Michael B. Blackburn

The identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta.

Two new myoinhibitory peptides, Mas-MIP I and Mas-MIP II, were identified from the ventral nerve cord of the adult tobacco hornworm, Manduca sexta. Sequences obtained by a combination of automated Edman degradation and electrospray mass spectrometry were, respectively, AWQDLNSAW and GWQDLNSAW. The native peptides were found to co-elute with synthetic C-terminal amides on a reverse phase HPLC system. When applied to isolated ilea (anterior hindgut) of adult M. sexta, both peptides were found to significantly reduce the rate of peristalsis, or abolish peristalsis entirely, at concentrations of 1 x 10(-9) M. Both peptides share sequence similarities with Lom-MIP, a previously identified myoinhibitory peptide from Locusta migratoria, and with the N-terminal portion of vertebrate peptides in the galanin family.

Photobactin: a Catechol Siderophore Produced by Photorhabdus luminescens, an Entomopathogen Mutually Associated with Heterorhabditis bacteriophora NC1 Nematodes

ABSTRACT The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.

THE ISOLATION AND IDENTIFICATION OF THREE DIURETIC KININS FROM THE ABDOMINAL VENTRAL NERVE CORD OF ADULT HELICOVERPA ZEA

Three novel diuretic peptides have been isolated and identified from the abdominal ventral nerve cord of the adult corn earworm, Helicoverpa zea. The peptides belong to the kinin family of myotropic and diuretic peptides and have the following sequences: YFSPWGamide, VRFSPWGamide, and KVKFSAWGamide. These peptides, which we refer to as the helicokinins, are potent stimulators of secretion by the Malpighian tubules of Manduca sexta. All three of the helicokinins stimulated secretion by M. sexta tubules at concentrations below 10−11 M. The helicokinins are the first kinins to be identified from a lepidopteran, and the first diuretic peptides to be identified specifically from the abdominal ventral nerve cord of any insect.

Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis

SUMMARY For normal development of Manduca sexta larvae, the ecdysteroid titer must drop following its sudden rise at the start of the molting cycle; this sudden decline in titer may be due to myoinhibitory peptide I (MIP I), which has an inhibitory effect on the release of ecdysone by the prothoracic glands of Bombyx mori in vitro. Using an antiserum to MIP, we have demonstrated secretion of an MIP-like peptide by the epiproctodeal glands of Manduca sexta, which are located on each proctodeal nerve, just anterior to the rectum. These MIP-immunoreactive glands are also present in B. mori. In fourth-instar larvae of M. sexta, the epiproctodeal glands show a distinct cycle of synthesis and sudden release of MIP that coincides with the time of the rapid decline in ecdysteroid titer. The function of the epiproctodeal glands appears to be the timely release of MIP during the molting cycle, so as to inhibit the prothoracic glands and thus to facilitate the sudden decline in ecdysteroid titer. In addition, we have found that MIP immunoreactivity is co-localized with that of crustacean cardioactive peptide (CCAP) in the 704 interneurons; these peptides appear to be co-released at the time of ecdysis. It is known that CCAP can initiate the ecdysis motor program; our results suggest that MIP may also be involved in activating ecdysis behavior.

Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies ( Trialeurodes vaporariorum )

A system of markers has been devised to track the development of 3rd and 4th instar/pharate adult greenhouse whiteflies. Instars were identified based on measurements of body width and body length. Depending upon the host plant, the product of the two measurements was exceptionally useful in distinguishing between instars. Body depth was used to divide the 3rd instar into eight stages and body depth and color and appearance of the developing adult eye were used to divide the 4th instar/pharate adult into nine stages. Under conditions of L:D 16:8 and a temperature of 26+/-2 degrees C, the body depth of 3rd instars reared on greenbean increased from 0.025 (stage 1) to 0.2mm (stage 8) and the instar duration was approximately 3 days. The body depth of 4th instars increased from approximately 0.1+/-0.02 (Stage 1) to 0.3+/-0.03mm (Stage 5) and then remained constant or decreased slightly during adult development. Ecdysteroid titers peaked at approximately 120fg/&mgr;g protein during Stages 3 through 6 of the 4th instar. Based on an external examination of developing 4th instars and the fluctuations in ecdysteroid titer, it appears that adult development is initiated in Stage 4 or 5 4th instars. Results from histological studies support this view. In Stage 4 nymphs, a subtle change was observed in the corneagenous cells of the eye. However, most Stage 4 4th instars possessed wing development characteristic of earlier, immature stages. In all Stage 5 insects, wing development had been initiated and the corneagenous cells had become quite distinct. In Stage 6 whiteflies, the wing buds were deeply folded and by Stage 7, spines were observed on the new cuticle, indicating that the adult cuticle was well-formed by this stage. Our study is the first to investigate the timing and regulation of the molt, to monitor ecdysteroid titers in precisely staged 4th instar whiteflies and to examine the internal anatomical changes associated with metamorphosis in these tiny homopteran insects.

Transcriptome of the Lymantria dispar (Gypsy Moth) Larval Midgut in Response to Infection by Bacillus thuringiensis

Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest.

Stimulation of Midgut Stem Cell Proliferation and Differentiation by Insect Hormones and Peptides

Abstract: Stem cells derived from midguts of the caterpillar, Spodoptera littoralis, can be induced to multiply and differentiate in vitro. Ecdysone (E) and 20‐hydroxyecdysone (20E) had a concentration‐dependent effect: E was more active in cell proliferation and 20E in differentiation. Ecdysteroid receptors in midgut stem cell nuclei were stained with the antibody 9B9. In addition, α‐arylphorin and four midgut differentiation factors (MDF) specifically stimulated proliferation and differentiation of stem cells, respectively. The activity of a panel of peptide growth factors and hormones on growth and metamorphosis of the insect midgut is discussed.

The distribution of PBAN (pheromone biosynthesis activating neuropeptide)-like immunoreactivity in the nervous system of the gypsy moth, Lymantria dispar

The production of sex pheromone in many moths is regulated by the neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). Studies in a number of species have shown that pheromone production can be linked to a hemolymph factor and that continuity in the ventral chain of ganglia is not required. However, it has recently been shown that production of pheromone in the gypsy moth, Lymantria dispar, is largely prevented in females with a transected ventral nerve cord (VNC). To begin to understand the cellular basis for this dependence on the VNC, we sought to determine the distribution of PBAN in the central nervous system and its neurohemal sites, including those associated with the VNC. Using an antiserum to L. dispar-PBAN in immunocytochemical methods, we have mapped the distribution of PBAN-like immunoreactivity (PLI). PLI is found in three clusters of ventral midline somata in the subesophageal ganglion (SEG), in three clusters of midline cells in each segmental ganglion, and in bilateral pairs of cells located posterolaterally in each abdominal ganglion. The SEG cells comprise both interneurons, with endings in the neuropil of each segmental ganglion, as well as neurosecretory cells, with endings in the retrocerebral complex and in an unusual neurohemal structure near the anterior aspect of the SEG. The latter structure, which we have named the corpus ventralis, receives axons from the two anterior clusters of cells in the SEG. In the abdominal ganglia, the posterolateral clusters of cells have immunoretroreactive axons exiting the ganglia via the ventral nerves. Endings of these axons reach the perivisceral organ in the next posterior ganglion and pass anteriorly into the median nerve, forming additional varicose endings. We did not detect PLI in the terminal nerve. Thus, our findings raise the possibility that the requirement for an intact VNC in pheromone production reflects a role for descending regulation of neurosecretory cells in the segmental ganglia. Arch. Insect Biochem. Physiol. 34:391–408, 1997. Published 1997 Wiley-Liss, Inc.1

Two New Bacterial Pathogens of Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Abstract Other than Bacillus thuringiensis Berliner, few bacteria are lethal to the Colorado potato beetle (Leptinotarsa decemlineata [Say]), a major pest of potatoes and eggplant. Expanded use of biologicals for the control of Colorado potato beetle will improve resistance management, reduce pesticide use, and produce novel compounds for potential use in transgenic plants. Using freeze-dried, rehydrated artificial diet in pellet form to screen bacteria lethal to other insects, we determined that strains of Photorhabdus luminescens killed Colorado potato beetle larvae. The LC50 for second instar larvae of strain HM5-1 was 6.4 ± 1.87 × 107 cells per diet pellet. In an attempt to find additional naturally occurring P. luminescens strains toxic to Colorado potato beetle larvae, we recovered, from soil, bacteria that produced a purple pigment. This bacterial strain, identified as Chromobacterium sp. by 16S ribosomal DNA sequencing, was also toxic to Colorado potato beetle larvae within 3 d. The LC50 for second instar larvae for these bacteria was 2.0 ± 0.79 × 108 cells per diet pellet, while the LC50 was approximately 1 log lower for third instar larvae. P. luminescens appeared to kill by means of a protein toxin that may be similar to the described lepidopteran protein toxins. Based on the heat and acid stability, the toxin or toxins that Chromobacterium sp. produces, while not fully characterized, do not appear to be typical proteins. In both bacteria, the toxins are made after exponential growth ceases.

Identification of a coffee berry borer-associated yeast: does it break down caffeine?

Two yeasts isolated from laboratory reared adult coffee berry borers [Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae)] and from insects collected in the field in Colombia were identified as Pichia burtonii Boidin and Candida fermentati (Saito) Bai, based on sequencing of the nuclear large subunit 26S rDNA variable D1/D2 domain. Liquid culture experiments using P. burtonii in media containing different caffeine levels indicated that caffeine levels in a range found within coffee seeds can retard yeast growth. HPLC analysis shows that P. burtonii does not break down caffeine.