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. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/15805 |
| Date | January 2015 |
| Creators | Voua Otomo, Laetitia |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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