The Life Cycle of Aurelia aurita Depends on the Presence of a Microbiome in Polyps Prior to Onset of Strobilation.

Aurelia aurita's intricate life cycle alternates between benthic polyp and pelagic medusa stages. The strobilation process, a critical asexual reproduction mechanism in this jellyfish, is severely compromised in the absence of the natural polyp microbiome, with limited production and release of ephyrae. Yet, the recolonization of sterile polyps with a native polyp microbiome can correct this defect. Here, we investigated the precise timing necessary for recolonization as well as the host-associated molecular processes involved. We deciphered that a natural microbiota had to be present in polyps prior to the onset of strobilation to ensure normal asexual reproduction and a successful polyp-to-medusa transition. Providing the native microbiota to sterile polyps after the onset of strobilation failed to restore the normal strobilation process. The absence of a microbiome was associated with decreased transcription of developmental and strobilation genes as monitored by reverse transcription-quantitative PCR. Transcription of these genes was exclusively observed for native polyps and sterile polyps that were recolonized before the initiation of strobilation. We further propose that direct cell contact between the host and its associated bacteria is required for the normal production of offspring. Overall, our findings indicate that the presence of a native microbiome at the polyp stage prior to the onset of strobilation is essential to ensure a normal polyp-to-medusa transition. IMPORTANCE All multicellular organisms are associated with microorganisms that play fundamental roles in the health and fitness of the host. Notably, the native microbiome of the Cnidarian Aurelia aurita is crucial for the asexual reproduction by strobilation. Sterile polyps display malformed strobilae and a halt of ephyrae release, which is restored by recolonizing sterile polyps with a native microbiota. Despite that, little is known about the microbial impact on the strobilation process's timing and molecular consequences. The present study shows that A. aurita's life cycle depends on the presence of the native microbiome at the polyp stage prior to the onset of strobilation to ensure the polyp-to-medusa transition. Moreover, sterile individuals correlate with reduced transcription levels of developmental and strobilation genes, evidencing the microbiome's impact on strobilation on the molecular level. Transcription of strobilation genes was exclusively detected in native polyps and those recolonized before initiating strobilation, suggesting microbiota-dependent gene regulation.

Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

Diapause in Univoltine and Semivoltine Life Cycles.

Although it is generally more adaptive for insects to produce additional generations than to have longer life cycles, some insects produce one or fewer generations per year (univoltine or semivoltine life cycles, respectively). Some insects with the potential to produce multiple generations per year produce a univoltine life cycle in response to environmental conditions. Obligatory univoltine insects have a single long diapause or multiple diapauses in different seasons. Semivoltine insects have multiple diapauses in different years, a prolonged diapause for more than a year, or diapause controlled by a circannual rhythm. Diapause in these insects greatly varies among species both in the physiological mechanism and in the evolutionary background, and there is no general rule defining it. In this review, we survey the physiological control of univoltine and semivoltine insects' diapause and discuss the adaptive significance of the long life cycles. Although constraints such as slow development are sometimes responsible for these life cycles, the benefits of these life cycles can be explained by bet-hedging in many cases. We also discuss the effect of climate warming on these life cycles as a future area of research.

Plasmodium falciparum CRK5 Is Critical for Male Gametogenesis and Infection of the Mosquito.

Cyclin-dependent kinases (CDKs) and cyclins are critical cell cycle regulators in eukaryotes. In this study, we functionally characterized a CDK-related kinase (CRK5) of the human malaria parasite Plasmodium falciparum. P. falciparum CRK5 (PfCRK5) was expressed in asexual blood stages and sexual gametocyte stages, but showed male gametocyte- specific expression. In contrast to previous findings, we showed that gene deletion Pfcrk5- parasites grew normally as asexual stages and underwent normal gametocytogenesis to stage V gametocytes. However, Pfcrk5- parasites showed a severe defect in male gametogenesis, which was evident by a significant reduction in the emergence of male gametes (exflagellation). This defect caused a severe reduction of parasite transmission to the mosquito. Genetic crosses performed using sex-specific sterile transgenic parasites revealed that Pfcrk5- parasites suffered a defect in male fertility but female gametes were fertile. Taken together, these results demonstrate that PfCRK5 is a critical sexual stage kinase which regulates male gametogenesis and transmission to the mosquito. IMPORTANCE Gametocytes are parasite sexual stages which differentiate from asexually replicating parasites. These stages are necessary for the completion of sexual phase of the parasite life cycle. Inside the mosquito midgut, gametocytes rapidly get activated to form fertilization competent gametes. These stages present a bottleneck in the parasite life cycle. In this study, we demonstrate that PfCRK5 is important for male gametogenesis and therefore regulates parasite transmission to the mosquito. Our study identifies PfCRK5 as a potential target for the development of drugs to block malaria transmission.

Population regulation throughout a complex life cycle.

Research Highlight: McIntosh, A. R., Greig, H. S., & Howard, S. (2022). Regulation of open populations of a stream insect through larval density dependence. Journal of Animal Ecology. https://doi.org/10.1111/1365-2656.13696. Despite decades of research on population regulation through density dependence, it remains challenging to identify and understand the relative importance of mechanisms governing open populations of organisms with complex life cycles. McIntosh et al. (2022) manipulated density of aquatic invertebrates in the field, and then followed populations for 2 years to track the effects on abundance through multiple life-history stages. The authors found that their density manipulation, performed on larvae that were about to pupate, had minimal effects on the number of emergent adults collected several months later. The manipulation had a similarly negligible influence on the number of egg masses laid at study locations. The authors attribute this to stochasticity around dispersal of flying adults through the terrestrial environment. However, later in the study, the authors found evidence of density-dependent population regulation among larval stages, seemingly controlled by resource availability. These results suggest that population dynamics depend on multiple mechanisms operating at different points in organisms' life history, which could either help or hinder population persistence with disturbance or environmental change.

Distinct Histone Post-translational Modifications during Plasmodium falciparum Gametocyte Development.

Histones are the building units of nucleosomes, which constitute chromatin. Histone post-translational modifications (PTMs) play an essential role in epigenetic gene regulation. The Plasmodium falciparum genome encodes canonical and variant histones and a collection of conserved enzymes for histone PTMs and chromatin remodeling. Herein, we profiled the P. falciparum histone PTMs during the development of gametocytes, the obligatory stage for parasite transmission. Mass spectrometric analysis of histones extracted from the early, middle, and late stages of gametocytes identified 457 unique histone peptides with 90 PTMs, of which 50% were novel. The gametocyte histone PTMs display distinct patterns from asexual stages, with many new methylation sites in histones H3 and H3.3 (e.g., K14, K18, and K37). Quantitative analyses revealed a high abundance of acetylation in H3 and H4, mono-methylation of H3/H3.3 K37, and ubiquitination of H3BK112, suggesting that these PTMs play critical roles in gametocytes. Gametocyte histones also showed extensive and unique combinations of PTMs. These data indicate that the parasite harbors distinct transcription regulation mechanisms during gametocyte development and lay the foundation for further characterization of epigenetic regulation in the life cycle of the malaria parasite.

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2'-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.

Embryonic development of the moon jellyfish Aurelia aurita (Cnidaria, Scyphozoa): another variant on the theme of invagination.

Background: Aurelia aurita (Scyphozoa, Cnidaria) is an emblematic species of the jellyfish. Currently, it is an emerging model of Evo-Devo for studying evolution and molecular regulation of metazoans' complex life cycle, early development, and cell differentiation. For Aurelia, the genome was sequenced, the molecular cascades involved in the life cycle transitions were characterized, and embryogenesis was studied on the level of gross morphology. As a reliable representative of the class Scyphozoa, Aurelia can be used for comparative analysis of embryonic development within Cnidaria and between Cnidaria and Bilateria. One of the intriguing questions that can be posed is whether the invagination occurring during gastrulation of different cnidarians relies on the same cellular mechanisms. To answer this question, a detailed study of the cellular mechanisms underlying the early development of Aurelia is required. Methods: We studied the embryogenesis of A. aurita using the modern methods of light microscopy, immunocytochemistry, confocal laser microscopy, scanning and transmission electron microscopy. Results: In this article, we report a comprehensive study of the early development of A. aurita from the White Sea population. We described in detail the embryonic development of A. aurita from early cleavage up to the planula larva. We focused mainly on the cell morphogenetic movements underlying gastrulation. The dynamics of cell shape changes and cell behavior during invagination of the archenteron (future endoderm) were characterized. That allowed comparing the gastrulation by invagination in two cnidarian species-scyphozoan A. aurita and anthozoan Nematostella vectensis. We described the successive stages of blastopore closure and found that segregation of the germ layers in A. aurita is linked to the 'healing' of the blastopore lip. We followed the developmental origin of the planula body parts and characterized the planula cells' ultrastructure. We also found that the planula endoderm consists of three morphologically distinct compartments along the oral-aboral axis. Conclusions: Epithelial invagination is a fundamental morphogenetic movement that is believed as highly conserved across metazoans. Our data on the cell shaping and behaviours driving invagination in A. aurita contribute to understanding of morphologically similar morphogenesis in different animals. By comparative analysis, we clearly show that invagination may differ at the cellular level between cnidarian species belonging to different classes (Anthozoa and Scyphozoa). The number of cells involved in invagination, the dynamics of the shape of the archenteron cells, the stage of epithelial-mesenchymal transition that these cells can reach, and the fate of blastopore lip cells may vary greatly between species. These results help to gain insight into the evolution of morphogenesis within the Cnidaria and within Metazoa in general.

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication.

P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.

Transcriptomic Profile of Canine DH82 Macrophages Infected by Leishmania infantum Promastigotes with Different Virulence Behavior.

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.

The big potential of the small frog Eleutherodactylus coqui.

The Puerto Rican coquí frog Eleutherodactylus coqui is both a cultural icon and a species with an unusual natural history that has attracted attention from researchers in a number of different fields within biology. Unlike most frogs, the coquí frog skips the tadpole stage, which makes it of interest to developmental biologists. The frog is best known in Puerto Rico for its notoriously loud mating call, which has allowed researchers to study aspects of social behavior such as vocal communication and courtship, while the ability of coquí to colonize new habitats has been used to explore the biology of invasive species. This article reviews existing studies on the natural history of E. coqui and discusses opportunities for future research.

Analysis of sex-specific lipid metabolism of Plasmodium falciparum points to the importance of sphingomyelin for gametocytogenesis.

Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to the mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here, we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase genes points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are distinct from asexual stages, and that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Taken together, our results highlight the sex-specific nature of gametocyte lipid metabolism, which has the potential to be targeted to block malaria transmission. This article has an associated First Person interview with the first author of the paper.

Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses.

Dengue is a mosquito-borne viral disease (arboviral) caused by the Dengue virus. It is one of the prominent public health problems in tropical and subtropical regions with no effective vaccines. Every year around 400 million people get infected by the Dengue virus, with a mortality rate of about 20% among the patients with severe dengue. The Dengue virus belongs to the Flaviviridae family, and it is an enveloped virus with positive-sense single-stranded RNA as the genetic material. Studies of the infection cycle of this virus revealed potential host targets important for the virus replication cycle. Here in this review article, we will be discussing different stages of the Dengue virus infection cycle inside mammalian host cells and how host proteins are exploited by the virus in the course of infection as well as how the host counteracts the virus by eliciting different antiviral responses.

In vitro differentiation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes using a biphasic medium.

The pathogen Trypanosoma cruzi differentiates from epimastigotes (E) into infective metacyclic trypomastigotes (MTs) to invade the mammalian cell. This process, called metacyclogenesis, is mimicked in vitro by nutrient starvation or incubation with minimal media. Here, we describe an alternative protocol for metacyclogenesis by incubating E forms in a biphasic medium supplemented with human blood. Although time consuming, this procedure yields fully differentiated MTs without the presence of intermediate forms, even for cultures that have been maintained as E for years.

Identifying the potential of anadromous salmonid habitat restoration with life cycle models.

An investigation into the causes of species decline should include examination of habitats important for multiple life stages. Integrating habitat impacts across life stages with life-cycle models (LCMs) can reveal habitat impairments inhibiting recovery and help guide restoration efforts. As part of the final elements of the Habitat Restoration Planning model (HARP; Beechie et al. this volume), we developed LCMs for four populations of three species of anadromous salmonids (Oncorhynchus kisutch, O. tshawytscha, and O. mykiss), and ran diagnostic scenarios to examine effects of barrier removal, fine sediment reduction, wood augmentation, riparian shade, restoration of the main channel and bank conditions, beaver pond restoration, and floodplain reconnection. In the wood scenario, spawner abundance for all populations increased moderately (29-48%). In the shade scenario, spring-run Chinook salmon abundance increased the most (48%) and fall-run Chinook salmon and steelhead were much less responsive. Coho responded strongly to the beaver pond and floodplain scenarios (76% and 54%, respectively). The fine sediment scenario most benefitted fall- and spring-run Chinook salmon (32-63%), whereas steelhead and coho were less responsive (11-21% increase). More observations are needed to understand high fine sediment and its impacts. Our LCMs were region-specific, identifying places where habitat actions had the highest potential effects. For example, the increase in spring-run Chinook salmon in the wood scenario was driven by the Cascade Mountains Ecological Region. And, although the overall response of coho salmon was small in the barrier removal scenario (6% increase at the scale of the entire basin), barrier removals had important sub-regional impacts. The HARP analysis revealed basin-wide and regional population-specific potential benefits by action types, and this habitat-based approach could be used to develop restoration strategies and guide population rebuilding. An important next step will be to ground-truth our findings with robust empirically-based estimates of life stage-specific survivals and abundances.

Unexpected plasticity in the life cycle of Trypanosoma brucei.

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host's circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia.

Uncoupling of the Infancy Life History Stage.

BACKGROUND: The life history of Homo sapiens is unique in having a comparatively short stage of infancy which lasts for 2-3 years. Infancy is characterized by suckling of breast milk, the development of sensorimotor cognition, the acquisition of language, mini-puberty, deciduous dentition, and almost complete skull growth. Infancy ends with the infancy-childhood growth transition (ICT) and separation from the mother. In modern-day affluent societies, breastfeeding depends on the mother's decision and may happen at any age, and the characteristic traits of infancy have uncoupled. The data and theory for this contention are presented. SUMMARY: The biological traits of mini-puberty and ICT characteristic of infancy occur before age 1 along with language acquisition. The cognitive (sensorimotor) component occurs by age 2, and the social component of separation from the mother by any age from 1 to 3 years. Key Messages: Human life history is based on a coherent stage of infancy which assumes coupling between the biological, cognitive, and social maturation of a baby. This is no longer the case in industrial societies and might never be so again. The upbringing of an infant needs to consider the new biology of this dissociated infancy and a new timetable of the infant's life-history events.

Mating Systems in True Morels (Morchella).

True morels (Morchella spp., Morchellaceae, Ascomycota) are widely regarded as a highly prized delicacy and are of great economic and scientific value. Recently, the rapid development of cultivation technology and expansion of areas for artificial morel cultivation have propelled morel research into a hot topic. Many studies have been conducted in various aspects of morel biology, but despite this, cultivation sites still frequently report failure to fruit or only low production of fruiting bodies. Key problems include the gap between cultivation practices and basic knowledge of morel biology. In this review, in an effort to highlight the mating systems, evolution, and life cycle of morels, we summarize the current state of knowledge of morel sexual reproduction, the structure and evolution of mating-type genes, the sexual process itself, and the influence of mating-type genes on the asexual stages and conidium production. Understanding of these processes is critical for improving technology for the cultivation of morels and for scaling up their commercial production. Morel species may well be good candidates as model species for improving sexual development research in ascomycetes in the future.

NaCl Promotes the Efficient Formation of Haematococcus pluvialis Nonmotile Cells under Phosphorus Deficiency.

Natural astaxanthin helps reduce the negative effects caused by oxidative stress and other related factors, thereby minimizing oxidative damage. Therefore, it has considerable potential and broad application prospects in human health and animal nutrition. Haematococcus pluvialis is considered to be the most promising cell factory for the production of natural astaxanthin. Previous studies have confirmed that nonmotile cells of H. pluvialis are more tolerant to high intensity of light than motile cells. Cultivating nonmotile cells as the dominant cell type in the red stage can significantly increase the overall astaxanthin productivity. However, we know very little about how to induce nonmotile cell formation. In this work, we first investigated the effect of phosphorus deficiency on the formation of nonmotile cells of H. pluvialis, and then investigated the effect of NaCl on the formation of nonmotile cells under the conditions of phosphorus deficiency. The results showed that, after three days of treatment with 0.1% NaCl under phosphorus deficiency, more than 80% of motile cells had been transformed into nonmotile cells. The work provides the most efficient method for the cultivation of H. pluvialis nonmotile cells so far, and it significantly improves the production of H. pluvialis astaxanthin.

Life history and nesting ecology of a Japanese tube-nesting spider wasp Dipogon sperconsus (Hymenoptera: Pompilidae).

To clarify the life history of the Japanese spider wasp Dipogon sperconsus, bionomical studies using bamboo-cane trap nests were carried out in Japan. Based on weekly and consecutive daily surveys of trap nests and rearing of broods from collected nests, we evaluated the production of cells and eggs per day, prey spiders, and seasonal patterns of nesting activities. We found a relatively short critical period of switching from the summer generation into the overwintering generation. We also found that the voltinism is affected by the timing of egg production of the second generation in relation to this critical period. The developmental period for each generation and sex, voltinism and cell production per day were determined based on data for a large number of individuals for the first time.