FLs_readme.txt

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\begin{document}

\hypertarget{timing-the-start-of-fertilisation-in-littorina-saxatilis}{%
\section{\texorpdfstring{Timing the start of fertilisation in
\emph{Littorina
saxatilis}}{Timing the start of fertilisation in Littorina saxatilis}}\label{timing-the-start-of-fertilisation-in-littorina-saxatilis}}

BACKGROUND\\
It is not uncommon for closely related animals and plants to have
diverged in the way that sperm and pollen are transferred. This form of
reproductive isolation is known as gametic or gametophytic isolation
(Dobzhansky 1951) and it represents one barrier that can be responsible
for the nonrandom union of gametes from two parental populations (Markow
1997). For example, it has been shown between different species of
\emph{Drosophila} that heterospecific sperms were less motile and stored
in smaller quantity in the female organs than the conspefic sperms
(Patterson 1947). Gametic islation can also occur between distinct
populations of the same species and potentially contribute to the
formation of new species. Self-incompatibilty is one major mechanism
acknoledged in plants and there are a few studies that have focused on
inbreeding in animals (Markow 1997; Ober et al.~1992) and fewer that
have studied the consequences of a fertilisation barrier to the
likelihood of speciation (for an example of such a study, see Larson et
al.~2011).\\
\ldots{}\\
Fertilisation involves mutliple steps due to the complex interactions
between male and female post‐copulatory reproductive traits (reviewed in
Wolfner 2009).\\
\ldots{}\\
Females and males of the intertidal snail species \emph{Littorina
saxatilis} adopt a characteristic mating position that can be clearly
observed in the wild as well as in the lab. Typically, the male
approaches the female and crawls on top of her shell until he stops at
the front-right side of the female shell. At this specific mounting
position, the male can insert the penis under the female shell and
initiate transferring the sperms. When the penis is inserted is
difficult to establish but it has been found a strong correspondece
between male mounting position and copulation attempt (Hollander et
al.~2005). What has not been investigated in \emph{L. saxatilis} is the
starting point at which the sperms are being transferred into the female
reproducitve tract.\\
There are two main reasons why we examined the time of sperm transfer in
the \emph{Littorina saxatilis} system. The first one is that the rough
periwinkle is a brooder and the females can carry up to \ldots{} (ref.
for number of offspring) that at the end of their embryonic development
(ref. for development time) crawl away from the mother as juveniles.
This means that we can infer whether the sperms have been transferred by
identifying the stage of the eggs after dissection of the female storage
organ. The second reason is connected with the results by Panova and
colleagues (2010). They found an extreme level of promiscuity and a
deviation from the expected number of offspring per sire. However, they
were uncertain whether it was a result of postcopulatory sexual
selection (e.g., cryptic female choice and/or sperm competion), a
process often involved during the build-up of reproductive isolation. To
test for this requires the knowledge about how and when the sperms start
to move into the female reproducitve tract. Here, we focus on the when,
essentially the time at which the fertilisation begins in \emph{L.
saxatilis}. Under laboratory conditions, mating pairs of the marine
snail species \emph{L. saxatilis} have been observed to mate for
different durations. Interestingly, the relationship between number of
matings and duration contains two peaks, one at approximately five
minutes and one at around 30 minutes after the male had assumed the
characteristic mounting position (Fig. 1). We aborted copulations at
one, five and 30 minutes in order to understand fertilisation and
offspring production in \emph{L. saxatilis}. Short copulations (one
minute) are expected to be inadequate for sperm transfer whereas longer
copulations (30 minutes) are more likely to yield over time an effective
transfer of sperms. Finally, intermediate copulations (five minutes)
will be relevant for the description of postmating prezygotic patterns.
These may show whether females will produce offspring and if so whether
they will produce fewer than the females mated for 30 minutes.

\textbf{Figure 1.} Count of embryos in the control and three different
mating time groups of a pilot study.

HYPOTHESIS\\
Females of other littorinid species can receive a quantity of sperms
that is proportianal to the mounting duration (Hollander et al.~2018).
An equivalent relationship is likely to apply to \emph{L. saxatilis} and
we expect that very short matings (one minute long) will not be
sufficient for the sperms to travel through the female reproductive
organs and eventually fertilise the eggs.

QUESTION\\
When does sperm transfer start in \emph{L. saxatilis}?

MATERIALS AND METHODS (for now, look at the schedule file)\\
Wild males were sampled from a rocky shore on the island of Saltö which
hosts regularly a suitable variation of shell size. Females were instead
reared under laboratory conditions isolated from males as unobserved
matings will confound the level of paternity. Females and males were
sized and then each female was matched once with a male according to the
estimated optimal size ratio (ref.). If no copulation attempt had been
observed throughout the length of the experiment (TBD), the same female
was used the next day and paired with another male under the same
method.\\
Virgin Crab and Wave ecotype females will be placed individually in
plastic spheres (diameter) and let them mate with slightly smaller males
for one, five or 30 minutes. Aborted copulations will be performed for
all the three groups at the respective times. Reproductive events that
will last less than the pre-assigned time will be also recoded and
females included in dissections. Dissections of the females will be
planned two-three weeks after the breeding session of the experiment
when embryogenesis will have reached suitable stages for the
discrimination between uncleaved eggs and developing embryos.

\hypertarget{references}{%
\section*{References}\label{references}}
\addcontentsline{toc}{section}{References}

\hypertarget{refs}{}
\leavevmode\hypertarget{ref-dobzhansky1951}{}%
Dobzhansky, Theodosius. 1951. \emph{Genetics and the Origin of Species}.
3rd ed. Vol. 11. Columbia University Press.

\end{document}