DNA barcoding of different earthworms' species and their response to ecotoxicological testing
Voua Otomo, Laetitia
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The ecotoxicological literature reveals that countless researchers worldwide rely upon informally identified commercial earthworm stocks for laboratory bioassays. The primary aim of this study was to investigate laboratory and commercial stocks of Eisenia species used in South Africa in order to confirm their taxonomy, assess their levels of genetic richness and differentiation. To do so, populations of potential Eisenia andrei and Eisenia fetida were purchased/obtained from vermiculturists and laboratories from four provinces of South Africa. DNA barcoding was used to investigate these taxonomic uncertainties. The COI gene was partially amplified and sequenced in selected earthworms from eight local populations (focal groups) and two European laboratory stocks (non-focal groups). Only nine COI haplotypes were identified from the 224 sequences generated. One of these haplotypes was found to belong to the Megascolecidae Perionyx excavatus. The remaining eight haplotypes belonged to the genus Eisenia although only a single Eisenia fetida haplotype, represented by six specimens, was found in one of the European populations. The other seven haplotypes, all occurring in South Africa, were Eisenia andrei. No Eisenia fetida was found in the South African based populations. One of the commercial stocks from South Africa and a laboratory culture from Europe were mixes of E. andrei - P. excavatus and E. andrei – E. fetida respectively. COI haplotype numbers were limited to two to three distinct sequences within each of the local groups. This translated into a haplotype diversity (H) lower than 0.45 in all the populations, which is very low when compared to other such earthworm studies in which COI polymorphism has been investigated. Of all the local populations investigated, only the lone field population included was genetically divergent from the other populations. This was explained by the haplotype distribution across the populations which indicated that this population was the only one not harbouring the haplotype which represented 75% or more of the COI sequences within the local populations. Because research suggests that earthworm populations with limited genetic diversity may suffer inbreeding depression which could affect traits such as reproduction and survival, the secondary aim was to test whether metal-sensitive earthworms were overly present in the populations investigated. To do so, the three most common COI haplotypes identified between the 8 local populations of E. andrei (called Hap1, Hap2 and Hap3) were paired up and exposed to cadmium. A total of six couples were exposed to 0, 25, 50 and 100 mg Cd/kg for 4 weeks at 20ºC. The survival, biomass variation, cocoon production and cocoon hatching success were assessed for all the couples. The results indicated that couple 6 (Hap3xHap3) was the most sensitive for three of the endpoints assessed whereas couple 4 (Hap1 x Hap3) was the least sensitive. Cocoon hatching success could not help differentiate the couples. The analysis of Cd tissue contents revealed that with increasing Cd concentration, Cp6 (Hap3xHap3) could accumulate significantly more Cd than any other couple (p ≤ 0.01). These findings indicate that earthworm populations may carry intrinsically metal-tolerant and metal-sensitive genotypes. In the context of ecotoxicological testing, the present results underline the importance of using genetically diverse populations in laboratory testing as Cp6 (Hap3xHap3) could have suffered from the deleterious effect of inbreeding. Because E. fetida could not be found in the local populations assessed, it is recommended that further earthworm DNA barcoding studies, covering a more representative geographical area of South Africa and including more field populations of Eisenia spp. be conducted. Because of the occurrence of genetic homogeneity in the populations studied, it is suggested that captive breeding initiatives be established using specimens obtained from several geographically distant field and reared populations. Further research investigating patterns of Cd accumulation/excretion kinetics between the Cd-tolerant and Cd-sensitive individuals reported in the present study, should be conducted to help determine whether inbreeding is the sole factor explaining the observed genotypic responses to Cd. Finally, the necessity of a standardised earthworm barcoding protocol that could help both to properly identify laboratory earthworm stocks and to select genetically diverse stocks suitable for laboratory testing, is discussed together with the relevance of the present work to ecotoxicological testing in general.