levels between host generations supports the idea thatE. puttleri adults require an in-field carbohydrate source such as aphid honeydew to reproduce. In Massachusetts, aphid populations in potato typically do not develop until the end of the 1^st larval generation. The recruitment method ofVan Driesche & Bellows (1988) proved to be a satisfactory approach for determining results of augmentative parasitoid releases.      

Evolutionary radiation of Asteromyia carbonifera (Diptera: Cecidomyiidae) gall morphotypes on the goldenrod Solidago altissima (Asteraceae)

Population divergence of phytophagous insects is often coupled to host‐plant shifts and is frequently attributed to the divergent selective environments associated with alternative host‐plants. In some cases, however, divergence is associated with the use of alternative host‐plant organs of a single host species. The basis of within‐host radiations such as these remains poorly understood. In the present stusy, we analysed the radiation of Asteromyia gall midges occurring both within one host plant species and within a single organ on that host. In this system, four morphologically distinct Asteromyia gall forms (morphs) coexist on the leaves of goldenrod Solidago altissima. Our analyses of amplified fragment length polymorphism and DNA sequence data confirm the genetic differentiation among midges from three gall morphs and reveal evidence of a genetically distinct fourth gall morph. The absence of clear gall morph related clades in the mitochondrial DNA derived phylogenies is indicative of incomplete lineage sorting or recent gene flow, suggesting that population divergence among gall forms is recent. We assess the likely history of this radiation and use the results of phylogenetic analyses along with ecological data on phenology and parasitism rates to evaluate potential hypotheses for the mode of differentiation. These preliminary analyses suggest that diversification of the Asteromyia gall morphs is likely shaped by interactions between the midge, a symbiotic fungus, and parasitoid enemies. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 840–858.     

Evaluation of cold-stored eggs of Dolycoris baccarum (Hemiptera: Pentatomidae) for parasitization by Trissolcus nigripedius (Hymenoptera: Scelionidae)

Trissolcus nigripedius Nakagawa (Hymenoptera: Scelionidae) is an egg parasitoid of Dolycoris baccarum L. (Hemiptera: Pentatomidae), a polyphagous insect pest of many crops including soybean. As a method for mass-rearing to augment the parasitoid, cold storage of host eggs were evaluated in the laboratory. After 0 (control), 8, 20, 60, 90, and 120 days of refrigeration, host eggs were given to adult female T. nigripedius. Host acceptance behaviors of the parasitoid, categorized as drumming, oviposition, and marking, on the refrigerated eggs and biological attributes of offspring were assessed. Most of the attributes examined were affected by the refrigeration of host eggs. But host eggs could be refrigerated for up to two months without significant change in emergence rate and sex ratio and with 90% of parasitism. In addition, the second generation of the parasitoid was not negatively affected at all. However, frozen eggs of D. baccarum can not be used for rearing the parasitoid since parasitism rate decreased to 44% on host eggs frozen for 8 days. Refrigeration of D. baccarum eggs could be useful for mass-rearing and augmentation of T. nigripedius to control D. baccarum without reduction in the quality of parasitoid’s progeny. Furthermore, refrigerated eggs could be supplemented in the field to boost the population of T. nigripedius since immature D. baccarum can not hatch after 20 days of refrigeration.