Hugh D. Loxdale

Molecular markers in entomology

A diverse range of novel molecular (DNA) markers are now available for entomological investigations. Both DNA and protein markers have revolutionized the biological sciences and have enhanced many fields of study, especially ecology. Relative to DNA markers, allozymes are cheap, often much quicker to isolate and develop, even from minute insects (aphids, thrips, parasitic wasps, etc.), and subsequently easy to use. They display single or multi-locus banding patterns of a generally easily interpretable Mendelian nature, and the statistics for their analysis are well established. DNA markers are also suitable for use with small amounts of insect material and can be used with stored, dry or old samples. They have an expanding range of applications, many involving intra- and interspecific discriminations. Like allozymes, they can be single or multilocus, whilst methods for their statistical analysis have recently been published. However, they can be considerably more expensive than allozymes, require more complex preparatory protocols, expensive equipment, may involve lengthy development procedures (e.g. isolating cloned oligonucleotides to develop primers to detect microsatellite regions) and some have complex multi-locus banding patterns which may be of a non-Mendelian nature (e.g. RAPDs, randomly amplified polymorphic DNA), and are in some cases, not easily repeatable. In this review, we hope to inform the general reader about the methodology and scope of the main molecular markers commonly in use, along with brief details of some other techniques which show great promise for entomological studies. Thereafter, we discuss their applications including suitability for particular studies, the methods used to load and run samples, subsequent band detection, band scoring and interpretation, the reliability of particular techniques, the issues of safety involved, cost effectiveness and the statistical analyses utilized.

THE RELATIVE IMPORTANCE OF SHORT- AND LONG-RANGE MOVEMENT OF FLYING APHIDS

1. Aphids are notorious pests of world agriculture. Even so, uncertainty persists as to their capacity for successful aerial dispersal. Evidence exists that, under some conditions, aphids can be wind‐borne over long distances, i.e. hundreds of kilometers over desert or sea. It has been argued, in the recent past, that this phenomenon may be part of a strategy to locate fresh host plants in new distant areas. However, the proportion of these insects successfully colonizing new hosts is unknown.

Behavioural manipulation in a grasshopper harbouring hairworm: a proteomics approach.

Abstract The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insects responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshoppers central nervous system (CNS) by producing certain ‘effective’ molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the hosts CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.

The nature and reality of the aphid clone: genetic variation, adaptation and evolution

1 When aphid clones and clonality are discussed, it is still often said that they are ‘genetically identical’, a statement for which there is no direct evidence, and certainly not for the entire genome. By contrast, there is a growing body of empirical data from the application of high resolution molecular (DNA) markers that aphid asexual lineages rapidly mutate and that, in some documented cases, this variation is selectable, either positively or negatively.

Do distantly related parasites rely on the same proximate factors to alter the behaviour of their hosts

Phylogenetically unrelated parasites often increase the chances of their transmission by inducing similar phenotypic changes in their hosts. However, it is not known whether these convergent strategies rely on the same biochemical precursors. In this paper, we explored such aspects by studying two gammarid species (Gammarus insensibilis and Gammarus pulex; Crustacea: Amphipoda: Gammaridae) serving as intermediate hosts in the life cycle of two distantly related parasites: the trematode, Microphallus papillorobustus and the acanthocephalan, Polymorphus minutus. Both these parasite species are known to manipulate the behaviour of their amphipod hosts, bringing them towards the water surface, where they are preferentially eaten by aquatic birds (definitive hosts). By studying and comparing the brains of infected G. insensibilis and G. pulex with proteomics tools, we have elucidated some of the proximate causes involved in the parasite-induced alterations of host behaviour for each system. Protein identifications suggest that altered physiological compartments in hosts can be similar (e.g. immunoneural connexions) or different (e.g. vision process), and hence specific to the host–parasite association considered. Moreover, proteins required to alter the same physiological compartment can be specific or conversely common in both systems, illustrating in the latter case a molecular convergence in the proximate mechanisms of manipulation.

Genotypic variation among different phenotypes within aphid clones

Most aphid species (Hemiptera : Aphididae) are parthenogenetic between periods of sexual reproduction. They are also highly polyphenic, with different adult morphs occurring in the life cycle, viz. winged, wingless, asexual and sexual. It is assumed that aphids born in a parthenogenetic clonal lineage are genetically identical regardless of the final adult form (with the exception of sexual forms). Using the randomly amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR) we have found that different asexual adult phenotypes (winged and wingless) of some clones of two cereal aphid species (the grain aphid,Sitobion avenae (F.) and the bird–cherry aphid, Rhopalosiphum padi (L.)) may be distinguished by the presence or absence of one or more RAPD–PCR bands. In three of nine clones examined, such differences were found, and Southern blotting and hybridization of the discriminating bands confirmed these to be of aphid origin, rather than due to endosymbiotic bacteria or contaminating fungi. The main 248 and 296 bp bands, in the two species, respectively, were sequenced and found to be A/T rich. The smaller band showed 57 per cent homology with white striated muscle over a stretch of 90 bp. Genomic DNA treated with dimethyl sulphoxide to remove secondary structures still showed differences in RAPD–PCR profiles between winged and wingless morphs within the unusual clones. This discovery may be widespread and therefore it is important to understand the phenomenon in relation to clonal organisms.

Host–parasite molecular cross-talk during the manipulative process of a host by its parasite

Summary Many parasite taxa are able to alter a wide range of phenotypic traits of their hosts in ways that seem to improve the parasite’s chance of completing its life cycle. Host behavioural alterations are classically seen as compelling illustrations of the ‘extended phenotype’ concept, which suggests that parasite genes have phenotype effects on the host. The molecular mechanisms and the host–parasite cross-talk involved during the manipulative process of a host by its parasite are still poorly understood. In this Review, the current knowledge on proximate mechanisms related to the ‘parasite manipulation hypothesis’ is presented. Parasite genome sequences do not themselves provide a full explanation of parasite biology nor of the molecular cross-talk involved in host–parasite associations. Recently, first-generation proteomics tools have been employed to unravel some aspects of the parasite manipulation process (i.e. proximate mechanisms and evolutionary convergence) using certain model arthropod-host–parasite associations. The pioneer proteomics results obtained on the manipulative process are here highlighted, along with the many gaps in our knowledge. Candidate genes and biochemical pathways potentially involved in the parasite manipulation are presented. Finally, taking into account the environmental factors, we suggest new avenues and approaches to further explore and understand the proximate mechanisms used by parasite species to alter phenotypic traits of their hosts.

Rapid genetic changes in natural insect populations

1. The insects represent around 75% of the worlds fauna and as such provide especially good examples of the evolutionary process in action, aided by their often rapid generation time and high rate of reproduction.

Genetic variability within and between English populations of the damson–hop aphid, Phorodon humuli (Hemiptera: Aphididae), with special reference to esterases associated with insecticide resistance

The damson‐hop aphid, Phorodon humuli (Schrank), is a serious pest of hops in England. It is holocyclic (with obligatory sexual phase) and host alternating. From suction trap data, P. humuli aerial densities are known to be greatest in the main hop growing regions of Herefordshire and Kent (mid-west and south-east England, respectively), some 260 km apart. The aphid is now resistant to several insecticides. This is in part conferred by elevated carboxylesterase activity, ranging from low in susceptible to high in very resistant strains. Enzyme markers, including carboxylesterases (EST-4 to -7), separated electrophoretically from individual insects, have been used to examine the degree of genetic heterogeneity among P. humuli sub-populations on both its hosts ‐ Prunus spp. (primary overwintering host) and hops, Humulus lupulus (secondary summer host). The esterase data revealed heterogeneity among subpopulations collected from wild, unsprayed hosts in regions less than 30 km in area, with a higher mean frequency of elevated esterase variants in the commercial hop growing regions of Herefordshire and Kent, compared with samples from a non-commercial region around Rothamsted. Esterase distributions remained similar over consecutive years. Similarly, allele and genotype frequencies for another enzyme (6-phosphogluconate dehydrogenase, 6-PGD) were also heterogeneous among subpopulations sampled at less than 30 km apart (especially from Prunus) in each of the three regions surveyed, whilst allele and genotype frequencies sometimes remained stable over a number of summers. In addition, 6-PGD genotype frequencies were mostly congruent with Hardy-Weinberg expectations, even for parthenogenetically-reproducing aphids colonizing hops. These data suggest that the 6-PGD alleles tested are selectively neutral; that gene flow (= migration) is restricted between aphid populations, even within a single region ( 30 km) and, that the autumn migration from hops to Prunus is probably of shorter range (perhaps less than 20 km) compared with the spring migration from Prunus to hops.

Tracking movement in small insect pests, with special reference to aphid populations

Insects are among the greatest pests of agriculture, horticulture and forestry world-wide, inflicting damage and economic costs both directly and by transmitting plant viruses. Many kinds of insects are now resistant or cross-resistant to pesticides. In order to combat these pests, including for purposes of immediate control or to follow movements in order to better understand pest biology, tracking studies are important. In turn, such studies become useful for modelling and forecasting outbreaks. This is especially so for flying insects, which constitute the greatest threat in terms of the dissemination of plant pathogenic viral diseases. Tracking the aerial displacement of small insects over large spatial scales is difficult mainly because of their size. Dilution effects upon take off, together with displacement by air currents, makes the recapture of individuals tagged e.g. by fluorescent or radio-labelling, highly unlikely and hence estimates of flight direction, speed, duration and distance are hampered, more especially over larger distances. This is generally true also for larger insects. Consequently, molecular markers have been employed in attempts to resolve population structure and dynamics by inference from estimates of gene flow. In this article, we describe some of the methodologies devised that have provided invaluable information relating to the movement of small insect pests, particularly aphids, and explain how these techniques can be added to by detail gained from molecular marker studies. We outline a national survey-system that could be managed by specialist centres and involving members of the Public, that would enable invasive pest insect populations to be tracked efficiently. The logistics of such a survey are discussed along with the benefits. Overall, an insect tracking mandate is described that will promote the standardisation of population movement measurements. Adopting this approach should allow more accurate intra- as well as interspecies comparisons.