Lertluk Ngernsiri

Cloning and expression analysis of the Bombyx mori α-amylase gene (Amy) from the indigenous Thai silkworm strain, Nanglai

Abstract &agr;-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), &agr;-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding &agr;-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect &agr;-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut.

Analysis of the Vitellogenin gene of rice moth, Corcyra cephalonica Stainton.

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721-bp long and contained 5five introns and six exons that together formed a 5,382-bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16-amino-acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C-terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed that CceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, the CceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, the CceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection of CceVg double-stranded RNA into early-emergent females caused severely abnormal ovaries.

Meiotic Chromosome Analysis of the Giant Water Bug, Lethocerus indicus

Abstract The giant water bug, Lethocerus indicus (Lepeletier and Serville) (Heteroptera: Belostomatidae), a native species of Southeast Asia, is one of the largest insects belonging to suborder Heteroptera. In this study, the meiotic chromosome of L. indicus was studied in insect samples collected from Thailand, Myanmar, Loas, and Cambodia. Testicular cells stained with lacto-acetic orcein, Giemsa, DAPI, and silver nitrate were analyzed. The results revealed that the chromosome complement of L. indicus was 2n = 22A + neo-XY + 2m, which differed from that of previous reports. Each individual male contained testicular cells with three univalent patterns. The frequency of cells containing neo-XY chromosome univalent (∼5%) was a bit higher than that of cells with autosomal univalents (∼3%). Some cells (∼0.5%) had both sex chromosome univalents and a pair of autosomal univalents. None of the m-chromosome univalents were observed during prophase I. In addition, this report presents clear evidence about the existence of m-chromosomes in Belostomatidae.

Identification of Puffer Fish of the Genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, Using Multiplex PCR

A multiplex PCR assay was developed to identify widespread poison-containing puffer fishes of the genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, using a novel KUGEN_ILSspec primer set which is specific for 12S ribosomal RNA (12S rRNA) and Cytochrome b (Cyt b) genes. KUGEN_ILSspec set is composed of fish positive-amplified primers that produced a 289-bp fragment while L. lunaris-, L. spadiceus- and L. inermis-specific primers produced 123-, 196- and 493-bp fragments, respectively. The sensitivity of this primer set was found to be high as it was capable of amplifying targeted DNA at concentrations as low as 1 ng/μL. Moreover, the primer set was shown to amplify intact DNA and species-specific fragments from heat-processed food. Consequently, multiplex PCR technique in combination with KUGEN_ILSspec primer set could offer an effective measure for detecting poisonous puffer fish contamination in both fresh and processed fish products, which will greatly aid in public health control and law enforcing endeavors.

The transformer2 gene of the pumpkin fruit fly, Bactrocera tau (Walker), functions in sex determination, male fertility and testis development

The insect transformer2 (tra2) gene has a prevalent role in cooperating with the sex‐determining gene transformer (tra) to direct female differentiation. Here, we report the identification and characterization of Btau‐tra2, the tra2 orthologue of the pumpkin fruit fly, Bactrocera tau, an invasive agricultural pest. The Btau‐tra2 gene produces three transcript variants. However, only two transcripts can be examined; one is present at all developmental stages in the soma and germline of both sexes and the other one is specific to the embryo and the germline. Knocking down the function of Btau‐tra2 produced a male‐biased sex ratio and some intersexes. Consistent with a role in sex determination, the obtained intersexual and male sterility phenotypes express a mix of male and female splice variants of the tra and doublesex (dsx) orthologues, indicating that Btau‐tra2 has a conserved splicing regulatory function and acts together with/upstream of tra and dsx. In addition, some males obtained from the knock down are fertile but their fertilities are extremely reduced. Moreover, almost all surviving RNA interference (RNAi) males harbour testes having some defects in their external morphologies. Most notably, the body size of a few surviving RNAi flies was two‐to threefold increased with respect to the normal size. Our findings suggest that Btau‐tra2 is involved in male fertility and may also have an unprecedented role in body size control besides its conserved role in sex determination.

ten‐a overexpression causes abnormal pattern in the Drosophila compound eye

Abstract Ten‐a is one of the two Drosophila proteins that belong to the Ten M protein family. This protein is a type II transmembrane protein and is expressed mainly in the embryonic CNS, in the larval eye imaginal disc and in the compound eye of the pupa. Here, we investigate the role of ten‐a during development of the compound eye by using the Gal4/UAS system to induce ten‐a overexpression in the developing eye. We found that overexpression of ten‐a can perturb eye development during all stages examined. In an early stage, overexpression of ten‐a in eye primordial cells caused small and rough eyes and interfered with photoreceptor cell recruitment, resulting in some ommatidia having fewer or extra photoreceptor cells. Conversely, ten‐a overexpression during ommatidial formation caused severe eye defects due to absence of many cellular components. Interestingly, overexpression of ten‐a in the late stage developing ommatidial cluster affected the number of pigment cells, caused cone cells proliferation in many ommatidia, and caused some photoreceptor cell defects. These results suggest that ten‐a may be a novel gene required for normal eye morphogenesis.

Segmentation gene expression patterns in Bactrocera dorsalis and related insects: regulation and shape of blastoderm and larval cuticle

The oriental fruit fly, Bactrocera dorsalis, is regarded as a severe pest of fruit production in Asia. Despite its economic importance, only limited information regarding the molecular and developmental biology of this insect is known to date. We provide a detailed analysis of B. dorsalis embryology, as well as the expression patterns of a number of segmentation genes known to act during patterning of Drosophila and compare these to the patterns of other insect families. An anterior shift of the expression of gap genes was detected when compared to Drosophila. This shift was largely restored during the step where the gap genes control expression of the pair-rule genes. We analyzed and compared the shapes of the embryos of insects of different families, B. dorsalis and the blow fly Lucilia sericata with that of the well-characterized Drosophila melanogaster. We found distinct shapes as well as differences in the ratios of the length of the anterior-posterior axis and the dorsal-ventral axis. These features were integrated into a profile of how the expression patterns of the gap gene Krüppel and the pair-rule gene even-skipped were observed along the A-P axis in three insects families. Since significant differences were observed, we discuss how Krüppel controls the even-skipped stripes. Furthermore, we discuss how the position and angles of the segmentation gene stripes differed from other insects. Finally, we analyzed the outcome of the expression patterns of the late acting segment polarity genes in relation to the anlagen of the naked-cuticle and denticle belt area of the B. dorsalis larva.