Evaluation of Prezygotic Barrier Using Laboratory Crosses Between Three Species of Blue Mussels of The Genus Mytilus, Present Along the Chilean Coast

Marine mussels represent an important component of the
intertidal and subtidal communities and estuaries, in terms of
the number of individuals and of biomass and production and are
also of considerable economic importance to aquaculture in many
regions of the world...


Introduction
Marine mussels represent an important component of the intertidal and subtidal communities and estuaries, in terms of the number of individuals and of biomass and production and are also of considerable economic importance to aquaculture in many regions of the world [1,2]. The genus Mytilus is composed of at  galloprovincialis in Tongoy Bay [4], central [5] and in southern coast of Chile [6]. The presence of M. edulis has been described in DOI: 10.26717/BJSTR.2021.34.005505 the inner coast of the Chiloé Island [7] and in the Straits of Magellan.
Unfortunately, among some of the main features of these Mytilus species is the well-known capability to naturally hybridize among them in locations where their distributions overlap [8,9], therefore, these alien mussel species could have also the potential for natural hybridization with the native mussel M. chilensis.
In fact, there is clear evidence for a high fitness of hybrids mussels resulting in the F1 from the interbreeding between M. chilensis and M. galloprovincialis, produced under controlled laboratory conditions [10]. M. galloprovincialis is well known to be a very drastic invasive species and is recognized as one of the 100 worst invasive species in the world [11,12]. Therefore, to understand the ecological and evolutionary processes that promote the invasion is of fundamental importance to elucidate the fecundity success and/or the presence of prezygotic barriers between crosses under laboratory controlled factorial designs.
A factorial design was carried out to permit full reciprocal crosses.
Fifty-five mussels from each species were exposed to air for about 1h, then placed in filtered seawater at ambient temperature, one mussel per beaker, for induction of spawning using thermal shock.
Eggs and sperm were held for about 45m at 18 o C before use, to allow as many mussels as possible to spawn, thereby helping to synchronize the egg stage [13]. Eggs from each female were rinsed with filtered (1mm) UV-irradiated seawater (FSW) and then resuspended in 2000mL FSW at 18 o C for fertilization. The spermegg ratio used for fertilization was approximately 100:1 in all trials. Eggs from each cross and each pure line were rinsed after 10m to remove excess sperm and resuspended in two 3L glass flasks (replicates) containing 2000 mL at a density 90-120 eggs mL [6].
The larval cultures were then maintained at 17 o C and after 30 and 60 minutes, three samples of 15mL were taken from each culture and fixed with 95% ethanol. The fixed sample was later examined in order to determine the percentage of eggs which had developed into a embryo. Three samples from each replicate flask were used to count the numbers of embryos larvae within one field of view (100 magnifcation). Evaluations of fecundity (three for each cross) were carried out using a Petroff-Hausser counting chamber. The spawned mussels were typed to confirm the especies indentification, using two PCR-based markers: Me15/16 [14] and COIXba [15]. The fecundity percentage in each cross, were analyzed using first the Cochran's Q test for heterogeneity and then general linear models were applied.

Fertilization occurred in all crosses and fecundation (cleavage)
was observed within 25 to 60 m following mixing of eggs and sperm.
Between 72 and 100% of fecundation at 30 m and 90% to 100% at 60m were detected. Nested ANOVAs at 30 m and 60m showed significant differences (P<0.05) among all crosses in the percentage of fecundity, however not significant differences between replicates were found. Similar statistical analysis showed no significant differences (P>0.05) between pure crosses (same species) and hybrids crosses (between species) with regard to the percentage of eggs developed into embryos (Table 1). A few abnormal larvae (less than 1%) were detected by direct observation in the hybrid crosses, but the proportion of normal embryos during the early stages of growth showed no significant differences between pure and hybrid crosses.

Discussion
The results of this study provide evidence that there are no differences in the percentage of fertilized eggs between pure line crosses and hybrid crosses between the three blue mussels belogning to the Mytilus genus found in coastal Waters of southern Chile. These results suggest that there is little or no evidence of prezygotic barriers to fertilization between these species. Therefore, there is a great posibility that the two exotic mussels present in the chilean coasts may interbreed with the native chilean mussel, causing a tremendous damage to the aquaculture industry in Chile.
According to [16] there should be a prezygotic barrier between M.
chilensis and M. galloprovincialis because a difference in the sperm morphology of the latter mussel. However, this study showed that the interbreeding between these two blue mussels is completely possible in both reciprocal crosses. Further investigations should be done, including larval abnormalities and juvenile mortality caused by incompatibilities between mitochondrial and nuclear genomes, that may be causing a higher frequency of abnormalities and lower survival among F1 hybrid larvae [17].