Proleg retractor muscles in Manduca sexta larvae are segmentally different suggesting anteroposterior specialization

Manduca sexta larvae are an important model system for studying the neuromechanics of soft body locomotion. They climb on plants using the abdominal prolegs to grip and maneuver in any orientation and on different surfaces. The prolegs grip passively with an array of cuticular hooks and grip release is actively controlled by retractor muscles inserted into the soft planta membrane at the proleg tip. Until now, the principal planta retractor muscles (PPRMs) in each body segment were thought to be a single fiber bundle originating on the lateral body wall. Here, using high resolution X-ray microtomography of intact animals, we show that PPRM is a more complex muscle consisting of multiple contractile fibers originating at several distinct sites on the proleg. Furthermore, we show that there are segmental differences in the number and size of some of these fiber groups which suggests that the prolegs may operate differently along the anterior-posterior axis.

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS’s role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the UDP-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites’ defensive function and solves the autotoxicity problem of this potent chemical defense.

A collection of Serratia marcescens differing in their insect pathogenicity towards Manduca sexta larvae

We investigated the ability of Serratia marcescens to kill Manduca sexta (tobacco/tomato hornworm) larvae following injection of ca. 5 × 105 bacteria into the insect hemolymph. Fifteen bacterial strains were examined, including 12 non-pigmented clinical isolates from humans. They fell into 6 groups depending on the timing and rate at which they caused larval death. Relative insect toxicity was not correlated with pigmentation, colony morphology, biotype, motility, capsule formation, iron availability, surfactant production, swarming ability, antibiotic resistance, bacteriophage susceptibility, salt tolerance, nitrogen utilitization patterns, or the production of 4 exoenzymes: proteases, DNase, lipase, or phospholipase. There were marked differences in chitinase production, the types of homoserine lactone (HSL) quorum sensing molecules produced, and the blood agar hemolysis patterns observed. However, none of these differences correlated with the six insect larval virulence groups. Thus, the actual offensive or defensive virulence factors possessed by these strains remain unidentified. The availability of this set of S. marcescens strains, covering the full range from highly virulent to non-virulent, should permit future genomic comparisons to identify the precise mechanisms of larval toxicity.