Pupal Developmental Stages Research Articles

Orco-dependent survival of odorant receptor neurons in ants.

Olfaction is essential for complex social behavior in insects. To discriminate complex social cues, ants evolved an expanded number of odorant receptor (Or) genes. Mutations in the obligate odorant co-receptor gene orco lead to the loss of ~80% of the antennal lobe glomeruli in the jumping ant Harpegnathos saltator. However, the cellular mechanism remains unclear. Here, we demonstrate massive apoptosis of odorant receptor neurons (ORNs) in the mid to late stages of pupal development, possibly due to ER stress in the absence of Orco. Further bulk and single-nucleus transcriptome analysis shows that, although most orco-expressing ORNs die in orco mutants, a small proportion of them survive: They express ionotropic receptor (Ir) genes that form IR complexes. In addition, we found that some Or genes are expressed in mechanosensory neurons and nonneuronal cells, possibly due to leaky regulation from nearby non-Or genes. Our findings provide a comprehensive overview of ORN development and Or expression in H. saltator.

Proteomic Analysis of the Midgut Contents of Silkworm in the Pupal Stage.

The silkworm Bombyx mori, a lepidopteran insect, possesses an 8-10-day pupal stage, during which significant changes occur in the midgut, where it first condenses into the yellow body, and then undergoes decomposition. To gain insights into this transformation process, proteomics was performed on Bombyx mori midgut contents on day 2 and day 7 after pupation. The results revealed the identification of 771 proteins with more than one unique peptide. An analysis using AgriGO demonstrated that these proteins were predominantly associated with catalytic activity. Among the identified proteins, a considerable number were found to be involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleic acid degradation, and energy support. Additionally, variations in the levels of certain proteases were observed between the midgut contents on day 2 and day 7 after pupation. An in-depth analysis of the two-dimensional electrophoresis of the midgut contents on day 7 after pupation led to the identification of twelve protein spots with potential gelatinolytic activity. Among these, six proteases were identified through mass spectrometry, including the p37k protease, vitellin-degrading protease, chymotrypsin-2, etc. These proteases may be responsible for the digestion of the yellow body during the later stages of pupal development.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function.

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Midgut remodeling during the metamorphosis of Chironomus riparius, Meigen (1804)

The holometabolous insects go through a complete metamorphosis that includes four life stages: egg, larva, pupa, and imago (adult). Chironomus riparius is a suggested model organism by the Organization for Economic Cooperation and Development (OECD) that is used in acute and chronic tests of chemicals. Tissue morphology of healthy non-biting midge larval stage was already described but the faith of the midgut digestive cells and tissue organization during the metamorphosis is unknown. We here described histological alterations of the midgut during ecdysis to distinguish them from the ones caused by toxins’ negative effects. The present study showed differences in tissue architecture of the midgut in the larval, prepupal, and pupal stages of development of C. riparius. During ecdysis, larval digestive cells detached from the midgut epithelium and moved to the lumen. In the pupa, the larval midgut layer was replaced with an adult midgut that had considerably reduced width. These changes in the midgut tissue morphology and organization probably follow changes in the environment and feeding behavior of C. riparius at different stages of development.

Nutritional Composition of Honey Bee Drones of Two Subspecies Relative to Their Pupal Developmental Stages.

Simple SummaryDespite the use of honey bee brood as food among several communities of the world, the nutritional potential of drones remained unexplored for a long time. In the recent past some scientific endeavour, including our own previous work, has been undertaken to explore the nutrient quality of this food source. Due to their limited socio-biological role, honey bee drones would be a suitable candidate to compare their nutrient content with that of worker honey bees. We therefore investigated the nutrient composition of honey bee drones belonging to two subspecies, namely Apis mellifera carnica and A. m. mellifera covering their pupal developmental period. To possess information of the drones’ nutritional value during their development would help in choosing the most suitable developmental stage for the commercial production of drone brood as food.We examined the contents of nutritional importance, i.e., amino acids, fatty acids and minerals of different developmental stages of drones of two honey bee subspecies, namely Apis mellifera carnica and A. m. mellifera. The results revealed that, in general, individual amino acid amounts and therefore the total protein increased along with the developmental stages of the drones. No statistically significant differences were found between the same developmental stages of the two subspecies. The reverse, i.e., a decrease with developmental stage occurred in relation to the fatty acid composition. Most of the minerals were higher at advanced developmental stages. Overall, the high protein content (31.4–43.4%), small amount of fat (9.5–11.5%) and abundance of minerals such asiron and zinc, make drones a suitable nutritional resource. Even though nutrient content, especially protein, was higher in the pupae than the prepupae, we propose prepupae also as a commercial product based on their higher biomass production. Provided standard production protocols maintaining hygiene and safety will be adhered to, we propose that drone honey bees can be utilized as human food or animal feed.

Novel Characteristics of Immune Responsive Protein IRP30 in the Bumble Bee Bombus lantschouensis (Hymenoptera: Apidae).

Immune responsive protein 30 (IRP30) is a Hymenoptera-specific protein first identified from honey bee hemolymph in response to bacterial infection. However, its function remains elusive. Here, we cloned the full-length IRP30 gene and clarified its expression pattern in the bumble bee Bombus lantschouensis (Vogt). The full-length IRP30 gene measures 1443 bp and contains two exons and one intron. The length of the cDNA is 1082 bp, including a 36-bp 5′-UTR and a 218-bp 3′-UTR, and it encodes a putative protein of 275 amino acids. As expected, the sequence of the B. lantschouensis IRP30 protein was clustered with the bumble bee group, which appeared as a single clade next to honey bees. The family shared similar conserved protein domains. Moreover, bumble bee IRP30 belongs to a recently diverged clade that has four leucine-rich repeat (LRR) conserved domains. IRP30 is highly expressed in the worker caste, during pupal developmental stages, and in the head and thorax tissues. Interestingly, its expression increases 20- to 90-fold when female bumble bees (B. lantschouensis) and honey bees (Apis mellifera L.) begin laying eggs. Overall, based on the expression of IRP30 during development and egg laying in female bumble bees, this protein not only responds to immune challenge but also may play an important role in metamorphosis and reproduction.

A Study of the pupal developments of Sarcophaga argyrostoma (Robineau-Desvoidy, 1830).

Forensic entomology is a discipline in which insect evidence is used to determine the minimum postmortem interval (PMImin). The insect evidence may also provide valuable information about the movement of the body, cause of the death, drug use, or poisoning. The pupal period constitutes approximately 50% of immature development. Therefore, it provides a more accurate estimation of the PMImin via a detailed examination. In this study, the pupal development of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830), which larviposits on dead bodies, was examined in Turkey. The pupal development periods of S. argyrostoma and the range of the beginning hours (maximum and minimum) of these periods were determined at a constant temperature of 20, 25, and 30°C. When all of the temperatures were considered, it was determined that 23 pupal development stages were similar; however, the development times shortened as the temperature increased. Adult emergence was observed at 459 (±1.41), 289.5 (±1.52), and 227 (±1.89) h at 20, 25, and 30°C, respectively. With respect to the pupal period used to determine the PMImin, the development of S. argyrostoma at different temperatures was described in this study for the first time.

Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

E93 Integrates Neuroblast Intrinsic State with Developmental Time to Terminate MB Neurogenesis via Autophagy

Most neurogenesis occurs during development, driven by the cell divisions of neural stem cells (NSCs). We use Drosophila to understand how neurogenesis terminates once development is complete, a process critical for neural circuit formation. We identified E93, a steroid-hormone-induced transcription factor that downregulates phosphatidylinositol 3-kinase (PI3K) levels to activate autophagy for elimination of mushroom body (MB) neuroblasts. MB neuroblasts are a subset of Drosophila NSCs that generate neurons important for memory and learning. MB neurogenesis extends into adulthood when E93 is reduced and terminates prematurely when E93 is overexpressed. E93 is expressed in MB neuroblasts during later stages of pupal development only, which includes the time when MB neuroblasts normally terminate their divisions. Cell intrinsic Imp and Syp temporal factors regulate timing of E93 expression in MB neuroblasts, and extrinsic steroid hormone receptor (EcR) activation boosts E93 levels high for termination. Imp inhibits premature expression of E93 in a Syp-dependent manner, and Syp positively regulates E93 to promote neurogenesis termination. Imp and Syp together with E93 form a temporal cassette, which consequently links early developmental neurogenesis with termination. Altogether, E93 functions as a late-acting temporal factor integrating extrinsic hormonal cues linked to developmental timing with neuroblast intrinsic temporal cues to precisely time neurogenesis ending during development.

Identification, characterization and expression analysis of Anopheles stephensi double peroxidase

Peroxidases catalyze the reduction of peroxides and that, in turn, oxidize various substrates. They have been widely reported to play an important role in mosquito innate immunity against various pathogens. Here, we have characterized double heme peroxidase (AsDBLOX) gene from the Indian malaria vector Anopheles stephensi. It is a true ortholog of An. gambiae DBLOX. This 4209 bp AsDBLOX gene encodes for a protein of 1402 amino acids that has two duplicated peroxidase domains, domain I (from amino acid 61 to 527) and domain II (from amino acid 714 to 1252). The first domain has only substrate binding sites and lacks all other motifs of a functional heme peroxidase (e.g. heme binding site, calcium binding site and homodimer interface). Instead, it has two integrin binding motifs-LDV (Leu–Asp–Val) and RGD (Arg–Gly–Asp). The second peroxidase domain, however, has all the features of a complete heme peroxidase along with an integrin binding motif LDI (Leu–Asp–Ile). Thus, AsDBLOX gene is a unique type of peroxinectin as these groups of proteins are characterized by integrin binding motifs along with a heme peroxidase domain. We also observed that the AsDBLOX gene is expressed in all the life cycle stages of mosquito and is highly induced in the pupal stage of development which indicates its possible role in development.

Identification and expression profiles of twenty-six glutathione S-transferase genes from rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae)

The rice weevil, Sitophilus oryzae, is one of the most destructive pests in stored cereals products. In this study, 26 cDNAs encoding glutathione S-transferases (GSTs) were sequenced and characterized in S. oryzae. Phylogenetic analysis displayed the categorization of 26 GSTs into six different cytosolic classes, including two in the delta, twelve in epsilon, three in omega, six in sigma, two in theta, and one in zeta class. RT-qPCR assay illustrated that the relative expression of ten GST genes was significantly higher in adult stages than in larval and pupal developmental stages. Tissue-specific expression analysis revealed that the SoGSTe5, SoGSTe7, SoGSTe12, and SoGSTz1 were up-regulated in the midgut, SoGSTe2, SoGSTe6, and SoGSTs2 were up-regulated in the fat body, and three GSTs (SoGSTd1, SoGSTd2 and SoGSTe4) were up-regulated in Malpighian tubules. RT-qPCR indicated that five GST genes were over expressed after exposure to phosphine at various times and concentrations. The increase in GST gene expressions after phosphine exposure in S. oryzae may lead to an improved tolerance for fumigations and xenobiotics.

Dissection and Staining of Drosophila Pupal Ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of <em>Drosophila </em>Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Hormetic benefits of prior anoxia exposure in buffering anoxia stress in a soil-pupating insect.

Oxygen is essential for most animals, and exposure to a complete lack of oxygen, i.e. anoxia, can result in irreparable damage to cells that can extend up to the organismal level to negatively affect performance. Although it is known that brief anoxia exposure may confer cross-tolerance to other stressors, few data exist on the biochemical and organismal consequences of repeated intermittent bouts of anoxia exposure. In nature, the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae), is frequently exposed to heavy tropical rainfall while pupating in the soil, equating to multiple exposures to hypoxia or anoxia during development. Here, we tested whether prior anoxia exposures during pupal development can induce a beneficial acclimation response, and we explored the consequences of prior exposure for both whole-organism performance and correlated biochemical metrics. Pharate adults (the last developmental stage in the pupal case) were most sensitive to anoxia exposure, showing decreased survival and fertility compared with controls. These negative impacts were ameliorated by exposure to anoxia in earlier pupal developmental stages, indicating a hormetic effect of prior anoxia exposure. Anoxia exposure early in pupal development reduced the oxygen debt repaid after anoxia exposure relative to pharate adults experiencing anoxia for the first time. Lipid levels were highest in all pupal stages when exposed to prior anoxia. Prior anoxia thus benefits organismal performance and relocates resources towards lipid storage throughout pupal-adult development.

The initial analysis of a serine proteinase gene (AccSp10) from Apis cerana cerana: possible involvement in pupal development, innate immunity and abiotic stress responses.

Serine proteinases play important roles in innate immunity and insect development. We isolated a serine proteinase gene, designated AccSp10, from the Chinese honeybees (Apis cerana cerana). RT-qPCR and a Western blot analysis at different pupal development stages indicated that AccSp10 might be involved in melanin formation in pupae and promote pupal development. In adult workers, the expression of AccSp10 was upregulated by treatments mimicking harmful environments such as the presence of Bacillus bombysepticus, different temperatures (4, 24 and 42°C), HgCl2, H2O2 and paraquat; the exception was treatment with VC (vitamin C), which did not upregulate AccSp10 expression. Western blot confirmed the results. A disc diffusion assay indicated that recombinant AccSp10 accelerated E. coli cell death during stimulation with harmful substances (HgCl2, paraquat and cumene hydroperoxide). These findings suggest that AccSp10 may be involved in the pupal development of Chinese honeybees and protection against microorganisms and abiotic harms.

Temperature-dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval.

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMImin ) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT2 + dT3 equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMImin estimation.

Anatomy of adult Megaphragma (Hymenoptera: Trichogrammatidae), one of the smallest insects, and new insight into insect miniaturization.

The body size, especially in cases of extreme reduction, is an important characteristic that strongly determines the morphology, physiology, and biology of animals. Miniaturization is a widespread trend in animal evolution and one of the principal directions of evolution in insects. Miniaturization-related features of insect morphology have been subject to intensive studies during the last few years, but the structure of the smallest insects remains insufficiently known. It is especially important to study hymenopterans of the genus Megaphragma, which include the smallest flying insects and a species in which an almost anucleate nervous system was recently discovered. This article is the first detailed study of the external and internal morphology of adults of Megaphragma mymaripenne and M. amalphitanum using histological methods, 3D computer modeling and other techniques. It is shown that in spite of the extremely small size the organization of Megaphragma retains a considerkable level of structural complexity. On the other hand, miniaturization leads to re-organizations of several organ systems. Unique structural features related to miniaturization have been found in both species: lysis of cell bodies and nuclei of neurons at late stages of pupal development, absence of the heart, and considerable reductions in the set of muscles. Comparative analysis of structure in the smallest insects representing different taxa has revealed common features of the evolutionary process of miniaturization in insects.

Persistence of TwoSalmonella entericaser. Montevideo Strains Throughout Horn Fly (Diptera: Muscidae) Larval and Pupal Development

Strains of Salmonella enterica can be subdivided into clades that differ in their genetic composition, influencing microbial ecology and bacterial transmission. Salmonella serovar Montevideo strains 1110 and 304, representatives of two different clades, were used to evaluate interactions with the various stages of horn fly development. Sterilized cattle dung was inoculated with Salmonella monocultures, and horn fly larvae were exposed to 103, 105, and 107 colony-forming units (CFU)/g per strain. Salmonella supported horn fly development, and concentration-dependent differences in pupal survival suggested that Salmonella Montevideo 304 impacts adult emergence when larvae are reared in a high concentration. Viable bacteria of each strain were quantified from larvae, pupae, and newly emerged adults. Both strains were cultured from larvae at a mean ~105, regardless of concentration, and both strains survived pupation. Quantities of Salmonella 1110 were stationary through the midpupal stage, after which quantities declined in pupae reared in 105 and increased twofold in pupae reared in 107 CFU/g. Quantities of Salmonella 304 remained stationary throughout pupal development when reared in 105, yet increased 29-fold when reared in 107 CFU/g. At high densities, properties of Salmonella 304 may influence its interaction with horn fly larvae, enabling the bacteria to evade degradation during larval gut histolysis and to subsequently proliferate during the late stages of pupal development. This may account for the observed effect on adult emergence. The Salmonella strains were rarely cultured from newly emerged adults, indicating that transstadial carriage to the adult stage is inefficient.

FRESHWATER UNIONID BIVALVE SHELLS AS SUBSTRATA FOR TRICHOPTERA ATTACHMENT

ABSTRACT A variety of competitive advantages accompany colonization of hard substrates in aquatic settings, and as a result, many organisms adopt this mode of life. Similarly, adverse effects may occur to organisms that have themselves been encrusted. Many marine basibiont faunas (organisms that provide a substrate for populations of attached epibiontic organisms), including bivalve mollusks, are known to employ a range of chemical and physical deterrents to minimize the incidence of epibiotic attachment, with its attendant detrimental impacts. Freshwater basibiont and epibiont associations, however, are poorly documented. We collected an assemblage of dead, empty freshwater mussel (unionid bivalve) shells from the Saint John River, New Brunswick, Canada. Some unionid specimens exhibited colonization by attached Trichoptera (caddisfly) cases, in either their final instar larval or pupal developmental stages. The trichopteran assemblage, identified from case morphology, included abundant Neophylax sp. toge...

Molecular identification of glucose-regulated protein 78 (grp78) gene from the Indian meal moth, Plodia interpunctella, and its regulation by nutrient uptake

Glucose-regulated protein 78 (GRP78) is a member of the HSP70 family of proteins and is localized in the endoplasmic reticulum (ER) within cells. GRP78 and its gene has been identified in only a few species of insects, and its role is not clear. Here, we identified full-length grp78 cDNA from the Indian meal moth, Plodia interpunctella, and demonstrated the role of grp78 in developmental and physiological processes of the insect. The deduced amino acid sequence of GRP78 contained highly conserved functional motifs of the HSP70 family and the C-terminal motif of KDEL, which is characteristic of ER-localized HSP70. It also showed high identity (93–94%) with GRP78 and related HSP70 proteins of lepidopteran species. Gene expression analysis showed that grp78 mRNA levels were high in the egg, feeding larval, and adult stages, but low in the molting, wandering larval, and pupal stages of development. In a tissue comparison of fifth instar P. interpunctella, grp78 level was higher in the gut than in the integument or fat bodies. Grp78 level decreased greatly when fifth-instar larvae were starved for 48h, but recovered within 3–6h after re-feeding. Our data suggest that grp78 is highly associated with dietary energy conditions during development and may play an important role in the nutritional physiology of insects.