David Eduardo Vrech.

Laboratorio de biología reproductiva y evolución, Instituto de

Diversidad y Ecología Animal (IDEA), CONICET-UNC and Facultad de Ciencias

Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sársfield

299, CP 5000, Córdoba, Argentina. dvrech@gmail.com. Sequential male sperm

storage in Timogenes elegans (Arachnida, Scorpiones, Bothriuridae).

Olivero, PA; Mattoni, CI; Peretti AV. Laboratorio de biología reproductiva y evolución,

Instituto de Diversidad y Ecología Animal (IDEA), CONICET-UNC and Facultad de

Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez

Sársfield 299, CP 5000, Córdoba, Argentina.

VRECH ET AL. -SPERM PRODUCTION IN TIMOGENES ELEGANS.

Abstract

Sperm competition influences the evolution of many reproductive traits. In this field,
analyses are concentrated in testes and ejaculates. Testes mass is a good proxy of the
sperm competition risk. However, not all testes show the same dynamics of sperm
production. Among arachnids, scorpions constitute an intriguing taxon for analyzing
sperm production. In the family Bothriuridae Simon 1880, Timogenes elegans Mello-
Leitão, 1931 is an interesting species for analyzing sperm production because is a
promiscuous species, males produce spermatozoa continuously, and store them in an
elastic storage organs (i.e. two seminal vesicles plus two vas deferens) before
transferring to the female using a sclerotize spermatophore. Our main aim was to
analyze sperm storage and transfer in T. elegans males. We described the volume and
concentration of sperm in storage organs and ejaculates; we compared the volume of
both storage organs; and finally, we showed data of ejaculate volume and
concentration in remating experiences. Storage organs were always fully filled and
there was a variation in the total storage organ size. The total volume of the
spermatophore was larger than the volume of the ejaculate. The volume of the sperm
drop represented 38% of the volume that could fill completely the spermatophore. The
rest corresponded to a gel-like substance, filling the space around the sperm drop.
Ejaculate volume and concentration did not vary significantly between successive
matings. T. elegans shows excessive sperm production stored in two storage organs.
Sperm stock is divided equally between both storage organs, and ejaculates are diluted
in the spermatophore with the gel stored in the trunk. There was no effect of body
condition over any of the variables analyzed. Male storage and transfer are discussed
in light of sperm competition.

Keywords: Scorpion; Storage organs; Ejaculates; Sperm transfer; Sperm

concentration.
 Introduction

Sperm competition is a powerful evolutionary force in postcopulatory sexual selection,

common in polyandrous species, where the sperm of different males compete for the

access to fertilize a set of female’s ova (Parker 1970; Birkhead & Møller 1998; Parker &

Pizzari 2010). Sperm competition influences the evolution of many morphological,

physiological, and/or behavioral traits directly related to reproduction (Andersson 1994;

Birkhead & Møller 1998; Wigby & Chapman 2004; Simmons 2014). In this field, most of

the information regarding sperm competition is concentrated in testes, followed by

ejaculates (Parker & Ball 2005; Simmons 2014). Testes mass is generally accepted as

a good proxy of the sperm competition risk (Parker et al. 1997; Parker & Ball 2005).

However not all testes show the same dynamics of sperm production (Møller 1991;

Schärer et al. 2004; Vahed & Parker 2012). Continuous sperm production is difficult to

assess as the production rate of sperm may be difficult to evaluate (Shärer et al. 2004).

Many arthropods only produce sperm until they reach the adult stage (Barr 1974;

Chapman 1998; Manogem 2002; Boomsma et al. 2005; Bressac et al. 2008). This

means that they have a fixed stock of sperm, and theory predicts that males should

allocate sperm strategically (Simmons 2001). Among arachnids, Michalik & Rittschof

(2011) suggested that the lack of sperm production during adulthood is unusual in

spiders, and spermatogenesis ends before the final molt. Testis size decreases when

reaching adulthood and changes its structure. Therefore, the amount of sperm

available for mating is limited to the sperm inside the pedipalps, and males lose their

ability to fertilize eggs once it is used. Similarly, Schneider and Michalik (2011)

discussed the evolution of three species of Nephila with different mating tactics. They

suggested that males can reverse to polygamy in various traits but they failed to revert

to adult sperm production, and they developed the potential to economize their limited

sperm supply. In other arachnids, such as solpugids, it has also been suggested that

spermatogenesis is completed after the adult reaches adulthood (Klann et al. 2005). In
this scenario, males should allocate sperm strategically, because it is a limited

resource (Simmons, 2014).

Besides, sperm is costly because spermatozoa are delivered together with seminal

fluids (Dewsbury, 1982). These fluids may be involved in many tasks, like for example

maintaining or helping spermatozoa, or contributing to the generation of a genital plug

(Kaufman et al., 2008; Dennenmoser & Thiel 2015). These seminal fluids may be

generated in an organ or be a product of secretion tissues (Leopold 1976). In spiders,

Nephila clavipes Linneo, 1767, sperm comprise spermatozoa and secretion, produced

by the somatic cells of the testis (Michalik & Ritchsof 2013). In solpugids, the genital

chamber seems to produce a secretion that is thought to be important for the sperm

transfer. The vasa deferentia and the glandular part of testes contribute to the secretion

that helps to form the sperm droplet, similar to what happens in actinotrichid mites

(Alberti & Coons 1999; Klann 2009).

Seminal vesicles are storage organs usually found in males among many arthropods

(Wedell et al. 2002). Seminal vesicles are thought to influence the outcome of sperm

competition in many taxa, mainly through seminal products (Ramm et al. 2005), as

males of sperm-storing species seem to produce more sperm than species that do not

store sperm (Orr & Zuk 2013). This has been demonstrated in vertebrates (Scaggiante

et al. 1999; Ramm et al. 2005) and arthropods (Chapman 2001; Simmons 2001).

Among arachnids, scorpions constitute an intriguing taxon for analyzing sperm

production. Males produce sperm continuously in paired testes (Jespersen & Hartwick

1973; Alberti 1983; Michalik & Mercati 2010; Vrech et al. 2014), and store sperm in two

seminal vesicles (Polis & Sissom 1990; Peretti & Battán-Horenstein 2003; Vrech 2013).

Before sperm transfer, males of some species may produce seminal secretions from

different glands, that will accompany sperm to the female reproductive system (Peretti

& Battán-Horenstein 2003). In some species, the cylindrical gland may secrete

substances that could contribute to the formation of a genital plug inside the genital

atrium of inseminated females (Vachon 1953; Hjelle 1990; Peretti 2010). Sperm is
transferred indirectly inside a sclerotized spermatophore that is deposited on the

ground (Francke 1979; Hjelle 1990; Peretti 2010).

In the family Bothriuridae, Timogenes elegans is an interesting species for analyzing

sperm production because 1) is a promiscuous species, as females are polyandrous

(i.e. each female copulates with more than one male per reproductive season) (Peretti

1993; Vrech et al. 2011), and males generally accepts more than one female during the

entire reproductive season (Peretti 2003), and 2) males produce sperm continuously in

their pair testes and store them in two elastic storage organs (i.e. two seminal vesicles

plus two vas deferens). Contrary to what happens in other bothriurid species, males

lack seminal glands inside genital system, and there is no production of a visible genital

plug in T. elegans (Peretti & Battán-Horenstein 2003). In addition, this species is

suitable for laboratory mating experiences, as individuals show a considerable body

size and are abundant in the Chaco region of Argentina (Acosta 1995; Ojanguren-

Affilastro 2005).

Data on T. elegans suggests that although being a promiscuous species, males show

relatively small testes mass (Vrech et al. 2014). Reproductive works on T. elegans,

generally were focused on descriptions of the reproductive system (Peretti & Battán-

Horenstein 2003), sperm state and morphology (Vrech et al. 2011) and effect of

polyandry on testes mass (Vrech et al. 2014). However, there are no works that report

the volume of storage and ejaculates, evaluation of sperm production on successive

matings, or report of sperm concentration in males.

Our main aim is to analyze sperm storage and transfer in T. elegans males. To

accomplish this task, first we described the volume of storage organs and ejaculates.

Second, we compared the volume of both storage organs. Finally, we show data of

ejaculate volume and concentration in remating experiences. We postulate three main

hypotheses: 1) sperm production will be abundant since T. elegans is a promiscuous

species, and storage organs will store an excessive number of sperm; 2) sperm

concentration will not vary from storage organs to ejaculate, as this species lack
accessory glands; 3) sperm volume will not decrease in successive multiple matings,

due to continuous sperm production. To our knowledge, this is the first analysis of

storage organs, ejaculates, and successive production of sperm in scorpions.

METHODS

Storage organs and ejaculates.- Adult males were dissected dorsally, and paraxial

organs were removed (n = 20). Both testes were cut at the base of the vas deferens

(Fig. 1, black arrow). The cut through this zone avoided sperm loss. The vas deferens

values were included to those of seminal vesicles because these also store sperm and

its cut could have resulted in a massive sperm loss. Therefore, when we will refer to

storage organs, be aware this will involve seminal vesicles plus vas deferens. The

storage organs were isolated from the paraxial organs.

Spermatophores were obtained in laboratory matings (n = 16) performed in an arena

with proper sand as substrate containing barks and stones to resemble the organism’s

natural habitat (Maury 1982). The female was placed one hour before starting the

mating trial, for acclimation. Precopulatory courtship was allowed, and mating was

stopped after spermatophore deposition, but before sperm transfer. After each mating

trial, the arena was washed with Ethanol 70%, and substrate was changed. Pre-

insemination spermatophore was removed from the soil and manually deployed over a

slide, obtaining the entire ejaculate content.

Volume measurementAnalyzed variables.- Volume measurements. We measured

volume of sperm drop inside the spermatophore and outside de spermatophore

(ejaculate). For this we used two techniques. First, to estimate sperm drop inside the

spermatophore, we used the volume of a sphere (4/3 π r3). The radius was obtained

measuring the total width of the sperm drop in pictures of the spermatophore in a front

view, and divided in 2. It is important to notice that this proxy does not discriminate the

real volume of sperm in sensu stricto and the volume of invaginated capsule (i.e. lobes

that are evaginated during sperm transfer inside female’s genital atrium). This was
impossible to discriminate both values since in the pre-insemination spermatophore the

sperm is located around the capsular lobes and cover completely them.

Second, to estimate the volume of the sperm drop outside the spermatophore

(ejaculate), and storage organ volume, we used a technique based on Gage (1994).

We used two microscope slides separated by the height of a single coverslip.

Coverslips were put on each side of storage organs or ejaculate to avoid collapsing.

After deposition, a second slide was put to rest over the coverslips, producing a

controlled crush of the sample. The pressure was uniform with the aid of four binder

clips, each one put on each corner. Since storage organs are soft, they did not oppose

resistance and crushing was complete. The area (mm2) of the sample pressed between

both coverslips was measured with the Image J 64bits image-processing software

(Schindelin et al. 2015). Volume (mm3) was obtained multiplying the measured area by

the height left by the coverslips.

Right and left storage organs were removed. First, we calculated the volumes of each

vesicle, and then we evaluated the difference in volume between them with a Wilcoxon

signed-rank test. We used the wilcox.test function of the package stats in R (R Core

Team 2016).

Total potential volume of the spermatophore.- Values were obtained by measuring

pre-insemination spermatophores, obtained from mating trials (n = 16). The volume

was obtained as the volume of a cylinder (π r2 h). For this, we measured the diameter

(width of front spermatophore photograph) of three different zones in the

spermatophore, the widest at the top (zone of inflexion of spermatophore), the

narrowest at the bottom (base of trunk) and the width in the middle from these two

zones. These values were then averaged and divided by 2 to get the mean radius of

the spermatophore. Height was obtained measuring the base of the lamella to the end

of the trunk. Measurements were obtained and multiplied to get a proxy of the volume.
 Spermatozoa concentration.- Fresh storage organs were immersed in saline solution

(Nacl2 0.9 gr./ 100ml distilled water) and cut to liberate sperm packages. Sperm

packages were collected using a 1-10 µl micropipette (Finnpipette, Thermo Scientific)

and mixed with 100 µl of saline solution. Spermatozoa were released from sperm

packages after 5 minutes’ vortex. Spermatozoa number was computed using a

Neubauer counting chamber, following the basic protocol for counting cells (Bukowski

& Christenson 1997; Bastidas 2014). Spermatozoa were counted in the four big

squares at the corners of the chamber's grid. This value was multiplied by the

appropriate dilution factor to obtain an estimate of the concentration of spermatozoa

(spermatozoa/ml). Additionally, the variation coefficient was used to evaluate the

changeability in sperm supply in the storage organs as well as the ejaculate.

Male condition.- Variables of storage organ, ejaculate volume, and sperm

concentration were regressed against body condition to analyze the possible influence

of an energy trade off in sperm production. Body condition was obtained as the

residuals of the regression between body mass (weight) and prosome length (size)

(Moya-Laraño et al. 2008; Jakob et al. 1996). Data was modelled using GLM in R. For

each variable, the distribution was assessed graphically with a Cullen and Frey graph

(skewness–kurtosis plot), and statistically with a chi-square test. We used a gamma

distribution for vesicle volume and spermatophore volume, a Gaussian distribution for

ejaculate volume, and negative binomial for concentrations.

Sperm load in successive matings.- Adult males were captured in January 2011 and

2012 (n = 10). To obtain more than one spermatophore per male, mating trials were

weekly performed during the sexual season (January-February). Volume of sperm drop

inside the spermatophore, spermatophore volume, and concentration of spermatozoa

were measured in all the spermatophores produced by each male (following the
techniques already described). A Wilcoxon signed-rank test was used to evaluate

differences between all matings.

RESULTS

Sperm volume in storage organs and spermatophore.- In storage organs, the part

that corresponds to the seminal vesicle, was always fully filled and there was a

variation in the total size of the seminal vesicle’s size (Fig 1). The volumes of the

storage organs showed a coefficient of variation of 71% among males. Both storage

organs had similar volumes (Wilcoxon rank paired test; V = 76, P = 0.379).

The total volume that the spermatophore could store was larger than the volume of

the sperm drop contained inside, and the ejaculate that came out (Table 1). Sperm

contained inside the spermatophore did not statistically differ with the ejaculated that

came out of the spermatophore (Wilcoxon rank paired test; V = 71, P = 0.90). The

sperm drop contained inside the spermatophore represented 38% of the volume that

could fill completely the spermatophore.

The rest of the volume inside de spermatophore corresponded to a gel-like substance

observed at the bottom of the capsule, filling the trunk, around the sperm drop (Fig. 2).

The ejaculate was formed by the sperm drop inside the spermatophore, and part of the

gel-like substance, that mixes inside and comes out with sperm after activation. This

amount of gel could not be measured.

There was not statistical support for the influence of the male’s body condition over

the volume of the sperm drop inside the spermatophore, the ejaculate volume, the

volume of spermatophore or the volume of the storage organs (GLM volume sperm

drop inside F = 0.14, P = 0.71, GLM volume ejaculate F = 1.92, P = 0.19, GLM volume

of spermatophore, F= 0.0001, P = 0.99; GLM volume of storage organ volume, F =

0.16, P = 0.69).
 Spermatozoa concentration inside storage organs and spermatophores.- Sperm

concentration inside the storage organ was 1.32 ± 0.94 108 spermatozoa/ml, and inside

the spermatophore was 3.14 ± 2.2 107 spermatozoa/ml. The coefficient of variation was

0.71 and 0.72 respectively with no statistical differences (Two-tail Z-Test, Z= 0.10; P=

0.95). The number of spermatozoa contained inside the spermatophore represented

24% of the spermatozoa stored inside both storage organs. Sperm number in the

storage organ was 1,57 107, and 4,61 104 inside the ejaculate.

Male body condition did not influence sperm concentration in storage organs and in

ejaculates (GLM sperm concentration storage organs, F=0.04, P=0.83; GLM sperm

concentration ejaculate, F=2.58, P=0.11).

Volume and concentration of sperm along successive matings.- Four males

remated once (Table 2). Ejaculate volume and concentration, and spermatophore

volume did not vary significantly between successive matings (Wilcoxon rank paired

Test, (ejaculate volume) V = 3, P = 0.62; (spermatophore volume) V = 6, P = 0.875;

(ejaculate concentration) = 3.0, P = 0.62).

DISCUSSION

The present study, was focused in showing sperm storage and transfer in the male of

the neotropical scorpion T. elegans, by examining the total volume and concentration

of spermatozoa inside male’s reproductive system. These two variables show clearly

the pattern of sperm production in this species.

Storage organs and ejaculates.- Our data show that spermatozoa is stored in great

amount inside storage organs. From these organs comes the sperm drop contained in

the spermatophore. The size of the storage organs seems to limit the amount of sperm

that each male can store. In T. elegans, storage organs are full and vary in sizes, but in

other species as in Brachistosternus ferrugineus Thorell, 1876, storage organ size is

very similar among males and there is a high variation in percentage of filling (Vrech
2013). At first glance, each storage organ could store the equivalent volume of at least

four ejaculates. However, since spermatozoa were more diluted in the ejaculate,

storage organs may store more, and the total potential ejaculates that storage organs

produce might be higher. Therefore, we are underestimating the number of ejaculates

that storage organs could store. We did not expect a dilution in the ejaculate, as T.

elegans males lack accessory glands connected with the capsular region of the

hemispermatophores in the paraxial organs (Peretti & Battán-Horenstein 2003).

Nevertheless, in many taxa seminal fluids are not always produced by accessory

glands (Poiani 2006). We have noticed that the gel-like substance that fills the trunk,

comes out mixed together with the sperm drop. Among bothriurids, if insemination is

successful the spermatophore will appear completely empty after ending the sperm

transfer phase (Peretti, 1992, 1994; Peretti et al. 2000). In our analysis, the sum of

sperm drop volume and ejaculate volume are equivalent and smaller than the estimate

of volume of the spermatophore (i.e. capsule region+ trunk). Sperm seems to be

already mixed with the gel before coming out, this could explain the similarity in the

volume of the sperm drop inside the spermatophore and the ejaculate. The rest of the

gel that remains around it helps to extrude the ejaculate. Thus, the gel-like substance

may have multiple tasks. First it could be involved in the mechanism of sperm

expulsion of scorpion spermatophores (Francke 1979) by facilitating the transmission

towards the capsular region of the pressure exerted by de lamella over the trunk.

Second, if could play an important role for the dilution of sperm. Indeed, we observed

that sperm concentration was higher in ejaculate than in storage organ. In addition, it

may aid sperm inside the female like seminal fluids in insects (Avila et al. 2011).

However, we should bear in mind that some components of the ejaculate can be

produced in other parts of male’s reproductive tract with secretory tissues (Avila et al.

2011). In spiders, for example, seminal secretions are added to the sperm in the testes

and deferent ducts (Michalik & Lipke 2013). Further analysis searching secretion tissue

in the distal part of the storage organs, and the detailed analysis of the gel-like
substance may help to figure out how the dilution process occurs as well as the origin

of that substance

Previous studies assessed that sperm reserves, ejaculate or spermatophore size

covary positively with the size of the male in many arthropods (e.g. Wedell 1993;

Bissoondath & Wiklund 1996; MacDiarmid & Butler 1999; Gosselin et al. 2003; Jivoff

2003; Rubolini et al. 2006). However, in our study, none of the volumes analyzed were

associated with body condition storage organ in T. elegans.

Sperm counts.- Some works in arthropods have reported sperm counts in storage

sites and in ejaculates or spermatophores. For example, Brown & Knouse (1973)

reported sperm numbers in a fertilization study of sperm and egg interaction in the

horse shoe crab Limulus polyphemus Linneo, 1758. Their plan was to examine the

effect of sperm concentration on fertilization and development. They report the

ejaculate of sperm in L. polyphemus, approximately 1010 non-dilute spermatozoa using

an electrical stimulation method. In the spiny king crab Paralithodes brevipes Milne-

Edwards & Lucas, 1841 sperm count is reported in a sperm depletion and recovery

analysis (Sato et al. 2006), and the number of spermatozoa in the vas deferens of

virgin males were in the order of 108. In insects, Swallow & Wilkinson (2002) reviewed

about sperm heteromorphism in insects. Besides analyzing hypotheses for the

evolutionary origin, maintenance, and function of apyrene, they show a table where

data of sperm numbers of many taxa of insects are provided. Values of sperm number

ranges from 103 in lepidoptera to 107 in other lepidoptera and diptera. Moreover, an

analysis in a very basal hexapoda order, collembola, showed that males transfer a

sperm droplet that contain no more than 103 spermatozoa to the female (Dallai et al.

2009). Recently an analysis in desert ants using flow cytometry showed that sperm

numbers were in the range of 106 spermatozoa in the accessory testes (Aron et al.

2016).

Among arachnids, there are also many analysis in spiders. For example, Bukowski &

Christenson (1997) analyzed sperm release and storage in Micrathena gracilis

Walckenaer, 1805 an orb weaving spider, and sperm counts were in the order of 103

spermatozoa in the male´s palps. In a successive analysis in Gasteracantha

cancriformis Linneo, 1758 Bukowski and collaborators (2001) reported 104

spermatozoa also in the male´s palp. Snow & Andrade (2004) reported 105 within the

two palps of a theridiid spider, and a correlation between the number of sperm in the

right palp and the left palp showed that both pedipalps charged an equivalent number

of sperm, similar to what we reported here for the volume of right and left storage

organs. Ceballos and collaborators (2015) reported for the orb weaving spider N.

edulis Labillardière, 1799 an average of 104 sperm transferred to the female, and the

same order remaining in the male’s palp. Arnaud and collaborators (2001) report sperm

counts of ejaculates together with the coefficient of variation. They showed a lower

variation in comparison with our results. In addition, other studies also showed the

value of reproductive structures or ejaculate masses. For example, Vahed (2006)

reported values of sperm counts that ranges from 105-106 in 18 Tettiigoniid species.

However, these numbers are difficult to compare with the data we obtained in the

present article. Here we reported a sperm count of 107 in the storage organ and 104 in

an ejaculate. The problem is the lack of standard or absolute values that limit the

potential comparisons with other groups. Although we have also presented values of

the sperm count in relation to the storage organ and ejaculate, comparison is still a

problem as not every storage organ or ejaculate are comparable in volume across

taxa. For example, some species may show bigger counts as structures or ejaculates

are bigger and not because they store or allocate more sperm.

Successive matings.- Regarding our analysis of successive matings in T. elegans,

we did not observe a clear tendency in sperm drop volume and/or sperm count.

Previous studies in arthropods give some insight of how sperm depletion may influence

ejaculate allocation (Vahed & Parker 2012). For example, among insects, Cook &

Gage (1995) reported the number of ejaculated apyrene and eupyrene spermatozoa

associated with the risk of sperm competition in lepidoptera. Values were close to 105
for eupyrene and 104 for apyrene, and numbers declined over successive matings. A

similar analysis was developed by Watanabe et al. (1998). In this case the authors

evaluated the effect of repeated matings on apyrene and eupyren numbers in

successive ejaculates of the cabbage white butterfly, Pieris rapae Linneo, 1758.

Spermatophore mass decreased in successive matings, but eupyrene and apyrene

numbers increased from the first to the second mating but decreased towards the

fourth mating.

Some studies suggest that males could adjust sperm allocation in response to certain

clues given by the population and/or females as evidenced in other groups (Nakatsuru

& Kramer 1982; Pitnick & Markow 1994; Schärer & Vizoso 2007). However, in the

present study we did not evaluate female status, therefore we cannot state if males are

evaluating directly or indirectly females by their mating status, and allocating ejaculates

strategically accordingly. Unfortunately, the number of analyzed matings were not

enough to allow a fine discussion of this matter in this scorpion species, and future

works are needed on this area.

Sperm redundancy.- Like in other animals the question that arises in T. elegans is:

why do males produce such large number of spermatozoa? Indeed, spermatozoa stock

in animals is excessive to fertilize the female’s entire set of ova (Brown & Knouse 1973;

Parker 1982; Pizzari & Parker 2009). Males can adopt different ejaculate strategies to

overcome sperm competition (Parker 1970; Parker & Pizzari 2010). Timogenes

elegans shows a male biased sex ratio (Nime et al. 2014), female polyandry (Polis &

Sissom 1990; Peretti 2010), together with the female ability to store sperm (Volschenk

et al. 2008), and the lack of an effective genital plug (Peretti & Battán-Horenstein

2003). These characteristics show a mating system with a high risk of sperm

competition (Parker 2000). The need to produce a great number of spermatozoa would

be linked with the possibility of generating many spermatophores and thus being able

to inseminate many females (Parker & Pizzari 2010; Vahed & Parker 2012). The

alternative to this strategy would be to deposit a greater amount of sperm to fewer

females (Parker 1998; Simmons 2001; Wedell et al. 2002). Although the

spermatophore is a sclerotized structure, the volume of ejaculate may change as

shown by the variation coefficient of the ejaculate in our analysis.

The dilution of sperm observed in the ejaculate, allows the transfer of even fewer

number of individual spermatozoa to the female. This fact may help to fill more

spermatophores with a fixed stock of sperm. However, in scorpions, the production of

many spermatophores during the reproductive season should not affect sperm stock,

as scorpions have continuous sperm production (Jespersen & Hartwick 1973; Alberti

1983) and sperm reserves may not diminish. In contrast, this occurs in other

arthropods (Barr 1974; Chapman 1998; Manogem 2002; Boorsma et al. 2005; Bressac

et al. 2008; Klann et al. 2005; Michalik & Rittschof 2011; Schneider & Michalik 2011).

To visualize this, we could suggest the following explanation. If we average 110 days

as the total effective reproductive period (end of November to beginning of March,

Acosta & Maury 1990). Males in T. elegans need five days to regenerate the

hemispermatophores (Peretti & Acosta 1998), and approximately two more days to

regain willingness to mate (Peretti 1993). A full potential spermatophore production in

an outstanding scenario would be of approximately 16 spermatophores for the entire

reproductive season. Our data suggest that storage organs together may store the

volume of at least six spermatophores. Therefore, males could produce sperm to fill 12

spermatophores, provided that storage organs could get filled two times during the

reproductive season (i.e. one round of emptying and filling of sperm of storage organs).

Unfortunately, we do not have data of sperm dynamics inside the storage organs to be

precise in this point. Given the previous assumptions, the value may be closer to the

theoretical value (i.e. 12 spermatophores). Thus, the redundancy of sperm could be

related in part to this theoretical maximum spermatophore production. However, we

should consider that this large number of spermatophores may be difficult to reach in

nature, as there are other variables affecting this capacity. For example, the rate of

predation may vary daily (Nime et al. 2013). Besides, the operational sex ratio is biased
towards males, and the real female availability for mating can be reduced. Additionally,

males are vagrant only a few nights during the reproductive season (Nime et al. 2014).

Another explanation of the redundancy of sperm is related to fertility. Males apparently

need an excessive number of spermatozoa for a successful fertilization event (see

Brown & Knouse 1973; Schradin et al. 2009). Indeed, ejaculates with low sperm supply

are unsuccessful in fertilizing eggs (Schradin et al. 2009). It is unknown what sperm

concentration is required to consider a scorpion male as fertile.

An alternative explanation for sperm redundancy is that large production of

spermatozoa (like the one males face during the reproductive season) leads to a high

rate of errors and mutations in the spermatozoa that would, in time, affect viability,

motility or even the acrosome structure (Cohen 1973). To counter this source of error,

males benefit from having a larger stock of sperm, where by chance they will have an

acceptable amount of sperm that is capable of successfully fertilizing eggs (Blumenstiel

2007; Pizzari & Parker 2009). Unfortunately, we lack data for analyzing this explanation

in this species.

Recently, another hypothesis tested for some reptiles suggested that a male can have

an increased sperm production to counter interspecific sperm competition between two

closely related species that are living in sympatry. For example, there is a relationship

between the number of spermatozoa and body size that suggest sperm competition

and possibly a reproductive interaction between the species of Podarcis bocagei

Seoane, 1885 and P. carbonelli Pérez-Mellado, 1981 (Carretero et al. 2006;

Kaliontzopoulou et al. 2007). The analysis showed that sympatric males of both

species produced relatively more sperm, suggesting there may be a reproductive

interaction between them. Recently, Naretto et al. (2016) found in Salvator Duméril &

Bibron, 1839 lizards that in sympatry the presence of males of one species increased

sperm competition by increasing the relative number of competitors for males of the

second species. This fact only affected testes mass but not sperm count between

species. This growth in relative testes mass was explained by the presence of
conspecific and heterospecific rivals in sympatry, the difference in availability of mates,

or a combination of both mechanisms.

To our concern, T. elegans lives in sympatry with one sister species, T. dorbignyi

(Guérin-Méneville 1843). This species is smaller than T. elegans (Ojanguren-Affilastro,

2005). In this scenario, both species could compete for similar resources as suggested

in the previous paragraph for lizards. Although this hypothesis is interesting, different

characteristics may give insight that this is rarely the case between these sympatric

species of scorpions. The level of mating synchrony is apparently low (Mattoni, Peretti

pers. obs.). In addition, they differ strongly in body size (T. elegans is larger) and

mating heterospecific trials do not result in courtships (Peretti 2003; Vrech pers. obs.).

Finally, courtship displays are different between them (Peretti 2003).

Conclusion.- Timogenes elegans males show excessive sperm production that is

stored in two storage organs. Sperm stock is divided equally between both storage

organs, and spermatozoa are diluted when passing to the spermatophore, and the gel-

inside the trunk may be the diluent. Further studies in other species of Bothriuridae and

other families would be very useful to offer a comparative survey of sperm production

across the entire order.

Acknowledgements

Matias Izquierdo, Lucia Calbacho-Rosa, Fedra Bollatti, Silvana Burela, Monica Nime

[field sampling]. Martin Ramirez, Marga Chiraraviglio, Alejandro Guidobaldi, Peter

Michalik [suggestions previous versions of ms]. German Gonzalez [help statistic] N

anonimous reviewers [improve ms] CONICET Foncyt, Secyt-UNC [funding].

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 Tables

Table1. Absolute values of volume expressed in mm3. Here, values are shown as the
sum of both storage organs. Spermatophore relates to the total volume that a
spermatophore can contain. Values are given as mean± SD. cv: coefficient of variation

Vesicles Sperm drop Ejaculate Spermatophore

Mean 11.84± 8.83 2.35± 0.99 2.04± 0.74 6.23± 1.03
cv 75% 42% 36% 17%

Table 2. Volume and concentration of spermatozoa inside spermatophores of

successive matings. Bold indicates the highest values.

Spermatozoa
Spermatophore
Males Matings Ejaculate concentration
volume (mm³)
volume (mm3) (x107/ml)
Male 01 1 2,58 0,56 6,85
2 2,44 0.23 6,46
Male 05 1 2,41 1,75 7,96
2 3,56 3,25 8,40
Male 06 1 2,29 0,94 8,23
2 2,40 2,63 6,98
Male 09 1 3,13 0,56 6,43
2 3,14 0,47 6,54
Figures

Figure 1. a-b. Two paraxial organs from two different males. Storage organs:

Seminal vesicles (grey arrows). Notice that both vesicles are entirely filled with

sperm. The small pouches (black arrow) are the vas deferens and correspond to

the connections between seminal vesicles and testes.

Figure 2. Pre-insemination spermatophore of T. elegans. Three zones can be

observed, the lamella, the capsule, containing the ejaculate (dotted polygon), and

the trunk, filled with a transparent gel-like substance (black arrow).