Do earthworms help to sustain the slug predator Pterostichus melanarius (Coleoptera: carabidae) within crops? Investigations using monoclonal antibodies.

Earthworms provide a major potential source of alternative food for polyphagous predators, such as carabid beetles, that are natural enemies of slugs, aphids and other agricultural pests. Non-pest prey may foster larger numbers of natural enemies, which then help to control pests, or alternatively may help to divert the predators away from pest control. An earthworm-specific monoclonal antibody was developed to study carabid-earthworm interactions in the field and assess the role of earthworms as alternative prey. The antibody could identify as little at 7 ng of earthworm protein in an ELISA, and could detect earthworm remains in the foregut of the carabid beetle Pterostichus melanarius for 64 h after consumption. Thirty-six per cent of field-collected beetles contained earthworm remains. Quantities of earthworm proteins in the beetle foreguts were negatively related to total foregut biomass, suggesting that earthworm consumption increased as total prey availability declined. There was also a negative relationship between foregut biomass and beetle numbers, but both quantities and concentrations of earthworm proteins in beetle foreguts were positively related to beetle numbers. This suggests that as beetle activity-density increased, total prey availability declined, or, as prey availability declined, beetles spent more time searching. In these circumstances, beetles fed to a greater extent on earthworms, an acceptable but nonpreferred food item. Earthworms may, therefore, provide an ideal alternative prey for P. melanarius, helping to sustain it when pest numbers are low but allowing it to perform a 'lying-in-wait' strategy, ready to switch back to feeding on pests when they become available.

Amplified detection, using a monoclonal antibody, of an aphid-specific epitope exposed during digestion in the gut of a predator.

Monoclonal antibodies are invaluable tools for identifying and quantifying prey remains in the fore-guts of predators. However, they must be target-specific, detect an epitope that is well replicated within the prey (to enhance assay sensitivity) and, critically, recognise a site that can resist digestion. A monoclonal antibody is reported that proved to be aphid-specific and capable of detecting, and accurately identifying, as little as 16.5 ng of aphid protein within a heterologous mixture of invertebrate material. The antibody was selected by screening hybridoma lines for antibodies that bound with semi-digested aphid proteins. The antibody detected an epitope that was found, against expectation, to significantly increase in concentration with time (by approx. 50% over 6 h) in the gut of the carabid predator Pterostichus melanarius. The resultant extended antigen detection period and half-life, and the high specificity of this antibody, showed it to be an enhanced tool for studying interactions between aphids and their predators in the field. It was concluded that the antibody was initially generated to a surface epitope on a high molecular weight native protein (> 200 kD). This epitope, however, was then either replicated on internal sites progressively revealed by digestion, or new epitopes became available as the conformation of the protein changed during digestion.

Electroacoustic production of murine hybridomas.

The optimal conditions for the production of murine hybridomas by electroacoustic fusion of cells in sugar solutions and of cells in ionic strength media (up to 115 mM NaCl) have been investigated. In the electroacoustic fusion technique cells were brought into contact in a 3 MHz ultrasonic standing wave field and were fused by application of an electric pulse. Hybridomas of murine splenocytes and X63-Ag8.653 mouse myelomas were successfully produced in low ionic strength mannitol solutions and in a range of salt concentrations up to 115 mM. The yield of hybridomas by electroacoustic fusion of cells in mannitol was at least as good as that obtained when cells were fused following conventional dielectrophoresis induced contact. The electroacoustic fusion yield was also comparable to conventional yields when cells were exposed to a pulse in higher ionic strength media where dielectrophoresis induces heating effects. Hybridomas were produced at similar electric field strengths when cells were suspended in high ionic strength media (up to 115 mM NaCl) or in mannitol. The effective electric field strength for hybridoma production was close to that at which trypan blue tests indicated membrane damage.

Electroacoustic fusion of millilitre volumes of cells in physiological medium.

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.

Evidence for filaggrin as a component of the cell envelope of the newborn rat.

A substrate of transglutaminase, specific to the epidermis, was identified, by fluorescent and radioactive labelling with the lysine analogues dansylcadaverine and [14C]putrescine respectively, in newborn-rat epidermal homogenates and whole-skin organ cultures. The labelled analogues were preferentially incorporated into the stratum-corneum protein filaggrin in a Ca2+-dependent manner in both 'in vitro' systems. When filaggrin was labelled in vivo with [3H]histidine and then incubated with rat epidermal preparations, the label was rendered SDS/thiol-insoluble. Incorporation of [3H]filaggrin into the insoluble envelope fraction was Ca2+-dependent and inhibited by EDTA and exogenous amines. Antisera to newborn-rat filaggrin cross-reacted with purified newborn-rat cell envelopes, and this reaction was blocked by adsorbing the antiserum with purified filaggrin. Quantification of the 'envelope-bound' filaggrin showed it to be a significant component, accounting for approx. 10% of the cell-envelope protein.