Instar Larval Development Research Articles

Tyrosine hydroxylase involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

Genes Expressed in Silk Glands of Bombyx mori Show Differential Association With Nuclear Matrix During 5th Instar Development

Silk glands are highly regulated during 5th instar larval development of Bombyx mori. Several genes show up and down regulation in silk glands during 5th instar stage. Nuclear matrix tethers chromatin and marks them into active and inactive domains, thereby playing an important role in gene regulation. The association of differentially regulated genes of silk glands to nuclear matrix has not been studied earlier. The present study explored the association between binding of genes to nuclear matrix and their differential expression. The association to nuclear matrix of the selected genes was studied using in-silico methods. The MAR Finder analysis showed high MAR binding potential for all the genes selected. The number of MAR binding sites varied from day 1 to day 7. Differences were observed in MAR regions associated with the genes. Variation in number and MAR binding sites during development often coincided with differential expression of genes. This study gives a new perspective in the understanding of differential gene regulation in silk gland development.

Cloning and characterization of a riboflavin-binding hexamerin from the larval fat body of a lepidopteran stored grain pest, Corcyra cephalonica

In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat body of Corcyra cephalonica. The complete cDNA (2121bp) encodes a 706-amino acid protein with a molecular mass ~82kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is prominently expressed during the last instar larval development. Homology modeling and docking studies predicted riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect. However, JH in the presence of 20HE maintains the RbHex 82 expression which indicates the JH's role as a status quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran pest. Further, the possibility of RbHex 82 as a pest control target is discussed.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae)

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F1F0ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata–corpora-cardiaca (CC–CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC–CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development.

Effects of temperature on the growth and reproduction characteristics of Bemisia tabaci B-biotype and Trialeurodes vaporariorum

The development period, survival rate, longevity and fecundity of two whiteflies, Bemisia tabaci B-biotype and Trialeurodes vaporariorum (Homoptera: Aleyrodidae) were compared under different temperature laboratory conditions (15°C, 18°C, 21°C and 24°C). Egg development of B. tabaci B-biotype was significantly longer compared with that of T. vaporariorum at 15°C, 18°C and 24°C. Significantly longer pseudo-pupae development and lower survival rate were found in B. tabaci B-biotype at 15°C compared with those at 18°C, 21°C and 24°C. Significantly higher fecundity was found in B. tabaci B-biotype at 24°C compared with that at 15°C, 18°C and 21°C. However, the fecundity of T. vaporariorum was significantly lower at 24°C relative to that at 15°C, 18°C and 21°C. Significantly shorter 1st instar larval development was found in T. vaporariorum compared with that of B. tabaci at 15°C and 18°C. Significantly longer 2nd instar larval development was found in B. tabaci and T. vaporariorum at 15°C compared with that at 18°C, 21°C and 24°C. However, significantly shorter 3rd instar larval development was found in T. vaporariorum compared with that of B. tabaci at 15°C, 18°C and 24°C. The adaptive divergence of tolerance to relatively low temperature may be an important factor that results in the interspecific differentiation between the seasonal dynamics of these two whiteflies in China.

Oviposition Preference and Nutritional Indices ofPapilio polytesL. (Papilionidae) Larvae on Four Rutaceous (Sapindales: Rutaceae) Host Plants

Oviposition preference and larval developmental performance of the butterfly, Papilio polytes L. on four rutaceous host plants, Citrus aurantifolia (Chrism.) Swing, Citrus reticulata Blanco, Citrus hystrix DC, and Murraya koenigii (L.) Sprengle, were investigated in outdoor cages. Maxima of eggs were laid on C. reticulata, followed by C. aurantifolia, but the numbers laid on the two host plants were not significantly different from each other; however, these numbers differed significantly (F = 155.70; P < 0.01) from those laid on C. hysrix and M. koenigii; the last host plant was the least preferred for egg laying. Leaves were significantly the preferred site (and in turn the underside of leaves) over stems for deposition of eggs in all host plants; negligible number of eggs was also laid on plant pots. The duration of 5th instar larval development was the shortest on C. reticulata and significantly different (P < 0.01) from that on M. koenigii, but did not differ significantly between the three Citrus species. The nitrogen content in leaves of all four host plants differed significantly (P < 0.01), with C. reticulata containing 4.52%, followed by C. aurantifolia (4.37%), C. hystrix (4.29%), and M. koenigii (3.73%), while the water content of leaves was significantly the lowest in M. koenigii (71.72%), compared to the three Citrus species (76.38–79.12%) among which the water content did not significantly differ. Relative consumption rate (RCR) and fecal dry weight of 5th instar larvae feeding on M. koenigii were the highest and significantly different (P < 0.01) from the other three host plants. Pupal dry weight, relative growth rate (RGR), efficiency conversion of digested food (ECD), efficiency conversion of ingested food (ECI) of M. koenigii were significantly lowest compared to the three Citrus species. The values of RGR, ECD, and ECI of the 5th instar larvae were similar for the three Citrus species; the approximate digestibility (AD) values were similar for all four host plants. This study shows that C. reticulata was the best host plant among the three Citrus spp. and M. koenigii was the least preferred.

20-Hydroxyecdysone regulation of H-fibroin gene in the stored grain pest Corcyra cephalonica, during the last instar larval development

20-Hydroxyecdysone (20E) controls molting, metamorphosis and reproduction of insects. It binds to a heterodimeric complex of ecdysone receptor (EcR) and ultraspiracle (USP), and regulates the transcription of genes containing ecdysone response elements (EcREs). However, the 20E regulation of silk fibroin genes is largely unexplored. In most lepidopteran larvae, the silk fibroin primarily consists of a large protein, heavy chain fibroin (H-fibroin) that is associated with two small proteins, L-chain fibroin and P25. In the present study, we demonstrate that 20E regulates the expression of H-fibroin gene in Corcyra cephalonica, in a dose-dependent manner during the last instar larval development. Semi-quantitative and real-time PCR studies reveal that physiological doses of 20E do not alter the normal expression, whereas higher doses cause a significant decline in the expression. Luciferase activity assays and gel shift experiments further confirm the presence of a functional EcRE in the upstream region of H-fibroin which regulates the ecdysteroid dependent transcriptional activity of fibroin gene through EcR. In vitro treatment with 20E mimicking insecticides, RH-5849 and RH-5992 decreases the expression of H-fibroin in isolated salivary glands. Insects fed with similar concentrations of these insecticides, metamorphose abnormally. Differences are also observed in the ultrastructure of the silk fibers of control and insecticide fed insects providing additional insight into the disruptive effects of these non-steroidal ecdysteroid agonists.

Light chain fibroin and P25 genes of Corcyra cephalonica: Molecular cloning, characterization, tissue-specific expression, synchronous developmental and 20-hydroxyecdysone regulation during the last instar larval development

The biologically active ecdysteroid hormone, 20-hydroxyecdysone (20E), regulates various processes like molting, metamorphosis and reproduction in insects. However, its role in expression of silk genes is obscure. The silk core in insects is generally constituted of a complex of three proteins namely, H-chain fibroin (H-fibroin), L-chain fibroin (L-fibroin) and P25. In the present study, we report full-length cDNA cloning and characterization of L-fibroin and P25 genes from rice moth, Corcyra cephalonica. Northern analyses demonstrated 1.1 and 1 kb transcripts of L-fibroin and P25 respectively. The tissue expression pattern shows the presence of these transcripts specifically in the salivary gland. These two genes are developmentally regulated at transcriptional level and their maximum expression is observed during the late-last instar larval stage. Semi-quantitative and real-time PCR studies revealed that 20E regulates the expression of these genes in a dose-dependant manner. This study further shows that physiological dose of 20E does not alter the normal expression of these two genes, whereas treatments with higher doses cause a significant decline in the expression. This study clearly suggests the role of 20E in the regulation of L-fibroin and P25 at molecular level.

Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilisation by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus

Data on the hormonal regulation of the formation and mobilisation of fat body stores are presented and discussed in relation to general parameters of last instar larval development such as growth, food intake, and moulting. Crickets feed voraciously during the first half of the last larval stage. With the onset of feeding, fat body lipid synthesis increases, leading to increasing lipid stores in the fat body with a maximum reached on day 5. Lipid (42% of fat body fresh mass) is the main constituent of the fat body stores, followed by protein (6%) and glycogen (2%). During the second half of the last larval stage, feeding activity dramatically decreases, the glycogen reserves are depleted but lipid and protein reserves in the fat body remain at a high level except for the last day of the last larval stage when lipid and protein in the fat body are also largely depleted. The process of moulting consumes almost three quarters of the caloric equivalents that were acquired during the last larval stage. Adipokinetic hormone (AKH) inhibits effectively the synthesis of lipids in the larval fat body. Furthermore, AKH stimulates lipid mobilisation by activating fat body triacylglycerol lipase (TGL) in last larval and adult crickets. Both effects of AKH are weaker in larvae than in adults. This is the first report on the age-dependent basal activity of TGL in larval and adult insects. In addition, for the first time, an activation of TGL by AKH in a larval insect is shown.

Estradiol-17β: Tracing its metabolic significance in female fatbody of fifth instar larvae of silkworm, Bombyx mori L (race: Nistari)

In recent years, various vertebrate peptide and steroid hormones have been identified in invertebrates, estradiol-17β (E 2) being a major one. We have specifically shown NADP-malate dehydrogenase (NADP-MDH) activity in fifth instar larval fatbody of female silkworm, Bombyx mori, as an E 2 responsive parameter. Interestingly enough, estradiol-induced increase in the enzyme activity could be counteracted by simultaneous application of specific E 2-inhibitor, ICI-182780. Further, a nice correlation was obtained among the E 2 titre, specific *E 2 binding and expression of NADP-MDH activity in fatbody during different days of normal fifth instar larval development. Though the nature of the binding sites is quite similar to known steroid receptors of vertebrate, the reported absence of estrogen receptor gene in some insects poses a question. A recent finding regarding the presence of an estrogen-related receptor ortholog in fruit fly may provide some answers. The specific effects elicited by estradiol in the female fatbody of this insect support its possibility of having an important metabolic function. This role played by E 2, whether hormonal or not, is yet to be identified.

Essential roles for the Dhr78 orphan nuclear receptor during molting of the Drosophila tracheal system

The Drosophila Dhr78 orphan nuclear receptor has been proposed to play a role in molting of the tracheal cuticle and regulate gene expression during the third larval instar, possibly in response to a novel systemic hormonal signal. Here, we show that there are no essential maternal functions for Dhr78 during development, and that mutants missing both maternal and zygotic Dhr78 function die primarily during second and third instar larval development. We show that defects in the tracheal system can be observed as early as the first instar, manifested as regions of fluid in the dorsal tracheal trunks. In addition, Dhr78 mutant tracheae show a highly penetrant defect in gas filling at the first-to-second instar larval molt. Dhr78 expression in only the tracheal system is sufficient to rescue the lethality of Dhr78 mutants, and selective inactivation of Dhr78 function in the tracheae by targeted RNAi is sufficient to result in tracheal defects. Finally, we see no evidence for widespread activation of the Dhr78 ligand binding domain in third instar larvae using the GAL4-LBD system, arguing against a systemic hormone for the receptor at this stage in development. Taken together, our results indicate that Dhr78 exerts its essential functions during molting of the tracheal cuticle in Drosophila.

ECDYSONE 20‐MONOOXYGENASE ACTIVITY IN THE PROTHORACIC GLANDS OF LAST INSTAR LARVAE OF BOMBYX MORI*

Abstract Using cell free radioassay, activities of ecdysone 20‐monooxygenase (E‐20‐M) were determined in homogenates of prothoracic glands (PGs) and fat bodies at 24 h intervals during last instar larval development of Bombyx mori. It was found that the profile of E‐20‐M activity in PG homogenates was characterized by a basal line from day 0 to day 3 which begins to rise on 4th day and reaches a peak on 5th day. Nevertheless, in comparison with PGs, E‐20‐M activity in fat body was much higher.

Molecular Analysis of the Initiation of Insect Metamorphosis: A Comparative Study of Drosophila Ecdysteroid-Regulated Transcription

More than 50 ecdysteroid-regulated Drosophila genes have been described in the literature. These genes were identified using several different ecdysteroid-responsive systems and characterized under a variety of experimental conditions. The diversity of these approaches has made it difficult to compare results and identify common responses to the hormone. As a first step toward characterizing the temporal regulation of these genes by ecdysteroids, we have examined their transcriptional activity throughout third instar larval and prepupal development using a single collection of staged animals. We see four coordinate changes in ecdysteroid-regulated gene activity during third instar larval development, at 78-88 hr, ∼100 hr, 106-108 hr, and ∼114 hr after egg laying. A dramatic transition in gene expression occurs at puparium formation, after which the prepupal ecdysteroid pulse induces successive waves of transcription. Our results suggest that several increases in the ecdysteroid titer during third instar larval and prepupal development program a precise temporal progression of gene activity that could direct the appropriate behavioral and developmental changes at the onset of metamorphosis.

The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis

During Drosophila third instar larval development, one or more pulses of the steroid hormone ecdysone activate three temporally distinct sets of genes in the salivary glands, represented by puffs in the polytene chromosomes. The intermolt genes are induced first, in mid-third instar larvae; these genes encode a protein glue used by the animal to adhere itself to a solid substrate for metamorphosis. The intermolt genes are repressed at puparium formation as a high titer ecdysone pulse directly induces a small set of early regulatory genes. The early genes both repress their own expression and activate more than 100 late secondary-response genes. The Broad-Complex (BR-C) is an early ecdysone-inducible gene that encodes a family of DNA binding proteins defined by at least three lethal complementation groups: br, rbp, and l(1)2Bc. We have found that the BR-C is critical for the appropriate regulation of all three classes of ecdysone-inducible genes. Both rbp and l(1)2Bc are required for glue gene induction in mid-third instar larvae. In addition, the l(1)2Bc function is required for glue gene repression in prepupae; in l(1)2Bc mutants the glue genes are re-induced by the late prepupal ecdysone pulse, recapitulating a mid-third instar regulatory response at an inappropriate stage in development. The l(1)2Bc function is also required for the complete ecdysone induction of some early mRNAs (E74A, E75A, and BR-C) and efficient repression of most early mRNAs in prepupae. Like the intermolt secondary-response genes, the late secondary-response genes are absolutely dependent on rbp for their induction. An effect of l(1)2Bc mutations on late gene activity can also be detected, but is most likely a secondary consequence of the submaximal ecdysone-induction of a subset of early regulatory products. Our results indicate that the BR-C plays a key role in dictating the stage-specificity of the ecdysone response. In addition, the ecdysone-receptor protein complex alone is not sufficient for appropriate induction of the early primary-response genes, but requires the prior expression of BR-C proteins. These studies define the BR-C as a key regulator of gene activity at the onset of metamorphosis in Drosophila.

The regulation of the cell cycle during Drosophila embryogenesis: the transition to polyteny

The process of polytenization plays a crucial role in Drosophila development, and most of the larval tissues are polytene. By analyzing the pattern of DNA replication in embryos pulse-labeled with BrdU, we show that many larval tissues undergo a transition to begin becoming polytene late in embryogenesis. Our results demonstrate that in these larval tissues polyteny results from a modified cell cycle, the endo cell cycle, in which there is only an S (synthesis) phase and a G (gap) phase. A key regulator of the mitotic cell cycle, the product of the string gene (the Drosophila homologue of cdc25), is not required for the endo cell cycle. The developmental regulation of the endo cell cycle is striking in that tissue-specific domains undergo polytene DNA replication in a dynamic pattern at defined times in embryogenesis. During subsequent rounds of the endo cell cycle in late embryogenesis and first instar larval development, the domains are subdivided and the temporal control is not as rigid. The length of the G phase varies among different tissues. By quantifying DNA content, we show that during the early polytene S phases the genome is not fully duplicated.

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.

Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans

The concentration of free amino acids, total soluble protein, and haemoglobin in the haemolymph of fourth instar Chironomus tentans was investigated. The concentration of the free amino acid pool increases between the early (15.7 mM/l) and mid-(33.9 mM/l) fourth larval stages followed by a decline during the late (16.9 mM/l) fourth larval period. Alanine, serine, and the amides of aspartic acid and glutamic acid are the predominant free amino acids at all stages. Physiological fluid analysis of late fourth instar haemolymph detected 32 ninhydrin positive components including 18 common amino acids plus homoarginine, ornithine, citrulline, β-alanine, α-aminoadipic acid, α-aminoisobutyric acid, and sarcosine. The concentration of total soluble protein steadily increases during fourth instar larval development to a maximum of 9.3 g 100 ml followed by a decline during the pharate pupal period. A similar pattern of variation occurs in haemoglobin content which comprises from 51 to 66% of Chironomus tentans haemolymph protein. The mM percentage of individual amino acids of total haemolymph protein varies little during the fourth instar. At all stages alanine and aspartic acid are the predominant amino acids.

Changes in the structure of the DNA of Drosophila melanogaster during development and aging

The DNA and RNA content of the fruit fly, Drosophila melanogaster undergoes two cycles of synthesis and loss during the larval and pupal stages of development. The adult stages show little or no change with age in DNA content. Thermal melting curves, sedimentation velocities and CsCl buoyant density measurements have been used to show that the molecular weight of larval DNA is of the order of 10 4 daltons. However, a fraction of the larval DNA (about 25%) is of a molecular weight greater than 10 6. Molecular weights increase during the later larval and pupal stages until a maximum of greater than 10 6 is reached during the adult stages. The molecular weight of embryonic DNA is greater than 10 6 but is rapidly degraded after hatching during early first instar larval development. Several interpretations of these data are offered. Buoyant densities in cesium salts showed no developmental or age-related changes.