Stuart E. Reynolds

An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA− mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA− mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA− mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.

Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387 000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant‐derived nicotine. New lepidopteran gene families such as the β‐fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte‐mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP‐labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut‐active Toxin complex A (Tca) and an RTX‐like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.

The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta

Three out of 4 isolates of the Deuteromycete Metarhizium anisopliae were pathogenic for larvae of the tobacco hornworm, Manduca sexta. The most virulent isolate (ME1) grew sparsely in the insect prior to death and caused paralysis of its host. The other 2 pathogenic isolates killed Manduca larvae more slowly, grew profusely in the haemolymph and did not induce symptoms of toxicosis. Toxicosis is apparently due to the production by the fungus of several cyclodepsipeptide toxins, destruxins (DTX). ME1 produced large quantities of DTX in vitro, while other isolates produced less. Destruxin A (DTX A) was recovered from the haemolymph of paralysed, diseased insects infected with ME1, but not with other isolates. It is suggested that DTX may have a ‘pathogenic role’, when the toxins are active in causing disease, or an ‘aggressive role’, when they facilitate the establishment of the pathogen.

Integration of Behaviour and Physiology in Ecdysis

Publisher Summary This chapter presents a review that aims to bring together what is known of the physiology of ecdysis with what is known of the behavior of ecdysing insects and shows how one is integrated with the other. Ecdysial behavior is probably largely programmed within the CNS, but once initiated the details of its performance may be more or less influenced by sensory feedback according to the species, and which part of the behavior is being considered. The exceptional stereotypy of the abdominal movements involved in the preeclosion and eclosion behavior of H. cecropia and the confinement of eclosion to a narrow, temporal “gate” by a circadian clock in the brain are key factors that led to the discovery of the role of the eclosion hormone in initiating eclosion behavior. The chapter provides evidence that silkmoth eclosion behavior is initiated directly by eclosion hormone. H. cecropia and A. pernyi emerge from the pupal cuticle only at a particular time of day, during relatively narrow gates determined by a circadian clock. If the pharate adults are removed from their pupal cases during the few hours before the normal eclosion gate, then they continue to show predominantly pupal behavior. In the case of A .pernyi , this is particularly striking, the peeled moth showing very little spontaneous motor activity at all. However, at the time of the normal eclosion gate, the moths go through a pantomime emergence, despite having been free of the pupal case for several hours.

Food and water economy and its relation to growth in fifth-instar larvae of the tobacco hornworm, Manduca sexta

Abstract Fifth-instar larvae of Manduca sexta were reared on artificial diet at 25°C. Daily measurement of the wet and dry weights of insects, food eaten and faeces produced, allowed the construction of detailed budgets for food and water economy. Approximate digestibility (AD) was about 60% both in dry weight and energetic terms. This did not change significantly on successive days of the instar, despite the very considerable changes that occur in the size of the insect and the amount of food eaten during the same period. Two indices of conversion efficiency (ECI and ECD) also showed only insignificant changes during this time. ECI and ECD were both very high: at their maximal values, ECI was 43% (51% in energy terms) and ECD was 72% (81% in energy terms). Correction of the nutritional indices for the presence of food in the gut reduced their value slightly in all cases. Most of the water requirements of the larva were provided by the diet. The estimated gain from metabolic water was between 3.5 and 10% of the total water input. Faeces accounted for most of the water lost from the body; under our conditions transpirational losses were very small. Almost half of the water taken up with the food was retained in the body. The gut contents are more hydrated than the food. The faeces contain less water than the gut contents. It is suggested that the caterpillars recycle water by absorbing it from the faeces and returning it to the contents of the midgut. A quantitative model of such water movement within the body is presented. The data provide indirect evidence that caterpillars may optimise the retention time of food in the gut (and thus the AD) in order to maximise the absolute rate of nutrient uptake from it.

Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica

RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi.

Food intake, conversion efficiency, and feeding behaviour of tobacco hornworm caterpillars given artificial diet of varying nutrient and water content

ABSTRACT Fifth stadium tobacco hornworm caterpillars, Manduca sexta (L.), given artificial diet diluted to varying extents with either cellulose or water compensated for the foods reduced nutrient content by eating more of it. This compensation was, however, in most cases not sufficient to maintain normal growth rates. When the water content of the diet was reduced, the insects ate less than the usual fresh weight of food but maintained their intake of nutrients. Nevertheless, growth rate was impaired. The insects were better able to compensate for dilution of their food with water than with cellulose.

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.

The feeding behaviour of caterpillars (Manduca sexta) on tobacco and on artificial diet

ABSTRACT. Feeding behaviour of fifth instar tobacco hornworm caterpillars, Manduca sexta (Johansen) (Lepidoptera; Sphingidae), eating tobacco or artificial diet, is quantitatively described. The insects grow at the same rate on both foods. There is no daily rhythm of feeding behaviour. For most insects, feeding on either food occurs in bouts with the lengths of interfeed gaps and of feeding bouts appearing to be distributed randomly. However, in many insects there is a strong correlation between the length of a feeding period and that of the preceding non‐feeding period.