Excerpt
Table of contents
1. Abstract
2. Introduction
3. Material and methods
4. Results
4.1 Duration of behaviors
4.2 Chronological occurrence of behaviors
4.2.1 Comparison of the behaviors occurring in the different treatment groups
4.2.2 Separate analysis of occurrence in each individual behavior
5. Discussion
6. Acknowledgment
7. References
1. Abstract
Aggressive behavior is widespread among animals and crucial for survival and passing on their genes. Aggressive behavior therefore mostly occurs during competition for resources like mating partners, nutrition and territory. Hence the burying beetle Nicrophorus vespilloides also use aggressive behavior to protect its brood.
In previous studies the JH III (Juvenile Hormone III) titer in burying beetles could be identified to be causal for brooding behavior and therefore also causal for the increased aggression during breeding time.
The regulation of JH titer in insects is unclear to this moment. Peptides with a tetrapeptic ending like FMRFamide have been suggested in the past to play a role in the regulation. Hence the effect of FMRFamide as a potential JH releasing factor also was investigated in burying beetles. This effect could not be confirmed to this date however a correlation between aggression and FMRFamide was found.
In this bachelor thesis the behavior of N. verspilloides was further investigated through a detailed behavioral study. In this study fighting videos between N. vespilloides residents (females with larvae and a carcass) and intruders (females without larvae and a carcass) were evaluated. Half of the residents were treated with a FMRFamide solution and the other half there treated with a saline solution (control group). The FMRFamide treatment results in a more aggressive behavior in the females. This reaffirm the role of FMRFamide as an agent which has an influence in triggering aggressive behavior in burying beetles.
2. Introduction
Burying beetles as the examined Nicrophorus vespilloides are members of the family Silphidae, commonly known as carrion beetles which belong to the order Coleoptera. Nicrophorus is a northern hemisphere genus about 75 species. Both, population densities and species diversity are higher in northern localities where habitat generalists and habitat specialists occur in sympatry (Scott 1998).
Perhaps the most striking aspect of the behavior of burying beetles is their parental care (Trumbo 1996; Scott and Panaitof 2004). Extended maternal care is unusual and scarce in insects, and the substantial investment by males is even more surprising (Scott 1998). When a male has succeeded in attracting a mate, the two beetles bury the carcass together to the ground, prepare it for the larvae and then reproduce on it (Eggert 1992). Eggs are laid in the soil nearby (Scott 1998). The carcass is shaped into a ball and later the parents regurgitate predigested carrion to the young larvae on the carcass (Eggert et al. 1998). The reproductive period of N. vespilloides is between late April and September and this burying beetle species is multivoltine, that means they have several broods in one season (Scott 1998). In contrast to other burying beetle species N. vespilloides survives without parental feeding (Reinking and Müller 1990). Males of this species exhibit relatively long paternal care: the males stay between 8 and 9 of total 16 days of parental care (Scott 1998). Experimental studies of biparental care have not detected any benefits arising from biparental feeding in the laboratory, but have shown that biparental care may lower the risk of brood failures. Brood failures can occur when superior congeneric competitors (intra- or interspecific) detect the buried carcass and destroy the brood present on the carcass prior to establishing their own brood (Eggert et al. 1998). However, resident males and females attempt to drive off the intruders. The beetles are able to recognize their breeding partners and to distinguish them from intruding infanticide conspecifics (Müller et al. 2003). This discrimination depends on the breeding status of the encountered beetle: breeding beetles of the opposite sex are generally accepted as partners, whereas nonbreeding ones of both sexes are attacked (Müller et al. 2003). Escalated fights can lead to severe injuries; even fatal fights occur (Eggert 1992). Beetles undergo physiological changes during a breeding attempt on a carcass, which include a dramatic increase in Juvenile Hormone III (JH III) level until a peak is reached on the first two days on which larvae are present on the carcass, and the intensity of parental care is highest (Panaitof et al. 2004). The Juvenile Hormone III is therefore responsible for the induction of the complex behavior (Trumbo et al. 1995; Panaitof et al. 2004).
Previous studies suggest that cuticular hydrocarbons, specifically polyunsaturated hydrocarbons, signal breeding state (Steiger et al. 2007). The question of how these hydrocarbons could be reliable indicator of breeding or hormonal state remains unsolved. It is possible that the production of specific hydrocarbons is necessarily linked to a high JH III titer. Earlier theses studied the link between JH III titer and aggressive behavior in burying beetles (Steiger et al. 2008; Haberer et al. 2010).
Aggressive behavior is widespread among animals. It mostly occurs during competition for resources like mating partners, nutrition and territory. Aggressive behavior is not only expressed in actual fighting which is scarce due the risk of injury to both participants. The ferocity of aggression could be limited through communicative display like threatening behavior. Threatening behavior can save both contenders against serious injuries which would decrease the survival and reproduction rate (Huntingford and Turner 1987).
In this thesis typically aggressive and non-aggressive behaviors should be identified and clarified. Furthermore, the question should be answered to what extend the treatment with FMRFamide has an influence in the showing of aggressive behavior of N. vespilloides. Specifically, in which way the administration with FMRFamide changes the duration of the specific behaviors and in which way it changes the succession pattern.
3. Material and methods
This bachelor thesis founds on the master Thesis “Influence of RFamide on the aggressive behavior of female burying beetle – A behavioral, immunocytochemical and pharmacological study” written by Annika Verena Lipp in 2015. For further details, please refer to that thesis. The thesis wanted to differentiate the aggressive behaviors which are shown by Nicrophorus vespilloides during the defense of their larvae. Furthermore, it wanted to show potential differences between the behavior on day 4 and 7 of the breeding cycle as if FMRFamide have an influence on aggressive behavior in resident females.
To achieve this, the nest defenders were separated in a manipulated group, which was treated with 30 µl RFamide dissolved in saline with a concentration of 0.001 g/l on the 4. and 7. day after breeding start and a control group which was treated with 30 µl mosquito saline on the same days.
Videos which were recorded originally for the mentioned master thesis by Annika Verena Lipp were further analyzed by me. Each video showing a 10 minutes long fight between a defender (manipulated group/control group) and an intruder female of N. vespilloides in a fighting arena on day 4 and 7 after breeding start.
All recordings were observed utilizing the Media Player Classic – Home Cinema program (MPC-HC (64-bit), version 1.7.10 (d911f14)). Each in the results considerer defender fought 2 times. The first time 4 days after mating the second time 7 days after mating.
At the beginning of the bachelor thesis behaviors were selected which should be documented hereinafter. The chosen behaviors are listed in table 1.
Table 1 : All considered Behaviors
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An analysis was made of each video trough picture by picture run through, slow motion and real time replay. The in table 1 listed behaviors were written down in an excel file with the beginning time and ending time of each observed behavior. After that the differences of ending time minus beginning time plus one second were formed. Thereby a recorded behavior lasted at least one second and the time resolution was one second therefore. The duration time of the category backsitting and biting was formed with the times in which the behavior backsitting was recorded simultaneously along with biting. Furthermore, with the first beginning time of each behavior category in every fight the order of behaviors was formed.
During the bachelor thesis it was noticed that many of the individuals fought 3 times instead of the expected 2 times. To ensure the comparability the already evaluated videos in which the individuals fought only 2 times were excluded from the Results.
Afterwards the excel files were analyzed and illustrated using R x64 version 3.3.0 (2016-05-03). Wilcoxon Rank Sum and Signed Rank Tests were used for the paired data.
4. Results
The behavior was evaluated in two different ways. First, the durations of behaviors on day 4 and 7 with and without RFamide treatment were shown. Second, the chronological order of the first occurrence of these behaviors were compared.
The following figures demonstrate the durations of the observed behaviors. The sample size for each of the 4 different groups is n = 7. The data were tested with the Wilcoxon signed rank test. For a better overview only significant data is further named. Please refer to the attached CD for more Data.
4.1 Duration of behaviors
In the behavior entanglement no significant differences were observed between the individuals of the different groups. Nevertheless, the duration of the entanglement between the treated residents with the intruders on day 7 tend to be longer (Figure 1).
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Figure 1 : Duration of entanglement observed between residents with intruders on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the aggressive behavior entanglement. Entanglement is the behavior in which both individuals are turned towards each other and are intertwined. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Day 7 RFamide treated residents with intruders showed more entanglement.
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Figure 2 : Duration of backsitting of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior backsitting. Backsitting is the behavior in which an individual sitting on the other. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Backsitting was observed longer in untreated than in treated residents. Mostly in untreated residents on day 7.
On day 7 the non-aggressive behavior backsitting was observed longer in untreated than in treated residents (Figure 2). This is consistent with the time of occurrence, backsitting was the second earliest behavior observed in the untreated residents on day 7 (Figure 13) but the treated residents showed the behavior in seventh place (Figure 17).
The behavior backsitting and biting was observed significantly more often in treated and untreated residents of the day 7 than in the intruders of the same day. (Wilcoxon Signed Rank Test: T7 p=0.03125, UT7 p=0.04688). The residents showed compared with each other similar high durations, except the treated ones of day 7 which stand out with a much higher duration time (Figure 3). Further on day 7 the residents showed backsitting and biting as the fourth earliest behavior which is the earliest rank compared to the other groups (Figure 17).
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Figure 3 : Duration of backsitting and biting of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the behavior backsitting and biting. The behavior backsitting and biting is the behavior in which the behaviors backsitting and biting are shown simultaneously. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Asterisk indicates significant differences * = p < 0.05 ** = p < 0.025. Remarkably is that the treated residents on day 7 showed most often this aggressive behavior.
The behavior biting was observed significantly more often in treated and untreated residents than in the intruders of the same day. (Wilcoxon Signed Rank Test: T4 p=0.01562, T7 p=0.01562, UT4 p=0.04688, UT7 p=0.03125). Treated residents on day 7 showed the longest duration (Figure 4).
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Figure 4 : Duration of biting of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the aggressive behavior biting. Biting is the behavior in which one individual spread the mandibles and have contact with the other individual. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Asterisk indicates significant differences * = p < 0.05 ** = p < 0.025. Note that treated residents on day 7 showed most often this aggressive behavior.
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Figure 5 : Duration of bodymove of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior bodymove. Bodymove is the behavior in which one individual is stretching the legs unilaterally upwards and moving the body on the side. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Asterisk indicates significant differences * = p < 0.05 ** = p < 0.025. Note that the median in the residents is the lowest in treated residents on day 7.
The bodymove was observed significantly more often in treated and untreated residents than in the intruders of the same day. (Wilcoxon Signed Rank Test: T4 p=0.01562, T7 p=0.01562, UT4 p=0.03125, UT7 p=0.04688) (Figure 5).
Notably is that the treated residents on day 7 showed the lowest median of the residents groups, this coincided with the median of the rank of occurrence of this group, which is the highest of all resident groups (Figure 23)
The behavior stridulation showed no significant differences between intruder and residents. (Figure 6). Both, the treated residents and their intruders on day 7 showed the highest median of all residents respectively all intruders.
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Figure 6 : Duration of stridulation of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the aggressive behavior stridulation. Stridulation is the behavior in which an individual rapidly move the abdomen up and down to generate a noise. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Note that the treated residents on day 7 as their intruders, each have the highest median compared with the other groups.
The behavior avoidance was observed significantly more often in intruders than in the treated and untreated residents of the same day except in the group of the untreated residents of day 7. (Wilcoxon Signed Rank Test: T4 p=0.01562, T7 p=0.01562, UT4 p=0.01562) (Figure 7).
Remarkably is that the highest median of this defensive behavior is observed in the intruders which their paired with the treated residents of day 7 which seemed to be the most aggressive group.
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Figure 7 : Duration of avoidance of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior avoidance. Avoidance is the behavior in which an individual is running or rapidly move in the opposite direction of the other individual. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Asterisk indicates significant differences * = p < 0.05 ** = p < 0.025. Note that avoidance is drastically more often observed in the intruders than in the residents. Also the highest median is observed in the intruders which their paired with the treated residents on day 7.
The non-aggressive behavior cleaning showed no clear trends. The highest interquartile range were observed in the untreated residents on day 7 which seemed to be the most non aggressive group of all residents (Figure 8).
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Figure 8 : Duration of cleaning of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior cleaning. Cleaning is the behavior in which an individual is swiping with extremities over the body. Often over the antenna or the abdomen. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Note that the untreated residents on day 7 have the highest interquartile range.
The next behavior, flying was nearly never showed and therefore not further respected in this thesis (Figure 9).
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Figure 9 : Duration of flying of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior flying. Flying is the behavior in which on individual is flying through rapid wing beating. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7.
The behavior climbing was observed significantly more often in intruders than in the residents of the same day except in the group of the treated residents of day 4. (Wilcoxon Signed Rank Test: T7 p=0.01562, UT4 p=0.04688, UT7 p=0.01562). Moreover, this is the only observed behavior in which a significant difference could be observed among groups in the residents themselves. Namely the untreated residents of day 4 climbed significantly longer than the untreated residents of day 7 (Wilcoxon Signed Rank Test: p=0.04688) Furthermore also the treated residents tend to climbed less at day 7 as at day 4 (Figure 10).
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Figure 10 : Duration of climbing of resident and intruder on day 4 and 7 after breeding. The y-axis shows the ratio of durations of the non-aggressive behavior climbing. Climbing is the behavior in which one individual move on the wall. The x-axis displays the different groups T = treated UT = untreated 4/7 = experimental day 4/7. Asterisk indicates significant differences * = p < 0.05 ** = p < 0.025. Remarkably is that untreated and treated residents on day 4 showed much more climbing than on day 7. Also note that intruders climb more often than residents.
4.2 Chronological occurrence of behaviors
The rank of occurrence is described in the following in two different ways. Firstly, all behaviors are compared in the different beetle groups. Secondly each individual behavior is illustrated separately with the difference in the rank of occurrence between the 4 resident groups.
4.2.1 Comparison of the behaviors occurring in the different treatment groups
No significant differences were observed in none of the beetle groups through the Kruskal-Wallis rank sum test. Nevertheless, interesting trends could be observed which are highlighted in the following.
Table 2 Categorization of all observed behaviors in aggressive and non-aggressive behaviors
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All behaviors were sorted in aggressive and non-aggressive behaviors (Table 2). Therefore, only the untreated residents of day 4 and 7 were considered (Figures 11 and 13). All behaviors which were performed far earlier on day 4 as on day 7 were defined as aggressive behaviors. The behaviors which showed no clear difference in the rank of occurrence or showed earlier on day 7 are regarded as non-aggressive behaviors. The almost never observed behavioral fly was discarded (Table 2).
Remarkably is that the as highly aggressive suspected behavior backsitting with biting was observed as the latest behavior in all intruders (Figures 12,14,16 and 18).
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