A. Krishna Kumaran

The boll weevil vitellogenin gene: Nucleotide sequence, structure, and evolutionary relationship to nematode and vertebrate vitellogenin genes

SummaryBoll weevil (Anthonomus grandis) eggs contain two yolk proteins, YP47 and YP160. Using anti-YP160 antiserum as probe, a partial-length complementary DNA (cDNA) was isolated from a λgt11 adult female cDNA library. A second partial-length cDNA was isolated from a λgt 10 adult female cDNA library by differential screening with male vs. female cDNAs. Northern blot analysis showed that each cloned cDNA hybridized to a 6-kb female-specific transcript. These cDNAs were used to probe a genomic library, and two overlapping genomic clones were obtained that span the boll weevil vitellogenin gene. The entire transcription unit was sequenced, and introns were mapped by a combination of primer extension experiments, S 1 nuclease protection experiments, and polymerase chain reaction-mediated synthesis of two additional cDNA clones. Based on these data, the vitellogenin mRNA is 5511 nucleotides [plus a poly(A) tail of undetermined length] and specifies a provitellogenin of 1790 amino acids. The deduced protein has a Glu + Gln content of 16.3%, which is a relatively high value that is typical of most vitellogenins. Protein sequence similarities including Cys clusters conserved between boll weevil vitellogenin and Xenopus laevis A2 or Caenorhabditis elegans vit-5 vitellogenins indicated that the boll weevil protein is a member of the ancient nematode-vertebrate vitellogenin family. Moreover, the six introns in the boll weevil vitellogenin gene interrupt the coding region at positions closely or exactly corresponding to a subset of the position, of the 34 vertebrate vitellogenin introns, further supporting the argument for a common evolutionary relationship. This report represents the first complete nucleotide sequence and structural analysis of a nondipteran insect vitellogenin gene.

Correlations between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleria mellonella

Abstract Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

Abstract The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.

Control of juvenile hormone esterase activity in Galleria mellonella larvae

Abstract The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae. JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.

Nucleotide sequence and structure of the arylphorin gene from Galleria mellonella

Abstract The complete nucleotide sequence and the structure of the arylphorin gene of Galleria mellonella have been determined. This gene contains four introns, at positions exactly corresponding to the four introns in other reported lepidopteran storage protein genes. The gene encodes a preprotein of 702 amino acids, including a signal peptide of 16 amino acids and 131 aromatic residues in the mature protein. The deduced amino acid sequence has been confirmed by determination of the 26 N-terminal residues of purified Lhp76. Thus, Lhp76 is a typical member of the arylphorin family of lepidopteran storgae proteins and is here renamed as the Galleria arylphorin. The deduced arylphorin protein sequence includes two potential N-glycosyation sites, in keeping with previous data that Lhp76 is an N-glycosylated protein. Although Galleria arylphorin gene espression is repressed by ecdysone in vivo and in vitro , no exact matches were found between 5′ flanking DNA sequences and known Drosophila ecdysone response elements.

Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella

Abstract Galleria mellonella a group of four larval hemolymph proteins (LHP) (74, 76, 81 and 82 kDa), which had been earlier shown to be storage proteins, exhibit a stage-specific synthetic pattern. The 82 kDa LHP is synthesized only in day-3 to day-5 last instar larvae, while the other three LHPs are synthesized both in the penultimate (six) and the last instar larvae. None of these LHPs are synthesized in day-0 last instar. With a view to isolate one or more cDNA clones corresponding to these LHPs a cDNA library was prepared in pBR322 starting with poly(A) + RNA from day-5 last instar larval fat body. By differential screening of 714 clones with poly(A) + RNA 39 day-5 larval stage-specific clones were isolated. Two of these clones, designated as 26–38 and 17–36, had 1200–1300 base pair cDNA inserts. Their cDNA inserts did cross hybridize to each other, exhibited different restriction endonuclease digestion patterns and hybridized in northern blots to transcrips of different sizes, thereby suggesting that they represent two separate genes. In addition, the genomic fragments that hybridized in southern blots to the two cDNAs differed in their size. On translation, mRNAs hybrid selected by 26–38 and 17–36 cDNAs produced 76 and 79 kDa polypeptides respectively. Both these genes are expressed in the fat body but not in the midgut, silk glands, Malpighian tubules or carcass. While 26–38 was expressed both in the sixth and seventh (last) instars, 17–36 was expressed only in the last instar. On the basis of tissue and developmental stage specificity of their expression and the sizes of their hybrid selected translation products, these clones are tentatively identified as two LHP-specific cDNA clones. The genes coding for these LHPs appear to be single copy genes.

Some properties of the haemolymph juvenile hormone esterases in Galleria mellonella larvae and Tenebrio molitor pupae

Abstract The apparent molecular weight, the K m , and the sensitivity to different organophosphates of the haemolymph juvenile hormone esterase (JHE) in the larvae of Galleria mellonella was determined. The apparent molecular weight, determined by gel filtration methods, was estimated to be 60,000. The apparent K m of the enzyme was 1.79 μM. The enzyme was insensitive to diisopropylphosphofluoridate (DFP) but was sensitive to paraoxon and 0-ethyl-S-phenylphosphoramidothiolate (EPPAT). The sensitivity of JHE in pupae of Tenebrio molitor to organophosphates was also determined. It was found to be insensitive to DFP and EPPAT.

Effects of juvenile hormone, ecdysteroids and nutrition on larval hemolymph protein gene expression in Galleria mellonella

Abstract Using two cDNA clones which represent the 82 kDa and 76 kDa larval hemolymph protein (LHP) genes of Galleria mellonella, the effects of juvenile hormone (JH), 20-hydroxyecdysone (20-HE) and nutrition on the levels of the correspoding transcripts in last instar larvae were determined. The transcripts coding for LHP82 and LHP76 declined in intact day-2 or day-3 last instar larvae 72–96 hr after application of 20 μg of JH, but in prothorax-ligated day-3 larvae JH had no effect on the levels of these transcripts. Application of 5 μg of 20-HE either to intact or to prothorax-ligated day-3 last instar larvae caused a decline in the levels of both the transcripts in 30–60 hr. Starvation of larvae beginning 24 or 48 hr after the last larval ecdysis did not inhibit production of either of the LHP transcripts, but starvation during the first 24 or 48 hrs of the last larval stadium, followed by normal feeding, delayed production of the LHP transcripts for up to 6 days. Ligation of the starved larvae 2 or 3 days after resumption of feeding accelerated production of the LHP76, but not LHP82, complementary transcripts. These data suggest that the two LHP genes are independently regulated, and that both are repressed by ecdysteroids.

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Abstract Egg chambers from apterous 4 (ap 4 ) , a female sterile mutant of Drosophila melanogaster , show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oocyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oocytes of ap 4 flies. Within 12 hr after topical application of ZR515 to homozygous ap 4 females the oocyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oocyte also shows some changes. In vitro studies in which ap 4 ovaries were incubated in Schneiders Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oocyte membrane.

Silk gland specific cDNAs from Galleria mellonella L

Abstract Four classes of Galleria mellonella silk gland specific cDNAs, designated PG-1, PG-2, MG-1 and MG-2 , were cloned, sequenced and used as probes in Northern and Southern blots. Analysis of the results and their comparison with the data available for Bombyx mori reveal that the isolated cDNAs correspond to mRNAs for major silk proteins. Two small transcripts (1.1 and 1.2 kb), detected with PG-1 in the posterior region of Galleria silk glands, represent mRNAs for small proteins related to the light chain fibroin of Bombyx . A large transcript (⩾ 10 kb) homologous to PG-2 seems to be the mRNA for the heavy chain fibroin. Conceptual translation of available PG-2 sequence yields a product with similar amino acid composition as reported for the chemically analyzed fibroin. Hybridization to Southern blots of Galleria genomic DNA showed that PG-1 and PG-2 correspond to separate genes. In the middle silk gland region, multiple transcripts homologous to MG-1 are produced. They include 1.9 and 4.2 kb species, and in certain developmental periods also 3.2, 7.2 and ⩾ 10 kb products. Two different transcripts, one dominant (3.4 kb) and one rare (5.2 kb) were revealed with the MG-2 probe. MG-1 and MG-2 cDNAs contain somewhat similar repeats, and their deduced translation products resemble Bombyx sericins by the dominance of serine and the high content of glycine and asparagine. Southern blots indicated that MG-1 and MG-2 represent different sericin genes apparently corresponding to Ser-1 and Ser-2 genes of Bombyx .