Selective Oviposition of a Natural Population of Drosophila ananassae
Corresponding author: Emilio Pimentel, Departamento de Biología, Instituto Nacional de Investigaciones Nucleares (ININ), Carretera México-Toluca, S/N, la Marquesa, Ocoyoacac, CP. 52750, México, Tel: +55 5329 7200;
Reproductive capacity is a good index of fitness in organ- isms that go through repeated cycles of rapid population growth and it has evolved as a way to maximize the poten- tial of survival for species . The study of their behavior and the factors that modify it have helped to understand the evolutionary mechanisms of adaptation of the species [2-4]. It is known that fertility depends on factors such as temperature, humidity, feed quality, the texture, and color of substrate medium, size of the female or male [5,6], but also behavioral components caused by physiological changes in- fluence oviposition site selection. In Drosophila as in other insect choosing the best place to lay eggs is of profound im- portance to the survival of the future generation. Oviposition sites in nature vary in quality resulting in significant differ- ences in reproductive success, this is: best sites guarantee a greater number of offspring. Since the seventies, the genus Drosophila has been used to evaluate various factors that influence the selection by females of the best places to lay eggs, the genetic basis that controls the election and the “de- cision” laying eggs by females of Drosophila have been also investigated.
Most natural populations of Drosophila choose to lay eggs in a nutrient substrate to guarantee a supply of food for larvae [7,8]. These preferences are known to be highly polymorphic with considerable heritability and ability to evolve , espe- cially in the case of interspecific competition where site se- lection can serve as a mechanism for reducing Intra and in- ter specific competition [10,11]. Recently it has been shown that the behavior of site selection oviposition is the result of complex physiological mechanisms; Drosophila females probe the site before laying each egg, presumably to assess the quality of the site, after that, the females accept or reject a specific medium, and this may be a neural decision. Yang et al,  suggested that neurons ILP7 expressing an insulin- like neuropeptide share some homology with relaxin, a ma- jor reproductive hormone in mammals, this is important in Drosophila for correct “decision” process in egg-laying.
Drosophila species are strongly attracted by the smell of food , particularly to ripe or decaying fruit for its high content of yeast such as Saccharomyces cerevisiae, and often they gather around a food source to feed, mate and lay eggs [13,14]. Besides that, it is still unclear through which mecha- nism the flies choose the most suitable place to lay eggs; it
is also unclear whether the mechanism is controlled by one or several factors which act to take the decision. Dweck et al, , found that the preference of Drosophila by citrus is not based only on the smell, but also in the texture of the fruit,
e.g. the pericarp reduces the possibility that the fruit is para- sitized, therefore avoiding larvae to die. They conclude that flies detect terpenes characteristic of these fruits via a single class of olfactory sensory neurons, expressing odorant re- ceptor Or19a. The comparison of the oviposition site prefer- ence of wild D. melanogaster lines, free or forcedly exposed to food rich in menthol, after twelve generations, oviposition preference between the two groups diverged. The forced lines to food with menthol showed persistent aversion to it, while the lines of free choice decreased aversion to food rich in menthol. The effect was specific to menthol because when exposed to caffeine or sucrose this did not alter their behav- ioral oviposition site selection. The study established that in the underlying process of oviposition site selection in Dro- sophila, genetic factors are influenced more quickly when flies can choose between several alternative substrates .
The aim of this study was to evaluate in a natural population
couples tested allowed to lay eggs. After that, the tracking flies were transferred to track another tube with fresh food. The couples were placed individually into the tube with tracked medium. Each pair remained laying eggs for 24 h, after that they were transferred to a new tracked tube and the number of eggs laid by each pair was counted. This pro- cedure was repeated ten days for each pair of each IF (see Figure 1). Tubes with male and female tracking, as well as tubes with egg-laying females, were kept in constant light in a culture room at 25 ± 1 °C and 60% RH. The females which did not lay eggs were not considered in the fecundity calcu- lations, eggs per female (h/f).
Count the number of eggs laid by each female
12 Isofemales and 18 couples for each
After 24 h
Females lay eggs for 24 h
of Drosophila ananassae the influence of the previous pres- ence of males in the female oviposition site. For this purpose we test three medium: unconditioned medium (UM), con- ditioned tracked by females medium (TF) and conditioned tracked by males medium (TM). The population of D. anan- assae was established as isofemale (IF) and the study was conducted with their offspring.
Material and Methods
females Tracked by males After 24 h
Biological material: Drosophila ananassae  were col- lected from Laguna Verde, Veracruz, Mexico, is a cosmopoli- tan and domestic species. It occupies a unique status among Drosophila species due to certain peculiarities in its genetic behavior . Different aspects of its biology have been studied: mating behavior, mating success, rare-male mating advantage, female and male remating, pupation site prefer- ence, oviposition site preference, sperm displacement, dura- tion of copulation, phototactic behavior etc., .
Each collected female was put into homeopathic glass tubes (9.5 cm high and 2.5 cm D) with culture medium, for inbred proliferation. The culture medium used in this study con- sisted of cornmeal (4%), attenuated yeast (1.9%), sucrose (2.2%), dextrose (1.6 %), agar (0.8%), and propionic acid (0.3%) as preservative. Twenty females were collected, from which twelve of them were mating in nature and their off- spring remained under laboratory conditions at 25 ± 1 °C and 60% relative humidity (RH) as IF line. Each IF was used for this study within four generations after the collection.
Egg-laying selection analysis: Three days old females and males from each IF were isolated and mass crossing. Later three groups of 18 couples were separated to allow them to lay eggs: one group in UM; the second group in TF and a third group in TM. The conditioning of the medium consisted in leaving five females or five males (same age and IF) in ho- meopathic glass tubes with fresh medium for 24 h before the
Figure 1. From each IF, 18 couples were isolated. They were placed individually in homeopathic tubes with fresh food previously tracked by five virgin females or five virgin males from the same IF. Each pair remained in the tracked medium for 24 h, then they were transferred to another tube with freshly tracked medium, and the eggs laid were counted. This procedure was repeated ten days for the 18 couples from each IF.
Egg-to-adult survival analysis: From F4 and for each IF, 75 mated females (7 days old) were put to lay eggs for 6 h in groups of 25, in Petri dishes (5 cm D) with fresh food, previ- ously tracked by 25 virgin females or males. After that from each IF, 240 eggs were tested in groups of 80 by homeopath- ic glass tubes. All tubes with eggs were kept at 25 ± 1 ºC and 60% RH conditions until their development was completed. After twelve days, all emerged adults were counted. The t- test was used to assess differences between groups.
Table 1 shows the fecundity data as the mean of lied eggs per female (h/f) in ten days. Based on the h/f, the IF were dis- tinguished and separated as (a) low fecundity IF’s with 6.28
± 0.8 h/f and (b) high fecundity IF’s with 11.85 ± 2.0 h/f to analyze and compare their fecundity behavior. In these form, the low fecundity IF increased their oviposition 49.52% (from 6.28 ± 0.8 to 9.39 ± 0.4 h/f) in the TF, but in contrast, the fecundity rose up 231.68 % in TM. The comparison be- tween the numbers of females which lied eggs in TF vs. TM showed that
Table 1. Fecundity of a natural population of Drosophila ananassae after medium was being tracked by females or males.
|UM TF TM|
|Isofemales with low fecundity|
|Isofemales with high fecundity|
Footnote: The fecundity data showed in the Table 1 are the total eggs laid by the females which really lay eggs from the eighteen tested for ten days. IF: Isofemale; UM: unconditioned Medium; TF: medium tracked by females; TM: medium tracked by males; No.♀♀: it refers to the number of females tested for 10 days; No.♀♀ Ovip: no. of females that really laid eggs for the ten days; Fr.♀♀Ovip: Eggs laid of female that really oviposit; No. of eggs: Total of laid eggs by females for the ten days. Eggs per♀: no. of eggs per female that really laid eggs.
Tracked by females Tracked by males
Tracked by females Tracked by males
Average No. of eggs / female + SEM
Average No. of eggs / female + SEM
3 4 5 6 7 8 9 10 11 12 13 14
Age of females (Days)
3 4 5 6 7 8 9 10 11 12 13 14
Age of females (Days)
Figure 2. This represents fecundity behavior during the ten days of monitoring. a) Female with low fecundity and b) with high fecundity.
TM stimulates significantly (p < 0.001) a greater fraction of females to oviposit rather than TF (0.54 ± 0.07 in UM; 0.53 ±
0.03 in TF and 0.83 ± 0.03 in TM).
Regarding the high fecundity IF, their fecundity decreased 25.14 % (from 11.85 ± 2.0 to 8.87 ± 1.58 h/f) in the TF although not significantly (p = 0.2). In contrast, the fecundity rose up 96 % in TM. The numbers of females which lied eggs in TF compare with TM, showed that TM stimulates signifi- cantly (p = 0.003) a greater fraction of females to oviposit rather than TF (0.66 ± 0.03 in UM; 0.60 ± 0.06 in TF and 0.83
± 0.03 in TM).
The fecundity behavior of both IF types during the ten days monitoring is shown in Figure 2: (a) refers to low fecundity IF and b) high fecundity IF, and represents the mean of h/f deposited in ten days ± SEM. The comparison between low and high fecundity IF’s curves showed significant differ- ences (p = 0.04) with a -0.2 and -0.6 laid eggs rate per day respectively, and a significant negative Pearson regression (r
= -0.73 and p = 0.01 for low and r = -0.81 and p = 0.01 for high fecundity IF). When females were allowed to oviposit in the TF or in the TM, no differences were found between both kind of IF, nor in the TF (p = 0.44) neither in the TM (p
= 0.27), however, contrary to the effect observed in controls groups, the laying eggs rates was positive 0.7 h/f per day for low fecundity IF (r = 0.89 and p < 0.001 ) and 0.9 h/f per day for high fecundity IF (r = 0.90 and p < 0.001) both in the TF. And 0.3 h/f in the TM for both types of IF, and a significant positive Pearson regression (r = 0.64 and p = 0.04 for low and r = 0.61 and p = 0.05 for high fecundity IF).
A dramatic increase in the fecundity was observed in both types of IF throughout the ten days monitored, when they laid eggs in a TM, the increase was therefore as many eggs laid (p < 0.001), as the number of females that laid eggs (p < 0.001), no differences were found between both kind of IF (p
= 0.27), see Figure 2. The viability comparison between eggs deposited in the UM, TF or TM demonstrated that the “trail” from males or females did not alter the survival of laid eggs, as shown in Table 2.
Table 2. Egg to adult viability laid on medium tracked previously by females or males of Drosophila ananassae from Laguna Verde, Veracruz, Mexico.
|Medium||No. of eggs||No. of adults||% of Viability ± SEM|
|UM||2880||1152||40.00 ± 2.2|
|TF||2880||1180||40.97 ± 1.5|
|TM||2880||1261||43.78 ± 5.3|
The culture medium was previously tracked for 24 h by twenty-five virgin females or males. From each IF, 240 eggs were put into ho- meopathic glass tubes with fresh medium in groups of 80 eggs for each. UM: unconditioned medium; TF: medium tracked by females; TM: medium tracked by males.
The results obtained with this study showed that female of D. ananassae laid eggs preferably in a medium tracked by males, and allowed to distinguish two IF types, although no experiments of polymorphism were made. Data obtained shows that TF medium provoked an increased rate of the laid eggs in both low and high fecundity IF related with the time. There are not previous studies pointing out this effect, so we can speculate that this result indicate that probably females also contribute to a signal component, which trig- gered a complex mechanism that provokes in fertilized fe- males to “decide” gradually to raise the fecundity in a partic- ular host site tracked by females from the same species, as to offer security to the progeny. Present data provide evidence supporting the “mother-knows-best” hypothesis, which is based on the idea that females will evolve preferences for oviposition sites in which offspring fare best  as a collec- tive effect between females from the same population. There are several factors known to influence female fecundity, fac- tors such as genes expressed in the male paragonial acces- sory gland in Drosophila, whose secreted products mediate the behavior and cause physiological changes in Drosophila females after mating. One is the PS-1, a peptide which causes a decrease of responsiveness in unmated females by males of the same strain, and the second one is the PS-2, a derivative of glycine carbohydrates which causes an increase in ovipo- sition when injected into unfertilized females [21,22]; either of the isolated components does not cause such a strong ef- fect as they do unfractionated.
However, because the increase in fecundity and female preference for oviposition eggs in a TM was overwhelming, “trail” released by males probably had a mechanism of ac- tion, which acted very differently from the “trail” released by females. Our results could support the Chen  work who found an increase in laid eggs through proteins trans- ferred by males and those found by Hoffman and Harshman
 showing that a male short-range or contact pheromone- stimulated oviposition in D. melanogaster females. It has been supported that in the behavior of the “decision” of the female, as to the most suitable site for oviposition, requires multiple sensory information, such as visual, olfactory, gus- tatory and own perception [8,25,26]. Studies have revealed that Drosophila male deposited the 9-tricosene pheromone when stimulated with the smell of food . This pheromone acts as a potent gregarious secretion and also plays a role of guidance cue for oviposition by the females; the pheromone is detected by an antenna receptor in female flies. This stim- ulates a specific type of brain cell causing the females to lay eggs in the place where the pheromone is deposited.
Lin et al,  showed that 9-tricosene activates the receptor Or7a present in antennas, which is a receptor activated by alcohols and aldehydes. Pheromone 9-tricosene, therefore, runs through the Or7a to link the odors of food perception, inducing the gregarious effect and finally taking the “deci- sion” to lay eggs. Lin et al  also suggest that 9-tricosene could temporarily mediate two different responses: one is the gregarious short-term lasting effect ~ 25 min, and the
second is the behavior of selecting oviposition sites to long- term and lasting hours. Detection thresholds for both be- haviors are different and dependent on the concentration; a higher concentration of 9-tricosene triggers the gregarious effect of females, whereas a low concentration affects the oviposition site selection.
The fact that there were no significant differences between low and high fecundity, confirms that the trait of the pref- erences for a certain place to lay eggs is a mechanism for reducing Intra and inter specific competition [10,11]. With our experimental design, where three types of culture medi- ums were tested, our results provided strong additional evi- dence that in a natural population of Drosophila ananassae the male “trail” plays a major role in the female “decision” to select the oviposition site. We also found evidence show- ing that females from natural populations of D. ananassae strong contribute on “decision-making” on the oviposition site. This topic is an opportunity to study which are the fac- tors involved in this mechanism.
We are grateful to Dr. Stanley Zimmering for encouraging this research. We would also like to thank Dora Luz Barron for technical help.
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