Assessment of fitness for purpose of an insect bioassay using the desert locust (Schistocerca gregaria L.) for the detection of paralytic shellfish toxins in shellfish flesh.

We have developed a bioassay using 5th instar desert locusts (Schistocerca gregaria L.) for the detection of saxitoxin-the paralytic shellfish poison in shellfish flesh. The bioassay procedure is to inject 10 locusts with a shellfish extract, and assess their conditions at time points up to 2h post injection, looking for an endpoint of paralysis. From the proportion responding, the equivalent dose of pure saxitoxin could be estimated. Performance characteristics of the bioassay were assessed using shellfish samples spiked with saxitoxin, and we found the bioassay could detect and quantify toxin levels in the range of regulatory relevance. Relative toxicities of selected saxitoxin analogues differed from those reported in mammalian systems. Variation for repeatability conditions was acceptable but variation was higher under reproducibility conditions. This was related to (a) batches of insects from different suppliers, (b) different operators, and (c) different observers assessing the endpoint. We also noted adverse reactions with some shellfish species. These problems may be resolved by further refinement of the method and operator training, before formal validation. Nevertheless, we suggest the method potentially offers a simple, ethically acceptable, broad-specificity functional bioassay, which is desirable in any toxin-monitoring programme.

Spontaneous and evoked quantal neurotransmitter release at the neuromuscular junction of the larval housefly, Musca domestica.

The release of neurotransmitter was monitored at the neuromuscular junctions of larval housefly ventrolateral muscles 6a and 7a, using intracellular recording, and a loose patch clamp technique to isolate discrete release sites. Transmitter release occurred spontaneously and could also be evoked by neural stimuli. Spontaneous discharges consisted of events which were randomly distributed in time and of bursts of temporally ordered events. Evoked and spontaneous release occurred in a quantal manner. The quantal content of evoked excitatory postsynaptic currents (EPSCs) was dependent upon the extracellular calcium concentration, increasing with a 3.8 power dependency. The relationship between the quantal content of a response and extracellular calcium concentration was offset by the presence of magnesium in the bathing saline. The rates of decay of miniature EPSCs (mEPSCs) and EPSCs were also found to increase with extracellular calcium concentration, consistent with a non-diffusion limited block of the glutamate receptor-channel complex by calcium ions (KB 2.5 x 10(4) s-1 M-1, P less than 0.01). The frequency of random mEPSCs (0.26 +/- 0.32 Hz, n = 24 cells) was independent of the extracellular calcium concentration. Random mEPSCs were not inhibited by 1 microM tetrodotoxin which blocked mEPSC bursts and neurally evoked responses. EPSCs evoked during mEPSC bursts had a significantly lower quantal content than those EPSCs recorded from the same nerve terminal between bursting, indicating that both of these forms of release recruited quanta from a common pool of transmitter. Following a neurally evoked EPSC the mEPSC frequency was potentiated severalfold, this delayed release was influenced by EPSCs with large quantal contents evoked in saline containing elevated calcium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Octopamine and proctolin mimic spontaneous membrane depolarisations in Lucilia larvae.

Octopamine and proctolin at concentrations below 10(-8) M reversibly induce a spontaneous rhythmic depolarization which occurs in body-wall muscles of Lucilia larvae. The effect appears to be postsynaptic and mediated by receptors specific for each substance.