Go local: Enhancing sustainable production of Tenebrio molitor through valorization of locally available agricultural byproducts.

Insects receive increasing attention as an alternative source of protein for animals and humans, and thus, the production of low-cost insects for meeting the dietary demand on sustained basis is an ever-growing concept. This study aims to design dietswith locally available agricultural byproducts from Greece as feed for larvae of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). This will considerably reduce the cost of insect feed and the environmental impact of insect farming by using locally available agricultural byproducts as economic insect feedstock. More specifically, five agricultural byproducts derived from the production of cereals and legumes were utilized to design twelve different diets at two protein levels, i.e., 17.4 and 22.5% protein content. All diets were evaluated both at laboratory scale, but also at pilot scale. Based on the obtained results, both bioassays revealed that the diets contained one legume and one cereal byproduct (i.e., lupin and triticale as well as lupin and oat) supported more efficiently the growth and performance of the larvae, irrespective of the protein level. Indicatively, individual larval weight of the best performed larvae from both groups ranged from 132 to 142 mg. Moreover, our results highlight the fact that data derived from laboratory scale bioassays are not always easy to be extrapolated to industrial production. For instance, the total harvest of larvae, a parameter assessed in the tray scale bioassay, exhibited a disparity between diet A2 (910 g) and diet A3 (749 g), despite both being deemed optimal in the laboratory-scale experiment. Our study aims to promote a circular approach for the industrial rearing of insects through integration of local agricultural byproducts into specific diets for T. molitor larvae.

Tribolium castaneum: A Model Insect for Fundamental and Applied Research.

Tribolium castaneum has a long history as a model species in many distinct subject areas, but improved connections among the genetics, genomics, behavioral, ecological, and pest management fields are needed to fully realize this species' potential as a model. Tribolium castaneum was the first beetle whose genome was sequenced, and a new genome assembly and enhanced annotation, combined with readily available genomic research tools, have facilitated its increased use in a wide range of functional genomics research. Research into T. castaneum's sensory systems, response to pheromones and kairomones, and patterns of movement and landscape utilization has improved our understanding of behavioral and ecological processes. Tribolium castaneum has also been a model in the development of pest monitoring and management tactics, including evaluation of insecticide resistance mechanisms. Application of functional genomics approaches to behavioral, ecological, and pest management research is in its infancy but offers a powerful tool that can link mechanism with function and facilitate exploitation of these relationships to better manage this important food pest.

Induced dormancy in Indian meal moth Plodia interpunctella (Hübner) and its impact on the quality improvement for mass rearing in parasitoid Habrobracon hebetor (Say).

A steady supply of hosts at the susceptible stage for parasitism is a major component of mass rearing parasitoids for biological control programs. Here we describe the effects of storing 5th instar Plodia interpunctella larvae in dormancy on subsequent host development in the context of host colony maintenance and effects of the duration of host dormancy on the development of Habrobracon hebetor parasitoids reared from dormant hosts. We induced dormancy with a combination of short daylength (12L:12D) and lower temperature (15°C), conditions known to induce diapause in this species, and held 5th instar larvae of P. interpunctella for a series of dormancy durations ranging from 15 to 105 days. Extended storage of dormant 5th instar larvae had no significant impacts on survival, development, or reproductive potential of P. interpunctella, reinforcing that dormant hosts have a substantial shelf life. This ability to store hosts in dormancy for more than 3 months at a time without strong negative consequences reinforces the promise of using dormancy to maintain host colonies. The proportion of hosts parasitized by H. hebetor did not vary significantly between non-dormant host larvae and dormant host larvae stored for periods as long as 105 days. Concordant with a prior study, H. hebetor adult progeny production from dormant host larvae was higher than the number of progeny produced on non-dormant host larvae. There were no differences in size, sex ratio, or reproductive output of parasitoids reared on dormant hosts compared to non-dormant hosts stored for up to 105 days. Larval development times of H. hebetor were however longer when reared on dormant hosts compared to non-dormant hosts. Our results agree with other studies showing using dormant hosts can improve parasitoid mass rearing, and we show benefits for parasitoid rearing even after 3 months of host dormancy.

Utilizing low oxygen to mitigate resistance of stored product insects to phosphine.

BACKGROUND: Data are provided on the utilization of modified atmospheres, at a commercial scale, against stored product insect populations that are resistant to phosphine. The method is evaluated on different populations of two major stored-product beetle species, Rhyzopertha dominica and Oryzaephilus surinamensis. The trials were carried out in commercial facilities, in which nitrogen was introduced through an embedded nitrogen generator. Each chamber contained three or four pallets of either currants or herbs. A computational model was developed to evaluate the nitrogen concentration. RESULTS: In most trials, 100% mortality was recorded for both beetle species and all populations, regardless of the temperature and exposure intervals tested. Control progeny production ranged between 20 and 45 adults per vial for R. dominica, and 29 and 27 adults per vial for O. surinamensis. Simulation results reveal that nitrogen can easily penetrate the currants, and its concentration is uniform (differences are below 1.5%) across the pallet. Additionally, the simulation model revealed that lower temperatures do not have an impact on the nitrogen concentration profiles. CONCLUSIONS: The modified atmosphere applications evaluated here were proved to be effective for all populations, regardless of the level of resistance to phosphine, and any survival could be attributed to the short exposure intervals. Modified atmosphere applications can be effective at a considerably short exposure interval, even at 2.5 days, which is an incontestable advantage for the use of this method against insects, at exposures comparable with those of commercial fumigations. © 2022 Society of Chemical Industry.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

'Insects as Food and Feed: If You Can't Beat Them, Eat Them!'-To the Magnificent Seven and Beyond.

The objective of this special issue is to highlight the current state of research in the field of insects as food and feed, but also other aspects on the exploitation of insect farming. In this editorial, we make a short introduction of the topic of the special issue, briefly present the contributions that are collected in it and offer some thoughts on the future research priorities and challenges that should be addressed. Regarding insect farming, there are additional applications, such as fertilizer, health-promoting products, and cosmetics, that can be produced and utilized, that go far beyond food and feed production.

Diatomaceous Earth for Arthropod Pest Control: Back to the Future.

Nowadays, we are tackling various issues related to the overuse of synthetic insecticides. Growing concerns about biodiversity, animal and human welfare, and food security are pushing agriculture toward a more sustainable approach, and research is moving in this direction, looking for environmentally friendly alternatives to be adopted in Integrated Pest Management (IPM) protocols. In this regard, inert dusts, especially diatomaceous earths (DEs), hold a significant promise to prevent and control a wide range of arthropod pests. DEs are a type of naturally occurring soft siliceous sedimentary rock, consisting of the fossilized exoskeleton of unicellular algae, which are called diatoms. Mainly adopted for the control of stored product pests, DEs have found also their use against some household insects living in a dry environment, such as bed bugs, or insects of agricultural interest. In this article, we reported a comprehensive review of the use of DEs against different arthropod pest taxa, such as Acarina, Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Ixodida, Lepidoptera, when applied either alone or in combination with other techniques. The mechanisms of action of DEs, their real-world applications, and challenges related to their adoption in IPM programs are critically reported.

Beetle-robot hybrid interaction: sex, lateralization and mating experience modulate behavioural responses to robotic cues in the larger grain borer Prostephanus truncatus (Horn).

Ethorobotics, a new fascinating field of biorobotics, proposes the use of robotic replicas as an advanced method for investigating animal behaviour. This novel research approach can also encourage the development of advanced bioinspired robots. In the present study, we investigated the pushing behaviour, a particular display occurring in several beetle species, such as the larger grain borer, Prostephanus truncatus, during both male-female and male-male contexts. We developed a robotic apparatus actuating female and male-mimicking dummies to study if sex, mating experience and asymmetries of robotic cues can modulate the escalation of pushing behaviour. Results showed that the time needed by P. truncatus to react to female-smelling biomimetic dummies was chiefly affected by their mating experience and the dummy odour. This was likely due to reduce waste of costly sperm in mated males during the subsequent sexual interactions. The pushing behaviour was performed longer and with a higher number of acts when virgin females were approached from their right side. More and longer pushing acts were noted when virgin males were approached from their left side. Dedicated neural circuits would likely act in opposite direction in females and males producing population-level lateralized sensory-motor displays, which may be evolved to promote male approaches from the left side of females, thus improving short-distance sex recognition. Overall, this study provides new insights on the behavioural ecology of stored-product beetles, as well as on self-organization and decentralized decision making that can be exploited to develop bioinspired algorithms for task optimization, involving real-world scenarios.

Biology and Control of the Khapra Beetle, Trogoderma granarium, a Major Quarantine Threat to Global Food Security.

The khapra beetle, Trogoderma granarium, is a voracious feeder of stored products and is considered one of the most important quarantine pests globally. Its ability to survive for long periods under extreme conditions facilitates its spread through international commerce, which has led to invasions of new geographic regions. The khapra beetle is an important quarantine pest for many countries, including the major wheat-producing countries the United States, Canada, Russia, and Australia, and has been classified as one of the 100 worst invasive species worldwide. This species cannot always be controlled by insecticides and other nonchemical methods that are usually effective against other pests of stored products, particularly owing to its diapausing late larval stage. It can rapidly develop at elevated temperatures and under dry conditions, which are not favorable for many major stored-product insects. We synthesize key published work to draw attention to advances in biology, detection and control of the khapra beetle, and directions to consider for future research.

Health Hazards Associated with Arthropod Infestation of Stored Products.

Insects and mites are common inhabitants and accidental invaders of food, including durable commodities, and their presence can have both direct and indirect effects on human health. The most common direct effect is contamination of food with arthropod fragments and related contaminants, which may be allergenic or even carcinogenic. The most important indirect effect is that their presence can change the storage microenvironment, making durable products suitable for the rapid development of fungi and other microorganisms. Some of these fungi can produce toxins (e.g., aflatoxins) that endanger human health. Insects may actively or passively contribute to the spread of microorganisms, increasing product contamination, and they may host bacteria that have developed antibiotic resistance, contributing to their spread in food. Several species also may host, attract, or transmit tapeworms, predators, or parasitoids that may affect health. This review synthesizes research on these topics and suggests directions for future research.

Influence of Temperature and Relative Humidity on the Insecticidal Efficacy of Metarhizium anisopliae against Larvae of Ephestia kuehniella (Lepidoptera: Pyralidae) on Wheat.

A series of laboratory bioassays were conducted for the evaluation of the insecticidal efficacy of an isolate of Metarhizium anisopliae (Metschnikoff) Sorokin (Ascomycota: Hypocreales) against larvae of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), under various temperature-relative humidity (r.h.) conditions. The fungus was applied at four doses (0, 8 × 106, 8 × 108, and 8 × 1010 conidia ml-1) on wheat and insect mortality was assessed after exposure of 1, 2, 7, and 14 d. Bioassays were conducted at three temperatures (20, 25, and 30 °C) and two r.h. levels (55 and 75%). Although complete control was not achieved in any case, the fungus provided a considerable level of insect control. Mortality of E. kuehniella larvae on wheat treated with M. anisopliae ranged between 41.1 and 93.3% after 14 d of exposure, whereas the respective mortality levels in control dishes never exceeded 28.3%. The increase of temperature resulted in most cases to higher efficacy, indicating that temperature is an important factor for the performance of the fungus. In contrast, in most cases r.h. did not significantly affect the efficacy of the fungus, at least for the humidity levels tested.

Applications of natural zeolites on agriculture and food production.

Zeolites are crystalline hydrated aluminosilicates with remarkable physical and chemical properties, which include losing and receiving water in a reverse way, adsorbing molecules that act as molecular sieves, and replacing their constituent cations without structural change. The commercial production of natural zeolites has accelerated during the last 50 years. The Structure Commission of the International Zeolite Association recorded more than 200 zeolites, which currently include more than 40 naturally occurring zeolites. Recent findings have supported their role in stored-pest management as inert dust applications, pesticide and fertilizer carriers, soil amendments, animal feed additives, mycotoxin binders and food packaging materials. There are many advantages of inert dust application, including low cost, non-neurotoxic action, low mammalian toxicity and safety for human consumption. The latest consumer trends and government protocols have shifted toward organic origin materials to replace synthetic chemical products. In the present review, we summarize most of the main uses of zeolites in food and agruculture, along with the with specific paradigms that illustrate their important role. © 2017 Society of Chemical Industry.

Capture of Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae) in Floor Traps: The Effect of Previous Captures.

The impact of prior captures on the trapping performance of floor traps was evaluated for the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), and the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), in laboratory conditions. The effect of trap seeding, adding adults of the same or different species, was evaluated in order to determine possible effects of prior captures in the trap on each species' behavioral responses. The presence of seeded beetles of the same species resulted in an increase in beetle captures for both T. castaneum and T. confusum, but when traps were seeded with the opposite species, there was no increase in beetle captures for either species, and for T. castaneum overall captures in both seeded and unseeded traps was reduced. Overall, T. castaneum tended to have greater captures than T. confusum regardless of the treatment. When the two species were released together, this negated the increased response to seeded traps observed in the single-species treatments. These findings suggest the potential that the presence of beetles in a trap may be influencing the response of beetles in a nearby trap and that T. castaneum and T. confusum when they occur together may influence each other's response to traps.

Long-Term Residual Efficacy of Spinetoram on Concrete and Steel Surfaces for the Management of Three Stored Product Beetle Species.

In this study, the residual efficacy of spinetoram on concrete and galvanized steel surfaces was evaluated under fixed laboratory conditions against the rice weevil, Sitophilus oryzae (L.), the confused flour beetle, Tribolium confusum Jacquelin du Val, and the sawtoothed grain beetle, Oryzaephilus surinamensis (L.). Spinetoram was applied at the dose rates of 0.025 and 0.1 mg (active ingredient)/cm(2), on steel surfaces that were stored in continuous darkness and on concrete surfaces that were stored either in continuous darkness or in 12:12 (L:D) photoperiod. The experimental period for the residual effect of spinetoram was 6 mo. Bioassays were conducted for all types of surfaces and storage conditions at monthly intervals starting from the initial application period (seven bioassays in total). For each bioassay, mortality of the exposed adult beetles was measured after 3 and 7 d of exposure. Among the tested species, T. confusum was the least susceptible, regardless of the surface type, storage conditions, and dose rate. Regarding the bioassays conducted in the surfaces stored in darkness, spinetoram proved very persistent and no reduction in the efficacy was noted throughout the experimental period. Moreover, there were no differences in spinetoram efficacy between the two types of surfaces. Conversely, in light [12:12 (L:D)] conditions spinetoram efficacy was notably reduced after the first month, but remained stable for the rest of the period. The results of this study indicate that spinetoram was persistent with long residual efficacy against major stored grain beetle species on the most common types of surfaces in continuous darkness, while the presence of light reduced its efficacy.

Efficacy of Deltamethrin Against Stored-Product Beetles at Short Exposure Intervals or on a Partially Treated Rice Mass.

Stored-product insects can potentially be exposed to grain protectants for variable time periods. Adults of three species, the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), the granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae), and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) were exposed for 1, 4, 8, and 24 h on brown rice treated with the pyrethroid deltamethrin at the label rate of 0.5 ppm, then removed and placed on untreated rice. Adults of these same species plus the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), and the warehouse beetle, Trogoderma variabile Ballion (Coleoptera: Dermestidae) were exposed on treated brown rice mixed with varying amounts of untreated rice to assess progeny production. Immediate and delayed mortality of exposed adults did not exceed 7% for any exposure interval, but progeny production for T. castaneum was generally lower in comparison with that for the other species. Increasing the amount of treated rice decreased progeny production of R. dominica but not for any Sitophilus species. Mixed results were obtained for T. castaneum and T. variabile. Results show that long exposure times and treatment of an entire rice mass may be necessary to give complete control of stored-product beetles.

Efficacy of Methyl Bromide for Control of Different Life Stages of Stored-Product Psocids.

The psocid species Liposcelis paeta Pearman, Liposcelis entomophila (Enderlein), Liposcelis decolor (Pearman), Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae), and Lepinotus reticulatus Enderlein (Psocoptera: Trogiidae) were evaluated in laboratory bioassays to determine their susceptibility to six concentrations of methyl bromide (0.027, 0.113, 0.280, 0.393, 0.452, and 0.616 g/m3) after 48 h of exposure at 27.5°C. The life stages that were evaluated were adults (for all species), nymphs (for all species except Lep. reticulatus), and eggs (for L. entomophila, L. decolor, and L. bostrychophila). Adults and nymphs were very susceptible, and complete mortality was recorded at concentrations between 0.027 and 0.280 g/m3. In contrast, eggs were by far more tolerant than adults and nymphs for all species tested. At 0.027 g/m3, mortality did not exceed 53%, while survival was high even at 0.113 g/m3. Complete (100%) egg mortality was recorded at 0.393 g/m3 for L. decolor and at 0.452 g/m3 for L. entomophila and L. bostrychophila; concentrations estimated to give 99% mortality for eggs of these three species were 0.710, 1.044, and 0.891 g/m3, respectively. These results show that stored-product psocids are susceptible to methyl bromide, but concentrations of ≥0.452 g/m3 should be used to control all life stages.

Knockdown and Mortality of Five Stored Product Beetle Species After Short Exposures of Thiamethoxam.

Laboratory bioassays were conducted to evaluate the effectiveness of thiamethoxam, against five major stored-grain beetle species, the lesser grain borer, Rhyzopertha dominica (F.), the rice weevil, Sitophilus oryzae (L.), the confused flour beetle, Tribolium confusum Jacquelin du Val, the larger grain borer, Prostephanus truncatus (Horn), and the sawtoothed grain beetle, Oryzaephilus surinamensis (L.). Adults of the above species were exposed on wheat (or maize in the case of P. truncatus) treated with thiamethoxam at 0.1, 1, and 10 ppm for 0, 2, 4, 6, 8, 16, 40, 72, and 96 h. After each of these intervals, mortality was recorded (immediate mortality) and the surviving individuals were transferred in untreated wheat (or maize), where mortality was recorded again 7 d later (delayed mortality). During both immediate and delayed mortality counts, the number of adults that were knocked down was also recorded. Immediate mortality was low in all exposures, with the exception of the highest dose rate and after 72-96 h. At these conditions, during this interval, most of the surviving individuals were knocked down. Delayed mortality was further increased with the increase of dose and the initial exposure, but knockdown was extremely low, with the exception of P. truncatus. The results of the present work show that O. surinamensis was the least susceptible species, while P. truncatus was the most susceptible. These findings show that, despite the increased mortality, recovery after short exposures is likely for all species tested here. In this regard, partially treated areas on which the insects are exposed only for short intervals may reduce thiamethoxam efficacy.

Residual efficacy of chlorfenapyr for control of stored-product psocids (Psocoptera).

The residual effect of chlorfenapyr (Phantom) was evaluated for residual control of three stored-product psocid species: Liposcelis bostrychophila Badonnel, Liposcelis entomophila (Enderlein), and Liposcelis paeta Pearman (Psocoptera: Liposcelididae). Chlorfenapyr was applied to individual arenas with a concrete surface at rates of 0, 2.8, 13.8, 20.6, 27.5, 55, and 110 mg active ingredient (AI)/m2. Adults were exposed on the treated arenas and mortality assessed after 1, 2, and 3 d. The procedures were repeated weekly on the same treated arenas for 3 wk to assess residual efficacy. At each week, mortality of all species was low after 1 d of exposure but notably increased after 2 or 3 d. L. entomophila was the most susceptible species, with 99-100% mortality at rates of 13.8 mg/m2 or higher. Similarly, mortality of L. paeta after 3 d of exposure at the same concentration ranged from 92 to 100%. L. bostrychophila was the least susceptible species, with mortality of < 60% during the third week after application at rates < or = 27.5 mg/m2. However, even for this species, mortality after 3 wk was 90% or higher at rates > 27.5 mg/m2. Complete mortality of all species occurred after 3 d exposure at the highest rate tested of 110 mg/m2. Thus, our results show that chlorfenapyr is effective against major psocid species at the application rates evaluated in this study.

Parasitoids and hyperparasitoids (Hymenoptera) on aphids (Hemiptera) infesting citrus in east Mediterranean region of Turkey.

The aphids, aphid parasitoids, and hyperparasitoids found in citrus orchards, the parasitoids' and hyperparasitoids' seasonal abundance, and the plant-aphid-parasitoid relationships in Hatay, Osmaniye, Adana, and Mersin provinces of the east Mediterranean region of Turkey are presented in the present 2-yr study. Aphidius colemani Viereck, Binodoxys angelicae (Haliday), and Lysiphlebus confusus Tremblay and Eady (Hymenoptera: Braconidae: Aphidiinae) were encountered as the most common parasitoids among 10 identified aphidiine and aphelinid taxa on different citrus species. Hyperparasitoids belonging to the genera Alloxysta, Phaenoglyphis, Asaphes, Pachyneuron, Syrphophagus, and Dendrocerus are reported for the first time emerging from aphids feeding on citrus in Turkey. Among them, Asaphes spp., Pachyneuron spp., and Syrphophagus spp. were recorded as the most common ones. Citrus reticulata Blanco and Citrus limon (L.) Burm. fil. were recorded as main hosts for the aphid parasitoids and their hyperparasitoids.

Effects of low temperatures on quiescence in Trichogramma evanescens Westwood and T. chilonis Ishii reared on Plodia interpunctella (Hübner): implications for mass rearing.

The egg parasitoids of the genus Trichogramma are important potential biological control agents for a wide range of lepidopteran pests. Cold storage of host eggs has been proposed as a valuable technique for ensuring the release of sufficient parasitoid numbers whenever it is needed. In this context, the impact of low temperatures to induce quiescence in T. evanescens Westwood and T. chilonis Ishii was studied using eggs of Indian meal moth Plodia interpunctella (Hübner). Prepupae of the parasitoids were stored for 15, 30, 45, 60 and 75 d at 4 °C, following a 7 d period of acclimation at 10 °C. Both parasitoid species seem to survive unfavorable temperature conditions by entering a state of quiescence. Parasitism, adult emergence, sex ratio and progeny quality were not affected by cold storage in either parasitoid species for up to 30 d of storage. Parasitized host eggs of P. interpunctella can be stored for up to 60 d at 4 °C for both parasitoids, but there was no emergence at 75 d. General productivity values gradually decreased as the duration of storage lengthened for both species. Our results clearly reveal that the eggs parasitized by these species can be stored for up to 30 d at 4 °C in a state of quiescence without much loss of their performance compared to the control eggs.