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The mite species, Brevipalpus phoenicus reproduces by thelytokous parthenogenesis, however occasional males are still produced and are seen to mate with females. The reasoning for this behavior is unknown. This paper therefore examines the hypothesis that these females are pseudogamous and in need of assisted fertilization in order to produce viable offspring. Colonies of Brevipalpus phoenicus were reared on Phaseolus vulgaris leaves and two sets of treatments were set up, leaves supporting 1 male and 2 females and leaves containing just 2 females. This was repeated twice under laboratory conditions (22 °C +/- 2 °C) and egg and larvae numbers counted every 2-3 days for approximately a month. Results showed that the average amount of eggs laid was lower in the female only colonies, however it was not significant with a p-value of 0.959. It was also found that the average number of larvae hatched was a little higher in the male and female colonies, but again the results were not significant with a p-value of 0.845. Whilst performing the preliminary experiment it was noticed that these mites died far more commonly when they were placed on their own, therefore an additional experiment was conducted to investigate the hypothesis that death rates decreased as colony size increased. Two females were placed on 3 leaves and 3 females on another 3 leaves. Egg and adult female numbers were then counted twice in the space of two-weeks. Results showed that 100% of females died on the single female leaves and over 50% survived on the 3 female leaves, although sample numbers were small. It is concluded that there is no significant evidence to suggest that males have a positive effect on the reproductive success of females. Although the discovery of potential social benefits in these mites has arisen and would therefore be a very interesting area of research to look into further.
Mites are hugely important in a wide range of areas, from agriculture to forensics. People have been interested in mites for a very long time and research into these creatures still continues today. The mite that this report focuses on is the species Brevipalpus phoenicus. After its discovery in Holland in 1939 this species has been reported worldwide (Haramoto, 1968). It can cause great agricultural losses directly by feeding on the plant, or indirectly by spreading diseases such as the citrus leprosies virus. Most damage can be seen in coffee (Coffea spp.) and citrus (Citrus spp.) plants in Brazil (Teodoro and Reis, 2006). Because of the economic impact of these mites, it is important that its biology, ecology and taxonomic relationships are fully understood in order to be able to understand and control this mite effectively. This species among other Brevipalpus species has a very complicated reproductive system. They are all asexual, however they still produce males. The main question that needs to be answered is when the female mates with the male besides being asexual are these females cheating or are they pseudogamous?
Most animal species reproduce sexually, whereby a male and female gamete is required in order to produce a new individual. The opposite of this would be asexual reproduction, whereby a female produces a daughter without the need for a male gamete (Groot, 2006). When looking at this it would seem obvious that sexual reproduction is the best way to reproduce, however it can be inefficient and costly. Costs can involve time and energy spent to find a mate, and males do not directly produce offspring (Groot, 2006). This conflict between sexual reproduction and asexual reproduction has become known as the paradox of sex and it a key issue in evolutionary biology (Groot, 2006). The Brevipalpus genus contains sexual and thelytokous species (Helle et al. 1980). However, B. californicus, B. phoenicus and B. obovatus, and are all said to reproduce by thelytokous parthenogenesis (Pijnaker et al., 1980). This form of reproduction is rare in the animal kingdom (White, 1984), and can be induced in hymeoptera by Wolbachia bacteria and Cardinium (Jeong and Stouthamer, 2005). Other species that this type of repoduction occurs in include, Cynipidae, Formicidae, Aphelinidae and Apidae (Suomalainen et al. 1987). It has been said that the females of B. phoenicus are haploid, as G banding patterns have been produced that suggest their chromosomes are not homologous (Pijnaker et al., 1980). This was backed up in 2001 when Weeks et al used fluorescent in situ hybridisation methods and microsatellites to prove that the chromosomes were not homologous.
Even though it has been found that these mites reproduce by thelytokous parthenogenesis, spanandric males have been reported many times in Brevipalpus (Pijnacker et al., 1980; Chigira and Miura, 2005). The reason for the production of these males is unknown. However, these males have been seen to mate with the thelytokous females (Groot, 2006), suggesting that there is a biological advantage of this act (Groot, 2006). This would in theory be very advantageous to the mites, because it would mean that the species could benefit from the advantages of sex at a small cost. This method of reproduction has been seen to happen in other species, for example aphids and daphnia (Innes and Singleton 2000; Vorburger et al. 2003). These species reproduce sexually in the autumn and when future changes in the environment are uncertain. This occurs because by reproducing sexually they produce more variation in their offspring and therefore if there are any sudden changes in environmental conditions there is more of a chance the offspring will contain individuals that are adaptable to the new environment and therefore increase their chances of survival (Groot, 2006). If occasional sex did occur in B. phoenicus in this way, it would mean that stress would be needed to induce occasional sex, this could be one of the reasons for the variation in outcomes of studies done in this area. For example an experiment done by Thomas Groot and Johannes Breeuwer (2006) failed to show successful occasional sex in Brevipalpus phoenicus. However another experiment done by Thomas Groot et al. (2006) showed that it might occur. The experiment that failed was performed in a room with stable temperature, humidity, and light regime (Groot, 2006). On the other hand it could be argued that the females were exposed to other kinds of stress, for example they were presented with different host species, treated with antibiotics and males harassed females. Because of these reasons it is unlikely that occasional sex happens due to stress (Groot, 2006). It is also said to be a possibility that the thelytokous Brevipalpus females receive sperm to fertilise their eggs (Lynch, 1984). This method of reproduction has been seen in other species, for example the thelytokous Trichogramma wasp (Stouthamer and Kazmer, 1994).
There are some studies that suggest that these males have no function for example Norton et al. (1993). However it cannot be ignored that this species still shows signs of sexual behavior, and it has been seen during this study that copulation occurs regularly, suggesting that these males are likely to be functional. Another example of how it is possible for these occasional males to have a function is that they are highly adaptable to a variety of different habitats. They can be found all over the world through the tropics and sub-tropics and have been reported from 486 plant species (Childers et al. 2003b). These mites have also been found to develop pesticide resistance very quickly (Campos and Omoto, 2002). This fast rate of adaptation and evolution is rare in asexual species (Colegrave, 2002) and therefore it seems highly feasible to assume that occasional males in the Brevipalpus phoenicus species do have a function.
Through the research and experimental methods performed during this study, the idea of the Brevipalpus mite being a social animal has arisen. Social behaviour has been seen in other Acari for example the suborder Prostigmata includes a high proportion of families that show different types of social behaviour (Choe and Crespi, 1997). The spider mite (Prostigmata, Tetranychidae) is an example of this and there are two species in particular that show highly developed subsociality. These species are Schizotetranychus longus and Schizotetranychus miscanthi (Choe and Crespi, 1997). Both these species have the same nesting habits and their complex social system can be seen in four different ways, two or three generations of mite live together in woven nests, they perform cooperative nest building, repairs and enlargement, they share nest space and resources and defend themselves from predators together (females defend brood and males against predators) (Choe and Crespi, 1997). However even though there is a sufficient amount of evidence to show subsociality there has been very little evidence of eusociality in Acari. An explanation of this is that their simple neural system decreases the chances of kin recognition (Choe and Crespi, 1997).
The aim of this study focuses on determining the presence of pseudogamy in asexual Brevipalpus and determines the degree of pseudogamy by comparing the number and quality of the offspring produced by mated and non-mated females. Pseudogamy is a form of parthenogenesis whereby females are required to mate with males in order to produce viable offspring, however no contribution to the offspring is made (Lanier and Kirkendall, 1986).
An additional experiment also aims to give an indication to whether this mite may be a social species. The results gained from this experiment will help us to understand why some asexual arthropods still produce males, and it will answer the question, do they really need males? It has been hypothesised that, in the male and female leaves, once the female has mated with the male, both the amount of eggs and number of viable offspring will be increased. A second hypothesis is that this species is a social animal and when singled out produces less eggs and viable offspring. Sexual and asexual reproduction both have their costs and benefits (De Meeûs et al. 2007) and if it is found that these males are functional then this mite would be very unique because they would have the ability to avoid the well known costs of asexual reproduction, but when needed, would gain all the benefits from sexual reproduction (Bourtzis and Miller, 2003). Very little work has been done into the social behavior of these mites and findings from this experiment will be a great help in developing a better understanding of this species.
Materials and Methods
Supply of Brevipalpus phoenicus
French beans (Phaseolus vulgaris)
11W G23 florescent light
Digital temperature/humidity gauge
Micro tools to move the mites
Planting the French Beans (Phaseolus vulgaris) and rearing colonies:
The first step to preparing the experiment was to grow a host, enabling colonies of mites to be reared on the leaves. The chosen host for this experiment was Phaseolus vulgaris as it is easy to obtain and grow. Small glass pots were collected and screwed up paper towels were placed in the bottom of the pots. Another sheet of paper then lined the pot and cotton wool was added, after this the paper towel was folder over the top of the cotton wool and water was added until saturated. Approximately ten beans where then pushed in around the paper towel and the Phaseolus vulgaris beans were left to germinate. They were watered every three days and this whole process was repeated every couple of weeks to ensure a constant supply of fresh leaves were available to maintain the colonies. Figure 1 shows the outcome of this process.
Figure 1: Set up of Phaseolus vulgaris beans. (Farmery, L. 2009)
Once the first set of leaves had grown they were cut off and placed in a plastic pot filled with water-saturated cotton wool, taking care not to get the top of the leaves wet. The stem of the leaf was covered with the cotton wool to enable it to take up water and consequently survive longer. A population of Brevipalpus mites, which originated from Brazil, was provided by a stock colony bred on bean leaves (Phaseolus vulgaris) and held in the Acarology Laboratory at Reading University. These leaves, where placed on top of the new leaves and the mites were left to move to the new leaf (figure 2). Again, this process was repeated approximately every two weeks throughout the experiment so the mites always has access to fresh leaves and a constant supply of mites was available for the experiment. Because these mites are used to living in hot climates (Denmark and Fasulo, 2006), and these mites are more productive at warmer temperatures (Sadana and Sharma, 1989) they were kept under a florescent lamp to encourage maximum production. This lamp was kept on for 16hours a day and turned off for 8 hours at night to ensure the photoperiod stayed constant. Development rates are also said to be affected by relative humidity (Chandra and Channabasavanna, 1974), therefore, temperature and humidity levels were recorded every 2-3 days to ensure conditions remained fairly constant (appendix D).
Results show that even though the average number of eggs laid per female is slightly higher on the male and female leaves, it is not significantly different. Therefore we do not have enough evidence to reject the null hypothesis that the presence of males has no effect on the production of eggs, and therefore these mites are unlikely to be pseudogamous. This is likely to be due to the high amount of variation seen within the same treatments. A higher sample number would be needed to counteract this large variation and in the experimental period allocated it was not possible to conduct more repeats. There were also other reasons for the small number of repeats which will be discussed later in this section. However, if these males do not effect the production of eggs, they must be there for another reason, and these reasons must also be discussed.
Interesting trends can be observed when breaking the data up into individual leaves, as seen in graph 8 and 9. It is clear to see that females die much quicker on the female only leaves compared to the male and female leaves. This would have ultimately caused the egg numbers to be lower on the female only leaves and would mean that it was not necessarily because the females were less productive. In fact when looking at the variation graphs, it can be seen that females that stay alive actually produce more eggs per female than on the male and female leaves. Therefore the only reason the female only leaves average out as lower than the male and female leaves is due to the high proportion of deaths. The fact that the surviving females on the female only leaves produced more eggs is an interesting point. It can be seen that the highest number of eggs laid per female on the male and female leaves was 20 eggs, however on the female only leaves the maximum of 24.5. This reinforces the conclusion that that these mites are unlikely to be pseudogamous and there is even a possibility that males could be detrimental.
It is inferred that the percentage of eggs that hatched into larvae is higher in the male and female colonies, however again the difference is so slight that they are not statistically different and therefore it is unlikely that the presence of males has a positive effect on the viability of eggs laid by the females. However, it was found during the experiment that all of the males had died by the end of the experimental period, therefore it is still possible that if the males were present throughout they could have some effect on the viability of the eggs. This however seems unlikely because when observing the mites it was clear to see that the females spent a large amount of time close together on the leaf and very near their eggs. The females were even observed stroking the eggs on a number of occasions. However, when the males were alive they were very rarely seen near the eggs or the females, suggesting that they would be unlikely to have had any effect on the development of the egg. It is not known whether the stroking behaviour seen by the females has any effect on the eggs development, however, it is more of a possibility that the deaths of the females would have reduced the viability of the egg. If the presence of these males is not affecting the viability of the eggs then, as mentioned before, there must be another reason for their presence.
The fact that females seemed to walk from the leaves and drown in the saturated cotton wool much more frequently when alone suggests that there maybe some advantage to being in a larger groups. When performing the preliminary experiment, a very high proportion of deaths were experienced. Changes were then made to the actual experiment to try and reduce these deaths. The leaves were made smaller and the females were put into pairs. These actions seemed to reduce the number of deaths. It was also observed that the mites were nearly always found close together on the leaves, this was the reason for the secondary experiment. The outcome of this experiment gave some very exciting results that showed, when a single female was placed on a leaf, 100% of them died by the end of the 2 week experimental period and when 3 females where placed on a leaf in the same conditions over 50% of them had survived. The presence of these extra females therefore seem to decrease the likelihood of them walking off the leaves into the cotton wool and supported our hypothesis that these mites are social species and that their survival and production rate is much greater when placed in groups. It could be possible that these mites release a pheromone when in groups which helps with their orientation on the leaves, reducing the chance of deaths by walking off the leaves into the cotton wool. However, as the sample number was small, further work would need to be done into this interesting area of research in order to investigate whether the survival of these mite species is effected by colony size.
A problem was experienced halfway through the experimental period that prevented more repeats from being conducted in the first and second experiment. This was caused by the heating being turned off on a cold night in the lab, this caused 90% of stock mites to die. Because of this the remaining mites had to be saved by placing them in a warm incubator at 24 ° C, however the mite populations would take over a month to recover, so due to time restrictions only a few of the surviving mites could be experimented on, causing the number of repeats to be lower than planned in the first experiment and the second experiment to be changed. The initial plan for the secondary experiment on social behavior was to collect 3 pots and fill them with cotton wool saturated with water, as done previously. Six leaves were then to be placed in each and in the first pot of six beans one female was to be placed on each, in the second two females were to be placed on each and in the third 5. These pots were then to be observed in exactly the same way as the previous experiment. However, it was only possible to look at 6 leaves, 3 with 1 female on and 3 with 2.
Other possible reasons why males are produced
From the results gained from this experiment it has to be concluded that the presence of males does not have a positive influence on the number of eggs produced, or the viability of these eggs. If this is the case then there must be another reason why occasional males are still present in this species of mite. It may just be that it is physically impossible for these mites to produce only females, however, this is somewhat unlikely as, from observations in this experiment it was seen that no males were produced for a period of over a month
Another reason for the presence of these males could be put down to recent evolutionary changes. Signs of mating behavior are still present, for example mate guarding was observed and mating occurred very soon after the males were placed onto the leaves containing the two females. This could suggest that this species used to be sexual and have recently become asexual, therefore still show this typical sexual behavior (Groot and Breeuwer, 2006b). If this is the case the genes that control this sexual behavior will eventually be phased out by deleterious mutations (Carson et al. 1982)
It is known that this species is feminised by the bacteria Cardinium (Kitajima et al. 2007). However it has been observed by Groot and Breeuwer (2006a) that males are only produced by very young females. This stimulates the idea that it is possible that when the females are very young the levels of bacteria contained within them are too low to suppress the production of males, and this is why males are still present in the population. This idea has also been put forward by another paper by Groot and Breeuwer (2006b).
Future changes that could be made to the experiment if it were to be repeated
When looking at the raw data it can be seen that the eggs took a fairly long period of time to hatch. The first larvae were observed approximately 2 weeks after the first eggs were laid. When comparing this to other experiments this is slightly longer. For example in a study done on tealeaves the developmental time of a B.phoenicus egg was approximately 10 days (Kennedy et al. 1996). However, Kennedy's experiment was conducted at 26 ° C and the approximate temperature throughout this experiment was 22 °C. It was mentioned before that if temperatures go below 20 ° C the eggs will not hatch (Haramoto, 1968) and the mites can die during their immature stages (Kessing and Mau, 1992). Therefore it could be that the low temperature in the lab slowed down the development of the eggs. However, heat lamps were used for 16 hours of the day in order to keep the photoperiod the same and a heater was left on, so the temperature, humidity and light was kept constant throughout the experiment (with exception to the uncontrollable circumstances noted previously, which involved the inadvertent turning off of the heating supply by a third party). It has been found that more eggs are produced during summer than winter and during day than night (Zhang, 2003). Due to time restraints in the experimental period it was not possible to conduct the experiment in the summer months, however, if this experiment were to be repeated, it would be suggested that it was done in the summer, as this is when the mites are at their peak. Another reason for this would be that during the winter period it was observed that not only do the females lay fewer eggs, but also the proportion of males produced is a lot lower. The reason for this is unknown as the conditions in the lab were kept constant, therefore the reduction in male production must be caused by factors other than temperature, humidity and light. More work needs to be done in this area to make it clearer why egg and male production changes throughout the year. This resulted in our experimental repeats being limited as there were not enough males produced to conduct more repeats.
This study has revealed that these mites are not completely pseudogamous, as viable offspring were still produced by non-mated females. However it was found that on average the female only colonies did produce fewer eggs than male and female colonies. These results were not significant although this may have been due to the small number of repeats, thus it is still possible that a degree of pseudogamy may be present. Furthermore, it was discovered during this experiment that death rates and consequently the productivity of these mites appear to be very reliant on colony size. The results from the experiment conducted on this provided some very promising results, suggesting that these mites have a higher survival rate when placed in larger groups. This would therefore be a very interesting area of research to take further.
I would like to thank my supervisor Dr. Alejandra Perotti for her help, advice and support throughout this project. I would also like to thank Rebekah Barnard who worked with me on the research for this report.