Abstract
Mytilus species are used as sentinels for the assessment of environmental health but sex or stage in the reproduction cycle is rarely considered even though both parameters are likely to influence responses to pollution. We have validated the use of a qPCR assay for sex identification and related the levels of transcripts to the reproductive cycle. A temporal study of mantle of Mytilus edulis found transcripts of male-specific vitelline coat lysin (VCL) and female-specific vitelline envelope receptor for lysin (VERL) could identify sex over a complete year. The levels of VCL/VERL were proportional to the numbers of sperm/ova and are indicative of the stage of the reproductive cycle. Maximal levels of VCL and VERL were found in February 2009 declining to minima between July – August before increasing and re-attaining a peak in February 2010. Water temperature may influence these transitions since they coincide with minimal water temperature in February and maximal temperature in August. An identical pattern of variation was found for a cryptic female-specific transcript (H5) but a very different pattern was observed for oestrogen receptor 2 (ER2). ER2 varied in a sex-specific way with male > female for most of the cycle, with a female maxima in July and a male maxima in December. Using artificially spawned animals, the transcripts for VCL, VERL and H5 were shown to be present in gametes and thus their disappearance from mantle is indicative of spawning. VCL and VERL are present at equivalent levels in February and July–August but during gametogenesis (August to January) and spawning (March to June) VCL is present at lower relative amounts than VERL. This may indicate sex-specific control mechanisms for these processes and highlight a potential pressure point leading to reduced reproductive output if environmental factors cause asynchrony to gamete maturation or release.
Highlights
Common mussels (Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus) are sessile, intertidal, filter feeding, sedentary organisms that inhabit coastal areas over a wide geographical area
The relative expression of H5 in male was maximally 3% of matching female samples (May 2010) but more generally less than 0.1% (Table S2b) In the determinations of relative transcript levels, actin was used as the reference gene as it has been observed to be invariant in previous studies [24] and the same was found in the current study
Work has indicated the significance of gender in the metabolism of xenobiotics in digestive gland of mussel [21,22]
Summary
Common mussels (Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus) are sessile, intertidal, filter feeding, sedentary organisms that inhabit coastal areas over a wide geographical area. Their habitat and ecology result in the accumulation of contaminants within their tissues (pesticides, hydrocarbons, metals etc.) [1,2,3,4,5,6] and this can result in a ‘‘stress syndrome’’. In previous studies the gender of the mussels and the stage of the reproductive cycle, were not necessarily considered to be variables that would affect the outcome of the investigations. We have improved the approach by the introduction of a Quantitative, Real-time PCR assay and shown that the method is accurate and applicable through all of the times tested during the reproductive cycle [24]
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