Characterizing the Putative Disperser Morph in Naked Mole-

Rats

by

Ilapreet Toor

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Ecology and Evolutionary Biology

University of Toronto

© Copyright by Ilapreet Toor 2017

ii

Characterizing the Putative Disperser Morph in Naked Mole-Rats

Ilapreet Toor

Master of Science

Ecology and Evolutionary Biology

University of Toronto

2017

Abstract

Naked mole-rats are eusocial mammals; their hierarchy consists of one breeding female, 1 to 3

male consorts, and their reproductively suppressed siblings and offspring. A potential “disperser”

subcaste has been suggested. The purpose of the current study was to better characterize the

putative disperser morph. I hypothesized that dispersers and workers would exhibit different in-

colony and out-colony behaviors, colony characteristics would affect the presence of dispersers,

and dispersers would reproduce quicker than workers. The results found that naked mole-rats of

both sexes consistently dispersed. Male dispersers and workers had minimal differences in in-

colony behaviors and both preferred familiar colony odor. Queen aggression increased presence

of female dispersers only. Dispersers were heavier and fatter than workers. Dispersers and

workers produced litters at a similar rate to each other. These results suggest that dispersers may

not be a separate subcaste, but rather a subset of the existing workers with increased exploratory

behavior.

iii

Acknowledgments

I would like to begin by thanking my supervisor and mentor, Dr. Melissa Holmes, for her

continuous guidance and support through my undergraduate and graduate career. Who knew a

little bit of bravery in approaching my professor would inspire what will be a life-long pursuit of

scientific discovery?

I would like to thank the Holmes lab and the UTM vivarium staff in helping me through this

project. I would specifically like to acknowledge Nagham Kaka and Rebecca Whitney for their

work in behaviour scoring. Also, a special thank you to Celina Baines from the McCauley lab in

helping me with my statistical analysis, and providing valuable advice on how to achieve success

in graduate school.

I would like to thank my graduate committee members Dr. Helen Rodd and Dr. John Ratcliffe

for their constructive feedback, ideas, and contributions to improving my project as well as the

resulting documents and presentations. I would also like to thank my defense committee, Dr.

John Ratcliffe, Dr. Darryl Gwynne, and Dr. Marla Sokolowski for taking time to read this

document and attend my defense. It is an honor to discuss my findings with professors many

only get to hear about in textbooks and journals. Finally, special thanks to Dr. Helen Rodd in

helping me navigate the EEB program.

Lastly, I would like to thank my family and friends for their continuous encouragement in my

pursuit for higher education. A special thank you to my best friend and trilingual literary genius

Palweet Kaur Dhaliwal for taking the time to meticulously read and edit this document.

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Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

Introduction ......................................................................................................................................1

Objective ........................................................................................................................................11

Methods..........................................................................................................................................12

Results ............................................................................................................................................19

Discussion ......................................................................................................................................27

References ......................................................................................................................................36

Appendices .....................................................................................................................................44

1

Introduction

Eusociality –

Eusociality is the most extreme form of social organization defined by 1) reproductive

division of labor, 2) overlapping generations within a colony, and 3) cooperative brood care

amongst colony members (Wilson, 1971). Reproductive division of labor refers to cases where

some animals are reproductively active, while others are suppressed or sterile. The origins of

eusociality have often been attributed to kin selection, an evolutionary strategy favoring the

reproductive success of relatives other than offspring. Eusociality is most commonly and

famously found in the order of Hymenoptera which includes ants, bees, and wasps, and much of

what we know about eusociality has been learned from these animals. In the past, the leading

hypothesis of how eusociality evolved in Hymenoptera was their unique haplodiploid genetic

system. Haplodiploidy was described as a genetic sex determining system where males develop

from unfertilized haploid eggs, and females develop from fertilized diploid eggs. In this system,

females inherit the full genome of their haploid father and half of the genome of their diploid

mother, thus making sisters 75% related to one another. In contrast, sisters are only 25% related

to their brothers because males inherit 50% of their mother’s genome and none of their father’s

genome. This makes it highly beneficial in reproductive fitness terms for female Hymenoptera to

help raise their sisters rather than establish their own colony, where they would be 50% related to

their offspring (Hamilton, 1964; Wilson, 1971). Recent literature has proposed that monogamous

pairings are a precursor to genetic relatedness which drove the evolution of eusociality. If kin

selection is a precursor to genetic relatedness, then monandry (mating with only one male) would

be a precursor to kin selection as mating with a single male increases genetic similarities

between siblings. It has been shown that monandry is ancestral to polyandry for all eight

2

independent origins of eusociality. Totipotency, the flexibility to either mate or remain

reproductively suppressed, is also affected by the type of mating a certain species follows.

Totipotency in worker caste animals is highly common in polyandrous species since there is

always a potential that siblings are not directly related, whereas monandrous species tend to lose

totipotency since all siblings are related by a single father. This suggests that ecological factors

and pair-type is linked to the evolution of eusociality, and to the variation of totipotency seen in

the working caste where kin selection works stronger in truly related siblings (Hughes et al,

2008). A recent phylogenetic study presented evidence of sex determination genetics not being

significantly correlated with sex ratio of worker caste animals, adding to the notion that ploidy

and genetic relatedness may be a byproduct of ecological pressures pushing the evolution of

eusociality. Indeed, this study presented findings of sex ratios of working castes being influenced

by the ecological advantage of social living where species with more brood care had a higher

number of female workers, and species with colony defense and brood care had a more equal sex

ratio (Ross et al, 2013).

Understanding the role of ecology aids in better understanding eusociality in diploid

animals, such as in termites. Being diploid means both males and females receive genetic

information from both parents. Thus, there is a lack of relatedness asymmetry and all termite

siblings are 50% related to each parent and to each other. Due to this, an ancestral genetic basis

for eusociality in termites was essentially overruled (Lin and Michener, 1972). The running

hypotheses for how eusociality evolved in termites turned to social factors, one of them being

maintenance of intestinal symbiotes. Termites must transfer cellulose digesting microorganisms

after hatching and after each molt, which is done via anal feeding (Bartz, 1979). It is proposed

that this necessity to be in close contact with kin is what pushed eusociality in termites (Lin and

Michener, 1972; Bartz, 1979). In addition, inbreeding resulting in relatedness asymmetry has

3

been proposed as a strong mechanism in the evolution of eusociality in diploid animals. Bartz

(1979) explained at a mathematical level how animals who inbreed between siblings or between

parent-offspring have offspring that are more genetically similar to their siblings than they would

be to their own offspring. This would create a relatedness asymmetry in diploid animals that

would be between both male and female siblings, not just between sisters as in Hymenoptera

(Lin and Michener, 1972). In relation to when and how inbreeding occurs in termites, it has been

noted that there are “supplementary reproductives” that exist within a termite colony. These

animals are the offspring of the initial established pair and will replace either member of the pair

if there is a mortality (Krishna and Weesner, 1971). The occurrence of supplementary

reproductives was also found to be associated with the occurrence of alates, winged termites that

disperse to create their own colony. It was hypothesized that alates are the offspring of

supplementary reproductives since they always co-exist within a colony, which would further

maintain inbreeding to a level where eusociality remains (Nutting, 1970). As for origins,

termites evolved from wood-feeding cockroaches which would have been found commonly in a

rotting wood environment (Hamilton, 1978). Forced congregation within a very specific

environment would have then enforced inbreeding, laying the foundation toward eusociality

(Hamilton, 1978; Bartz, 1979).

The hypothesis of kin selection favoring and aiding the evolution of eusociality has been

popular, but there have been hypotheses that argue against this notion. Namely, it was explained

by E.O. Wilson and B Holldobler that group selection of alleles is more likely to drive the

evolution of eusociality, and kin selection would only play a minor role (Wilson and Holldobler,

2005). Wilson and Holldobler explained that at the simplest level, there is an allele for altruistic

behavior and phenotypic flexibility that would be selected under appropriate environmental

pressures. Thus, there is group selection taking place for a specific allele. After this, there can be

4

kin selection taking place that increases relatedness and promotes altruism and eventual

eusociality, but it is not essential. This is because the focus is on phenotypic flexibility resulting

in a “worker caste” as well as a push for eusociality. Technically, eusociality can then develop in

unrelated individuals, but it is more probable that such alleles would be spread through relatives.

The hypothesis that the evolution of eusociality is via group selection instead of kin selection has

some empirical support (Wilson and Holldobler, 2005), but is controversial (e.g.; West et al,

2007a; West et al, 2007b; Pievani, 2014).

Dispersal –

Dispersal is described as a one-way trip away from natal groups, as opposed to migration

which is to-and-fro. Dispersal can be difficult to detect or even study since it occurs at a

relatively low frequency, just enough to counter-balance extinction (Semlitsch, 2008). However,

recent technologies such as genetic markers (microsatellites) make it possible to study dispersal

in increased detail (Peakall, Ruibal, Lindenmayer, 2003). Dispersal is an important contributor

for gene flow, distribution patterns, population regulation and stability, extinction, and

recolonization (Chepko-Sade and Halpin, 1987; Johnson and Gaines, 1990; Dieckman et al,

1999). There is a hump-shaped relationship between dispersal frequency and local diversity in

metacommunities, or patchy habitats that are interconnected via dispersing species. Specifically,

with an absence of dispersal there is low local diversity within metacommunities which can

ultimately lead to the extinction of species. As dispersal increases, local diversity also begins to

increase but only to an intermediate level. Beyond this level, an excess of dispersal will result in

domination of a single population, thus decreasing diversity again (Kneitel and Miller, 2003).

Dispersal is an adaptive trait in various situations. One of these situations is for inbreeding

avoidance; highly inbred offspring can experience a myriad of health problems and often do not

5

survive to adulthood (Pusey and Wolf, 1996). Dispersal is also adaptive in increasing inclusive

fitness and decreasing kin competition. By leaving their natal grounds, animals can spread their

“familial” genes thus increasing inclusive fitness, while avoiding engaging in mate competition

or resource competition with their own kin (Perrin and Mazalov, 2000). Ultimately, the leading

hypotheses fueling dispersal relate the process to competition for mates (Dobson 1982).

Sex biases are often found in dispersal and vary with mating systems. Birds are often

monogamous and have a female bias in dispersal (Greenwood, 1980; 1983; Greenwood and

Harvey, 1982). In contrast, mammals have a polygynous and promiscuous mating system and

males are more likely to disperse. For example, male ground squirrels often disperse from their

natal area to establish territories, to gain access to females for mating. Given that more dominant

males usually maintain their territories until death, male dispersal is necessary for reproduction.

Females on the other hand are much more likely to already be a part of a male’s territory and

remain close to the natal area (Evans and Holdenried, 1943; Dobson, 1979). Interestingly, in the

few mammal species that are monogamous, both sexes disperse (Dobson, 1982; Greenwood,

1980). Research has also found that in species that exhibit no parental care, such as some

reptiles, males are still more likely to disperse than females (e.g.; Tucker et al, 1998; Dubey et al,

2008; Rivera et al, 2006).

Eusociality in Mammals –

It was proposed by R.D. Alexander that if there were to be a eusocial mammal, it would

likely be a rodent, have a subterranean lifestyle, and rely on an abundant risk-free food source

(Sherman et al, 1992; Bromham and Harvey, 1996). Two eusocial mammals, members of the

Bathyergidae family, were discovered that matched this description. These two African mole-rat

species are the Damaraland mole-rat (Cryptomys damarensis) and the naked mole-rat

6

(Heterocephalus glaber) (Jarvis & Bennett 1993). The evolution of eusociality within the

African mole-rats occurred twice and independently of each other, providing evidence of

convergent evolution (Honeycutt et al, 1991; Jarvis & Bennett 1993; Faulkes et al, 1997; Faulkes

et al, 2004). It was believed that eusociality in the naked mole-rat (NMR), but not the

Damaraland mole-rat (DMR; Burland et al, 2002), is a product of inbreeding similar to how

Hamilton’s theory of kin selection was used to describe eusociality in insects. High relatedness

of NMRs to siblings would benefit them from a genetic fitness perspective (Hamilton, 1964;

Sherman et al, 1992).

It has also been proposed that environmental factors play a role in the evolution of

eusociality. African mole-rats range from solitary to social in behavior which is believed to be

influenced by climate. The Food Aridity Hypothesis (FAH) has often been used to explain the

variation in African mole-rat sociality, and the evolution of eusociality as well (Faulkes et al,

1997). Sociality is correlated with habitat aridity and food distribution. Solitary mole-rats inhabit

more mesic areas with predictable food sources comprised of geophytes, plants bearing

underground food-storage organs such as tubers. In contrast, social mole-rats are found more

often in arid regions that have unpredictable rainfall. Soil is much harder to dig through in arid

regions as well, resulting in higher energetic costs of burrowing in comparison to mesic areas.

Arid regions with low rainfall also have patchy food sources that increase the risk of

unsuccessful foraging. These factors make it difficult to maintain a solitary lifestyle in an arid

environment and promote sociality for survival. The discouragement of solitary behavior may

also result in very high costs of dispersal (Faulkes et al, 1997). Faulkes et al presented evidence

to support the FAH by looking at mole-rat group sizes in response to environmental factors.

Maximum group size and geophyte density was shown to be negatively correlated: as geophyte

density decreases, group size increases. Maximum group size and unpredictability of rainfall is

7

positively correlated: as unpredictability of rainfall increases, group size also increases. Rainfall

over 25 mm per month (minimum rainfall needed for soil to become penetrable) was negatively

correlated with group size (Faulkes et al, 1997). Since DMRs are considered almost obligate

outbreeders and have relatedness scores at par with normal diploid animals (R=0.5), their

evolution of eusociality is attributed to FAH, or other similar extrinsic factors that delay

dispersal or the encountering of foreign opposite-sex conspecifics (Jarvis and Bennett, 1993;

Bennett et al, 1996; Cooney and Bennett, 2000; Burland et al, 2002)

Comparing Social Bathyergids –

African mole rats have extreme variation in social structure ranging from solitary to

eusocial which has resulted in behavioral differences that begin at birth. Pups from solitary

genera mature and grow more rapidly than social mole-rat pups. Solitary genera pups also have a

higher K constant than social pups; the growth rate constant K is defined as the number of

generations over time. Although solitary genera physically mature quicker, social mole-rat pups

become more mobile and explore earlier than solitary animals with NMRs being an exception.

Solitary pups also begin sparring and play-fighting earlier than social pups (Bennett et al, 1991).

Despite both being eusocial, DMRs and NMRs have different behavioral traits (Jarvis et al,

1994). Damaraland mole-rats live in colonies with a maximum of about 40 individuals in 1-

kilometer burrows (Bennett and Jarvis, 1988; Jarvis et al, 1994). Naked mole-rats live in colonies

typically containing 70 to 80 but sometimes upwards of 300 individuals in an expansive burrow

system up to 3.5 kilometers long (Jarvis, 1981; Brett, 1991; Jarvis et al, 1994). Upon the death of

the breeding male or female, all DMRs within the colony will disperse at once. In contrast,

NMRs will fight often to the death to become a breeder, especially females for the position of the

breeding “queen”. Although field studies show that NMRs have left their colony (Braude, 2000),

8

it only occurs one at a time, not a full fragmentation like the DMR. Coupled with the fact that

NMRs are highly xenophobic, DMRs seem to have a higher chance of outbreeding than NMRs

(Jarvis et al, 1994). Direct reproductive success of the two bathyergids have been estimated at

1% for NMRs and 8% for DMRs (Burland et al, 2002).

The two species also differ anatomically. NMRs are small (≈30g) poikilothermic animals that

require body heat and a thermostable environment to maintain homeostasis. In contrast, DMRs

are larger (≈131g) and endothermic (Jarvis et al, 1994). Subordinate NMR ovaries show very

little follicular development whereas DMR ovaries have follicles but do not ovulate: they are

constantly in a state of pseudopregnancy when they are not in a breeding caste. In DMR males,

testosterone levels are similar in both breeders and non-breeders. In contrast, NMR non-breeders

have lower testosterone levels than breeders, but still have spermatogenesis (Jarvis et al, 1994).

The Naked Mole Rat –

A NMR’s strict social hierarchy consists of three castes: one reproductively active female

called the queen, one to three breeding males, and dozens of non-breeding subordinates of both

sexes (Jarvis, 1981; Brett, 1991). The subordinates can be divided into two subcastes, soldier and

worker, and can transition between the roles as adults (Mooney et al, 2015). Social hierarchies

seem to be maintained via aggressive interactions between queen and subordinates, as the queen

repeatedly shoves and attacks workers to stimulate work and maintain suppression (Reeve &

Sherman, 1991; Reeve, 1992; Faulkes et al., 1990; 1991).

NMRs were believed to be highly inbred due to colony formation via fission and thus mating

with kin (Brett, 1991). Evidence has shown this system may have evolved due to their

environment, where there are more risks in dispersing to find a mate due to patchy food sources,

9

predation, and harsh climate, referred to in the previous section as the food-aridity hypothesis

(Reeve et al., 1990; Alexander et al., 1991). Recently, a genetic field study demonstrated that

inbreeding occurs in the southern parts of Kenya due to a bottleneck effect, whereas there is

considerable genetic variation in northern Kenyan populations (Ingram et al, 2015). The southern

“inbred” population is separated from the northern population by two large rivers: The Tana

River and Athi River. Upon moving northward, there is an increase in allelic variation and levels

of heterozygosity. Inbreeding coefficients decrease when moving north, suggesting that southern

populations are more inbred.

Dispersal in The Naked Mole Rat –

In a 1996 study by O’Riain et al, evidence of a third potential subcaste called a “disperser

morph” emerged in captive bred NMRs. O’Riain et al found a subset of subordinates, almost

exclusively male with high luteinizing hormone levels and fat stores, who were highly motivated

to leave their natal colony. Disperser morphs were described as unaggressive with opposite-

sexed conspecifics and solicited sexual behavior. Non-dispersers in contrast rejected all

unfamiliar animals and spent more time engaging with their colony mates. Dispersers were also

shown to spend more time attempting to access unrelated opposite-sexed animals from a 3-way

chamber, and the opposite was observed in non-dispersers. O’Riain et al found that disperser

morphs did not engage in many working behaviors in the colony and spent more time feeding

and in locomotory activity compared to non-disperser subordinates. Disperser morphs were thus

described as subordinate animals that actively leave their natal colony to establish their own as

breeders (O’Riain et al, 1996).

A mark-and-capture field study was also conducted to see if the disperser morph is a

laboratory-exclusive phenomenon, or if a disperser morph exists in the wild as well. Dispersers

10

were indeed found within the wild, albeit with different characteristics than in colony. Braude

presented an almost equal sex ratio of dispersing NMRs and described them as being more

common than expected with a total of 21 female and 16 male dispersers identified. Dispersers

were described to disperse independently at night and founded independent colonies as well. 20

new colonies were captured from which 16 colonies consisted of previously tagged NMRs. 7

pairs were NMRs from different colonies, 2 pairs were NMRs from the same natal colony and 10

NMRs were found alone, apparently in wait of a mate. Only 1 male was found to have infiltrated

an already established colony and became the breeding male. A similarity between O’Riain and

Braude’s study was dispersers being fatter than non-dispersing conspecifics, although Braude’s

method of measuring fat was solely based on a length to weight ratio (Braude, 2000).

Characterization of the disperser morph has mainly been reliant on these two papers, and

O’Riain et al’s description is most often used by other scientists to discuss dispersal in NMRs.

There is increased research on the level of inbreeding in NMRs, especially using genetic markers

in wild populations (e.g.; Ingram et al, 2015) to add to the research that NMRs do indeed

disperse and are not as inbred as previously described. However, the definition of what

constitutes a disperser morph remains the same, many times even overlooking Braude’s findings

of equal sex dispersal (2000). Thus, I conducted this study to further characterize the disperser

morph, and to determine whether this indeed is a separate “morph” of naked mole-rat

subordinates or simply a behavioral trait.

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Objective

The purpose of this study was to characterize and compare disperser morph social

behavior to subordinate worker social behavior to determine if dispersers truly are a separate

subordinate “morph”. Furthermore, I examined colony conditions under which dispersers exist.

Colony conditions included population size, population density, queen behavior, presence of

pups, and in-colony sex ratios.

I hypothesized that dispersers will have different in-colony and out-colony behavior from

worker caste animals. For dispersers to be different from workers, dispersers would be less likely

to engage in working behaviors and more likely to be feeding within the colony as compared to

workers, resulting in dispersers having higher fat stores. I also predicted that most dispersers

would be male in accordance to the mammalian norm. As for out-colony behavior, I predicted

that dispersers would be less interested in their own colony’s scent and more interested in the

scent of a foreign colony. This prediction was based on the definition that dispersers are more

interested in foreign animals than colony members (O’Riain et al, 1996). Furthermore, I

hypothesized that the number of dispersers per colony would vary according to different colony

characteristics. I predicted that a higher population density and relaxed queen behavior would

result in a higher number of dispersers exiting a certain colony. Lastly, I hypothesized that

dispersers would be more motivated than workers to mate and establish their own colonies.

Dispersers should have higher prosocial interactions, perhaps even showing immediate mating

behavior when paired with an unfamiliar opposite-sexed conspecific. I predicted that dispersers

would become pregnant more quickly and would produce more litters than worker caste animals.

12

Methods

Animals –

Captive NMRs housed in the University of Toronto Mississauga vivarium were used in

this study. Animals were kept in polycarbonate cages of varying sizes (small:

30 × 18 cm × 13 cm high; medium: 46 × 24 cm × 15 cm high; large: 65 × 45 cm × 23 cm high)

depending upon the population of the colony and were connected using polycarbonate tubing

(See Appendix A). The rooms containing the colonies were kept at a temperature between 27-

28°C with 50% humidity, and were on a 12-hour light: dark cycle with lights on at 7:00am.

Animals were fed hydrated sweet potato daily, and wet Harlan mouse chow three times a week.

All NMRs had been microchipped at a minimum of 6 months of age with a rice-grain sized chip

for identification purposes. Animals ranged from 8 months to 5 years of age; considering that

NMRs reach adulthood approximately within 1 year and can live for over 30 years, these

experimental animals were relatively young adults (Buffenstein, 2008; O’Riain and Jarvis,

1998). Each animal was labeled with a letter or symbol using a Sharpie marker to allow

individual visual identification during video recordings, and its weight, age, sex, and chip ID

number was recorded. Animals were fed hydrated sweet potato ad libitum approximately 2 hours

prior to testing to minimize the effect of hunger as motivation to leave the colony. All testing

took place between 12:00 pm and 5:00 pm.

A total of 17 colonies were used throughout the study with an approximate total

population size of 593 animals. The study was broken down into three cohorts. Cohort 1A and

cohort 1B consisted of the following 9 colonies: Colony S (n=26), Colony F (n=50), Colony T

(n=45), Colony R (n=49), Colony O (n=27), Colony Y (n=39), Colony Da (n=15), Colony De

(n=29), Colony W (n=30). Cohort 1B was studied 12 months after cohort 1A. Cohort 2 consisted

13

of the following 8 colonies: Colony H (n=6), Colony G (n=51), Colony Dn (n=24), Colony E

(n=58), Colony Dh (n=36), Colony AS (n=17), Colony Z (n=44), Colony C (n=35).

Cohorts 1A and 2 were used to test disperser morph behavior and colony factors affecting

the presence of the disperser morph. Cohort 1B was used to evaluate anatomical differences

between disperser morphs and worker caste animals. All three cohorts were subjected to the

same experimental procedure, except for the last two tests which differentiated between

behavioral and anatomical studies of the disperser morph (Figure 1). This study was conducted

across a two-year span.

A) Cohort 1A (2015) and Cohort 2 (2017)

B) Cohort 1B (2016)

Disperser

Test

In-Colony

Videos

Out-pairing Olfactory

Preference

D W

Disperser

Test Repeat

Breeding Pairs

Week 0 Week 1 Week 2 Week 3 Week 4 Week X*

Disperser

Test

In-Colony

Videos

Out-pairing Olfactory

Preference

D W

DXA Scanning

Week 0 Week 1 Week 2 Week 3 Week 11

D W

D-D D-W W-W

14

Figure 1: Timeline outlining the order of tests for this study. The study was made up of 3 cohorts. Cohort 1A (A)

consisted of 9 colonies and tested disperser behavior and colony factors affecting the presence of dispersers.

Breeding pairs were set up 6 months after the disperser test repeat. Cohort 2 (A) also tested disperser behavior and

colony factors, but used 8 different colonies. Breeding pairs were set up 3 weeks after the disperser test repeat for

cohort 2. Cohort 1B (B) used the same colonies as cohort 1A, but were used to test anatomical differences between

dispersers and workers using a DXA scanner. These animals did not go through a disperser test repeat. The animals

were scanned 8 weeks after the olfactory preference test. “D” = disperser; “W” = worker”.

Disperser Test –

The testing procedure began with the “disperser test” to establish the presence of putative

dispersers, which was adapted from O’Riain et al (1996). Colonies taking part in the disperser

test had an extra hole on the side of the busiest cage in their colony. This hole was blocked with

a plastic stopper (see Appendix B). During testing, the plastic stopper was removed and a plastic

platform (22.86 x 30.48 cm) was placed directly under the hole so that the NMRs could easily

explore the opening and its surrounding areas. When an animal exited it was immediately placed

back in the colony in the cage furthest from the hole. The identification of all NMRs that exit,

along with how many exits, was recorded. Each trial lasted 30 minutes.

Three dispersal trials (one per day for three consecutive days) were administered. The

disperser test was administered twice, once as initial testing and then 4 weeks later as a repeat to

confirm stability of motivation to leave the colony. Animals that left three or more times during

each of the first and second set of trials were considered presumed dispersers and were then

further tested to confirm disperser status (see Out-pairing Test). Animals that failed to leave the

colony in all the trials were considered workers. Intermediately exiting animals were not

included in the study.

15

Out-pairing Test –

An out-pairing test was administered to remove any aggressive animals from the

presumed disperser cohort (i.e., animals leaving the colony); dispersers, by definition, would be

unaggressive towards unfamiliar conspecifics (O’Riain et al, 1996).

Presumed dispersers were each paired with an unfamiliar opposite-sex animal that was

similar or less in weight. The out-pairing was conducted in a 46cm x 24cm x 15cm high cage

(medium size). The animals were paired for 10 minutes and their interactions were recorded

using a GoPro Hero 3 camera. Interactions where one animal punctured the skin of another were

immediately stopped and the animals were removed from the presumed disperser list. Presumed

dispersers that also passed the out-pairing test (i.e., they were not aggressive towards the

unfamiliar animal) were classified as dispersers for the duration of the project.

In Colony Recordings –

Regular activity within the 17 colonies used for the study was recorded on 30-minute

videos within a week after the colony’s disperser test began. These videos were used to score

daily behavior of dispersers, workers, and queens found in each colony. Aggressive, sex

affiliative, non-sex affiliative, working, in-transit, feeding, and pup care behaviors were scored

(see Appendix C for a detailed ethogram highlighting scored behavior). In-colony behavior of

the 25 dispersers (10 males and 15 females) and 20 workers (8 males and 12 females) in Cohort

1A was scored. Cohort 2 animals have been tested but scoring of the videos is not yet complete.

Descriptive measures of each colony, such as population, colony area, presence of pups, queen

age, and colony sex ratios were also recorded to analyze factors affecting dispersal rate. Videos

16

were recorded using a GoPro Hero 3 camera, and were later scored using Observer XT software

(Noldus, Wageningen, The Netherlands).

Olfactory Preference Test (OPT) –

This test was conducted in lieu of a subject preference test where an animal is exposed to

conspecifics. This was done to minimize habituation between subjects and foreign animals, and

to minimize the opportunity of injury during interactions.

In cohort 1A, 25 dispersers and 20 workers completed the olfactory preference test

(OPT) to evaluate preferences towards familiar or unfamiliar conspecific odors. Cohort 2

animals have been tested but scoring of the videos is not yet complete. Animals were

individually placed in a medium cage containing two ceramic ramekins, one containing soiled

bedding from the animal’s natal colony, and the other containing soiled bedding from an

unfamiliar colony. The ramekins were placed equidistant from each other and the cage walls.

The duration of time spent sniffing each stimulus was recorded for 10 minutes using a GoPro

Hero 3, and was later scored using Observer XT software (Noldus, Wageningen, The

Netherlands).

Breeding Pair Set-Up –

After all other behavior testing was complete, pairs were set up between two unfamiliar

opposite-sexed animals: either two dispersers (disperser-disperser), a disperser and a worker

(disperser-worker), or between two workers (worker-worker). This was done to evaluate whether

disperser-disperser pairs reproduce more quickly than worker-worker pairs. In cohort 1A,

dispersers were removed from 4 of the 9 colonies. The remaining colonies had workers (non-

17

dispersers) removed. In cohort 2, dispersers and workers were removed from all 8 participating

colonies.

Breeding pairs were arranged in new medium caging between opposite-sex individuals of

similar weight. Worker animals removed from colonies were typically under 35g. Interactions of

the pairs were recorded using a GoPro Hero 3 and the initial 10 minutes were scored using

Observer XT software (Noldus, Wageningen, The Netherlands). Scored behaviors included

aggressive, sex affiliative, non-sex affiliative, working, in-transit, and feeding behavior (see

Appendix C for a detailed ethogram highlighting scored behaviors).

Relatedness –

The pedigree of each colony within the UTM vivarium is known. To calculate how

related animals are across separate colonies, the following website was used to calculate the

relatedness factor:

http://www.ihh.kvl.dk/htm/kc/popgen/genetics/4/5.htm

Once calculated, the relatedness factors were correlated with scored behavior from the

breeding pairs to analyze whether relatedness might contribute to affiliative behaviors when

unfamiliar animals are introduced. Relatedness was also used as a covariate for the breeding pair

analysis (see Statistical Analysis).

DXA Scanning -

This procedure was inspired by O’Riain et al (1996). Body fat composition, bone

composition, and muscle composition was determined using dual-energy x-ray absorptiometry

(DXA) scanning (QDR 4500; Hologic, Waltham, MA) in 25 dispersers and 23 workers from

18

cohort 1B. Animals were first anesthetized using 4% isoflurane per 1liter oxygen. They were

then placed on an elevated surface to be scanned, and upon scanning were put alone in a small

heated cage until re-gaining consciousness. These animals did not go through a disperser test

repeat (see Figure 1B).

Statistical Analysis –

All statistical analyses were conducted using SPSS software (IBM Corp. Released 2012.

IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp). MANOVAs were

used for the analyses of in-colony behavioral comparisons between dispersers and workers,

where behaviors were dependent on subcaste; males and females were analyzed separately. A

generalized linear model was conducted to analyze factors affecting the number of dispersers

found in a colony. For the OPT, a sex by subcaste by stimulus MANOVA was conducted, with

sex, subcaste, and stimulus type as the independent variables and time spent sniffing the stimulus

as the dependent variable. For analyzing interaction differences between breeding pair types, a

MANCOVA was conducted with pair type as the independent variable, behaviors as the

dependent variables, and relatedness score as a covariate. A MANOVA was also used to detect

any differences between pair types and the number of litters they produce, or time to their first

litter. When analyzing body fat differences between dispersers and workers using the DXA scan,

a two-way sex by subcaste ANOVA was conducted with fat score (mean fat or percent fat) as the

dependent variable, and subcaste and sex as independent variable. Each sex was analyzed

separately as well using a one-way ANOVA where fat score (mean fat or percent fat) was the

dependent variable, and subcaste was the independent variable.

19

Results

Disperser Test and Out-pairing Test–

Dispersers were classified as subordinates that exited three or more times during both the

initial and the 4-week repeat disperser test, were unaggressive during out-pairing test, and were

of adult body size (more than 20g in weight; Table 1).

Table 1: Disperser breakdown. The following table outlines the number of dispersers discovered

over the project, their sex, and the percentage of dispersers per colony. No.

Dispersers

Cohort 1A

(2015)

Dispersers

M F

No.

Dispersers

Cohort 1B

(2016)

Dispersers

M F

No.

Dispersers

Cohort 2

(2017)

Dispersers

M F

25 10 15 10 5 5 9 2 7

Rate of Dispersal in Cohort 1A

Colonies Y O W S F R De T Da

1 out

of 39

5 out

of 27

1 out

of 30

4 out

of 26

7 out

of 49

4 out

of 48

1 out

of 29

0 out

of 45

2 out

of 15

3% 19% 3% 15% 14% 8% 3% 0% 13%

Rate of Dispersal in Cohort 2

Colonies H G Dn E Dh AS Z C

0 out of

6

1 out of

51

0 out of

24

1 out of

58

1 out of

36

0 Out of

17

3 out of

44

2 out of

35

0% 2% 0% 2% 3% 0% 7% 6%

Olfactory Preference Test –

A significant main effect of stimulus where NMRs spent more time sniffing familiar

bedding than unfamiliar bedding was detected (F1,38= 6.949, p=0.012). No significant effects of

20

sex (F1,38=0.587, p=0.448) or subcaste (F1,38=0.002, p=0.963) were found. No significant

interactions were seen either.

Figure 2: Olfactory Preference Test Results for cohort 1A. The graph depicts the amount of time (seconds)

dispersers and workers spent sniffing familiar or unfamiliar bedding. N(dispersers)=10, N(workers)=8. NMRs,

regardless of caste or sex, spent more time sniffing familiar bedding on average. There were no subcaste or sex

differences found.

In Colony Behaviors –

Male dispersers spend more time in transit than male workers (F1,16 =6.260, p=0.024;

Figure 3A and B). Male dispersers also tend to work less than male workers (F1,16 =3.855,

p=0.067), although the difference was not significant. No significant differences were found

between female dispersers and female workers. No sex differences were found (F1,16 =1.407,

21

p=0.242), and no statistically significant difference in feeding was found in males (F1,16 =1.922,

p=0.185) or females (F1,16=1.691, p=0.206).

A)

22

B)

Figure 3: In-colony behavior analysis of dispersers and workers between males (A) and females (B). Male dispersers

spend significantly more time in transit than male workers. Males dispersers also tend to work less than male

workers, although the results are not significant. No significant effects were seen between female dispersers and

workers.

In Colony Factors Affecting Dispersal –

Various factors including presence of pups, queen age, population density, queen

aggression, and in colony sex ratio were used to analyze which factors affect the dispersal of

male or female dispersers. It was found that queen aggression had a positive significant effect on

number of female dispersers in a colony (B=0.013, SE=0.0067, df=1, p=0.044). However, there

were no significant effects found on number of male dispersers.

23

Breeding Pairs –

There were 9 disperser-disperser (DD), 11 disperser-worker (DW), and 9 worker-worker

(WW) breeding pairs. DW pairs exhibited significantly more aggression levels than DD or WW

pairs (DW-DD: MDifference=28.223, SE=10.634, DW-WW: MDifference=27.412, SE=10.634, F2,25 =

5.349, p=0.012) in a MANCOVA, with relatedness scores between pairs as a covariate. No

significant differences in aggression levels between DD and WW pairs were found. DW pairs

were inactive for a longer period of time than DD or WW pairs, but it was not statistically

significant (F2,25 = 3.096, p=0.063). There was also no significant difference in sex affiliative

behaviors between the three breeding pair types (F2,25 = 1.738, p=0.197).

24

Figure 4: Comparing initial behavior of different pair-types. Disperser-worker pairs exhibited higher aggression

levels than disperser-disperser or worker-worker pairs. Disperser-worker pairs also exhibited higher inactivity

levels than worker-worker pairs, but the results were not significant. Bars represent standard error.

Within 12 months of pairing, cohort 1A had numerous successful breeding pairs (Table

2). There were 2 successful disperser-disperser pairs, 3 successful disperser-worker pairs, and 2

successful worker-worker pairs. They had a total of 3, 4, and 4 litters respectively. There was no

significant difference in the number of litters or time to the first litter between the three pair

types.

Table 2: Established Breeding Pairs. A summary of successful pairs that yielded at least 1 litter

within 12 months of pairing.

Relatedness –

A linear regression analysis was done comparing each behavior during pairing and

relatedness scores of the animals. This was done to ensure that animals who are closely related

do not exhibit specific behaviors with one another due to recognition of kinship. All correlations

between were found to be very weak and statistically insignificant.

DXA Scanning and Anatomical Differences –

Established Breeding Pairs

Pair Type Pair Number Number of Litters Time to 1st Litter

(months)

Disperser-Disperser #3 2 4

#6 1 5

Disperser-Worker #14 1 10

#21 1 10

#22 2 4

Worker-Worker #102 2 6

#105 2 8

25

Mean disperser weight (33.03g) was significantly higher than mean worker weight

(24.02g; p < 0.005). DXA scanning revealed that dispersers had significantly higher mean fat

scores than workers (F1,44=4.890, p=0.032) (Figure 5 A). However, there was no significant

differences between mean percent fat between dispersers and workers (F1,44=1.535, p=0.222)

(Figure 5 B). There was no main sex effect for mean fat (F1,44= 0.583, p=0.449) or percent fat

(F1,44=0.368, p=0.547). Also, when looking at sex separately, male dispersers and male workers

had no significant difference in mean fat (F1,22=2.794, p=0.109) or percent fat (F1,22=0.234,

p=0.633). Female dispersers and female workers had no significant differences between mean fat

(F1,22=2.098, p=0.162) and percent fat (F1,22= 1.952, p=0.176) either. Significant differences

were only seen when the sexes were combined.

A)

26

B)

Figure 5: Comparing mean fat in grams (A) and mean percent fat in percentage (B) in dispersing and working

NMRs. Dispersers exhibit significantly higher mean fat scores than workers (A) however, there is no difference

between disperser and worker mean percent fat scores (B). Bars represent standard error.

27

Discussion

The purpose of this study was to characterize and compare disperser morph social

behavior to subordinate worker social behavior to determine if dispersers truly are a separate

subordinate “morph”. The disperser morph has previously been described as typically male, lazy,

having higher body fat, and eager to reproduce (O’Riain et al, 1996). This was contrasted

somewhat by Braude’s (2000) field study finding that although dispersing NMRs tended to be

fatter than non-dispersers, the sex ratios were equal. Although I had predicted that most

dispersers would be male, like Braude I found an almost equal number of male and female

dispersers in my study. In contrast to my prediction of dispersers preferring unfamiliar bedding, I

found that dispersers in fact preferred their own colony’s bedding in equivalence with worker

caste preferences. Initially I had predicted that dispersers would work significantly less than

worker caste animals within their colony environment, resulting in them having higher fat stores.

My results showed that male dispersers worked less than male workers, but the difference was

not statistically significant (p=0.067). Male dispersers spent more time in-transit than male

workers, but there were no in-colony behavioral differences between female dispersers and

workers. Interestingly, dispersers had higher mean fat scores than workers, but there was no

difference when looking at mean percent fat. I had predicted that relaxed queen behaviour would

result in a higher number of dispersers. However, a more aggressive queen equated to a greater

proportion of female dispersers in the colony, which was not true for males. Patterns reflecting

my prediction of high population density affecting number of dispersers were not seen. I had

predicted that dispersers would exhibit higher levels or prosocial behaviour and would be more

motivated to establish their own colonies. However, these predictions were not reflected in the

results. When paired with opposite-sex conspecifics, disperser-worker pairs exhibited higher

28

aggression levels than disperser-disperser and worker-worker pairs. Although disperser-disperser

pairs seemed to exhibit higher sex affiliative behavior levels, the results were non-significant.

The three pair-types also showed comparable timings of first litter birth and number of litters

birthed over a 12-month span.

Finding equal numbers of male and females is a very interesting result. Past literature on

mammals has shown that males are more likely to disperse than females, especially in a

polygynous mating system (e.g.; Evans and Holdenried, 1943; Dobson, 1979). However, a

mammalian eusocial mating system is somewhere in between monogamy and polyandry.

According to literature, this would imply an equal dispersal rate between the sexes (Dobson,

1982; Greenwood, 1980). Males being the dispersers was a somewhat convincing argument

though as anatomically males are more prepared to reproduce since even non-reproductive males

have spermatogenesis (Jarvis et al, 1994). It would be easier for males to become reproductively

active within their own colony as well, since there are often more male consorts and only one

breeding female. Females are thus obligated to leave the colony, or kill and replace the queen, if

they are to reproduce.

In-colony behavioral differences were only seen in male naked mole-rats. Male dispersers

spent significantly more time walking around the colony (in-transit) and seemed to exhibit less

working behaviors than male workers. O’Riain et al found similar results where dispersers,

which happened to be all male, exhibited heightened locomotory activities and minimal

maintenance tasks. It is interesting that these differences do not manifest in females. Decreased

working behaviors were interpreted to be a method to increase fat stores for when the

opportunity to disperse arises (O’Riain et al 1996). Why this would not be the case for female

dispersers is peculiar. Increased in-transit behavior, or simply walking around the colony, can

also be interpreted as scouting opportunities to exit the colony, although it is difficult to

29

understand why females would not use a similar strategy. However, I do err on the side of

caution, since O’Riain’s in colony behaviors were based upon the study of only one colony,

whereas I analyzed 17 colonies. However, in-transit behavior remains significant in males. This

sex difference is puzzling, but perhaps expected; Braude did speculate in his discussion that

males and females must be dispersing due to different environmental cues (Braude, 2000).

Further analysis into colony conditions provided some answers to this phenomenon.

Female disperser morphs have been mentioned in literature once in the wild (Braude, 2000).

My study gives a hint of why female dispersal may exist in NMRs by showing that increased

queen aggression is related to higher female, but not male, disperser frequency. Thus, it is

possible that female-female competition may be what is driving female dispersers out of the

colony. Female-female competition usually arises after the death or removal of the queen, where

many females experience heightened levels of progesterone and testosterone, weight gain, and

develop perforated vaginas. These females are also the oldest, heaviest, and most dominant in the

colony. Upon queen removal, sisters will fight amongst each other for the position of queen, and

sometimes the queen can be killed by her offspring as well (Clarke and Faulkes, 1997). There is

also mention of a “beta female”, a large female NMR who also has perforated genitalia, but does

not breed (Jarvis, 1981). Whether these specific females are primed for dispersal are not yet

known, but some disperser morph females within my study were very large and produced

multiple litters within a year of pairing. When fighting between two females, or a queen and a

subordinate female occurs, one of the females must be removed from the colony. These females

are often paired with an unfamiliar male once recovered from injury; the pairings are quite

successful and typically lead to newly established colonies (personal observation, Toor 2017).

Thus, it is likely that queen aggression towards particular females serves to maintain

reproductive suppression when they are beginning to reproductively activate.

30

Overall, the equal sex ratios in dispersing NMRs reported here and by Braude (2000) is

more likely than exclusively male dispersers reported by O’Riain et al (1996). If only male

NMRs dispersed, they would have to join an existing colony and become a breeding male.

Indeed, this has been noted once in field populations (Braude, 2000). Although this shows that

such a situation is possible, it is important to note that within 10 years only 1 male out of 20

mole-rats was successful in infiltrating an established colony (Braude, 2000). It has been found

that NMRs prefer to mate with unfamiliar animals than their own siblings (Ciszek, 2000). In that

study, experimental colonies were set up with a male and female from one colony, and a male

and female from a stranger colony, to test whether the siblings will mate or whether mating will

take place between strangers. In all cases, only the strangers exhibited mating behaviors within

one another; mounting was almost exclusively exhibited between unrelated NMRs (Ciszek,

2000). Coupled with evidence of NMRs frequently outbreed (Ingram et al, 2015), a stronger case

for equal sex ratios in disperser morphs resulting in unrelated pairings in the wild can be made.

Disperser morphs and worker caste animals, regardless of sex, spent more time sniffing

their own colony’s bedding than unfamiliar colony bedding. This overall trend is not surprising

as it has been shown at least twice. In a T-maze test, subordinate NMRs were presented 3

different scents, (1) foreign bedding, (2) familiar bedding from either their nest or toilet chamber,

and (3) unused bedding. NMRs consistently spent more time sniffing chambers that contained

familiar bedding (O’Riain and Jarvis, 1997). In a recent study with a similar olfactory preference

paradigm, subordinate NMRs preferred the ramekin with familiar bedding, even after main

olfactory bulb impairment using zinc sulfate (Toor et al, 2015). However, in these previous

reports, animals were not classified into different subcastes. I had hypothesized that dispersers

would have a reduced preference for their own colony’s bedding as dispersers are by definition

less aggressive towards unfamiliar conspecifics and more exploratory in nature (O’Riain et al,

31

1996). While the data reported here do not support that hypothesis, it is important to consider

that the olfactory preference paradigm may not have been the best test to use. Soiled bedding

contains the full colony odors; thus, it is very likely that the preference of their own colony is out

of fear or intimidation of a large number of animals. It would be safer to return to your own

colony than to be caught in an unfamiliar colony where there is increased danger. It will be

important to test olfactory preference between a familiar and unfamiliar individual, rather than

colony, odors in the future.

It was interesting to find that disperser-worker breeding pairs exhibited significantly

higher aggression levels than disperser-disperser or worker-worker pairs. Dispersers found in

O’Riain’s study were all motivated to mate with unfamiliar conspecifics, although it is not

mentioned how the non-disperser behaved in this interaction. In my study, I looked at the

summed behaviors exhibited by both animals, as the behavior of one animal will yield a reaction

in the other. Disperser-worker pairs also exhibited higher inactive behavior, albeit statistically

insignificant. Elevated inactivity in disperser-worker pairs could be a by-product of aggressive

interactions where the animals “freeze” in response to fear or aggression. It is also possible that

dispersers began by showing curiosity towards the unfamiliar animals, and the worker responded

in fear or aggression in return yielding an aggressive response in the disperser. This interaction

could have been avoided in disperser-disperser pairs as they exhibited higher (albeit

insignificant) sex affiliative behavior, and worker-worker pairs exhibited higher working and

lower inactivity levels (statistically insignificant).

Disperser morphs on average were heavier than workers and had higher mean body fat, in

line with the O’Riain paper (1996), but percent body fat between dispersers and workers was not

statistically significant in my study. A study on colony formation in NMRs had similar results

where NMRs with higher weight but smaller neck width were more likely to become breeders.

32

There was also no body fat correlation with breeder status (Ciszek, 2000). In contrast, Braude

(2000) found that dispersing NMRs in the wild had a higher weight-to-length ratio, which was

used as a measure of fatness. I would err on the side of caution with Braude’s methodology since

taking measurements on live NMRs is very difficult and possibly inaccurate due to their highly

active nature (personal observation, Toor 2017). Upon analyzing the results of time to first litter

and number of litters produced within 12 months, there are no differences between disperser-

disperser, disperser-worker, or worker-worker animals. Thus, although it may be more likely for

larger animals to become reproductively active within or outside of the colony, smaller animals

are also capable of the transition. The results of my study could also be influenced by the length

of time it took to conduct DXA scans, and the limited number of animals used. Unfortunately,

only cohort 1B’s results could be analyzed as the DXA machine was under repair for a large

portion of the study.

The disperser morph was described as rare (O’Riain et al, 1996; Braude, 2000), with

1.7% of O’Riain’s animals exiting 3 or more times. I found varying levels of dispersal within

each colony (cohort 1A: 0% - 19%; cohort 2: 0% - 7%), and the majority of colonies (13 out of

17) contained disperser morphs. In fact, the number of dispersing NMRs would have been even

higher if we only included a single disperser test as O’Riain et al did instead of our 4-week

repeat. It is intriguing that the disperser morph was found to be more common within my study

than was reported by O’Riain et al (1996). The rare occurrences of dispersers were attributed to

NMRs apparent preference for inbreeding, but this prediction was not supported by data as

discussed above (Ciszek, 2000). In fact, Ingram et al (2015) found that there is not as much

inbreeding between NMRs, and most of it only exists in the southernmost population in Kenya

due to the natural bottleneck of the Athi and Tana rivers. Most lab and zoo populations,

including our own, originated from the southern areas of Kenya. Thus, it is very possible that

33

disperser morphs are quite common in nature, especially during periods of high rainfall. The

vivarium environment is somewhat similar to that of sub-Saharan Africa during rainy season,

with soft bedding and a moist environment. This could explain why I found such an abundance

of dispersing animals in comparison to previous studies.

My study only investigated adult animals, but there were pups and juveniles (under 20g)

that also consistently exited and passed the initial stages of the disperser test (personal

observation, Toor 2017). This may hint that explorative behavior is something innate to

individual animals and may lead to developing characteristics of disperser morphs. It would be

interesting to study when these pups first start rough-and-tumble play in comparison to their less

explorative siblings. Whether these pups are the first to start crawling and leaving their nesting,

and when they begin working behavior are also important questions that can be answered.

Tracking the life history of a brand-new colony would be an excellent way to explore this topic.

Beginning at the first litter, how each animal develops and how its behavior changes or remains

constant through time and subsequent litters would help to understand whether disperser morphs

are more in line with a separate subcaste, or if they are derivatives of the current worker and/or

soldier castes. Upon reaching adult size, a colony’s hierarchy can be determined using a tube-test

paradigm by creating pass-over matrices (Toor et al, 2015). The disperser test can also be

administered at various point of the colony’s life to observe any consistencies or changes in who

disperses. This would answer many questions regarding how dispersers come about in the colony

and whether there are changes to dispersing frequency over time.

The purpose of this study was to better characterize the disperser morph, and to

determine whether the disperser is a true “morph” of the subordinate NMR castes. There were

indeed many animals that consistently exited the colony both in the initial trial and in the 4-week

repeat, and there were animals that consistently did not exit the colony even once. Many of the

34

exiting animals were also unaggressive towards unfamiliar opposite sexed conspecifics, thus

fitting the disperser morph definition. However, past this initial test there were few striking

differences between dispersers and workers. Although there was a small behavioral pattern in

male in colony behaviors and female dispersal rates with queen aggression, there were no

striking differences in breeding pair interactions. Surprisingly, there also were no difference in

birth of first litter or number of litters; workers were as successful as dispersers in establishing a

colony. Upon analyzing these results, I question whether the disperser is truly a distinct

subordinate morph and instead propose and alternate hypothesis. I now hypothesize that if there

are no remarkable differences between regular in-colony behaviors or mating behaviors, then

perhaps there is no distinct polymorphisms between subordinate caste workers and dispersers. I

do not doubt that a subset of animals is more motivated to leave the natal colony than others, but

I remain skeptical about whether we can appropriately classify these animals separately from

worker and soldier subcastes. Looking back at the O’Riain paper, I agree that a dispersing mole-

rat would have to be highly explorative and prosocial with unfamiliar conspecifics as logically

that is the only way it can mate. I do also agree that dispersing mole-rats, or at least successful

dispersing mole-rats, would be laden with fat as this would aid in survival. However, my results

do not reveal striking behavioral differences within the colony that separate dispersers from

workers apart from some animals being more curious than others. Although I required the NMR

colonies to be fed prior to testing, there still lies a possibility that these so-called dispersers are

simply foraging subordinates attempting to locate a food source or expand their colony. Despite

my hesitation regarding this specific polymorphism, I am unwilling to state that the heightened

explorative behavior in some subordinate animals is non-adaptive as increased foraging

behaviour benefits the colony and dispersal could eventually lead to outbreeding and

establishment of new colonies. Instead, I reason to redefine how the disperser morph fits into the

35

social hierarchy of a naked mole-rat colony. Although the preference to explore exists within

some naked mole rats, I would consider thinking of the disperser morph as a behavioral trait of

certain worker caste animals instead of a separate entity itself.

36

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Appendices

Appendix A – The 3 cage sizes that can house naked mole rats (a). These cages are connected in

various combinations using polycarbonate tubes (b).

(a) (b)

Appendix B – Colony set-up with plastic stopper removed (a) (To see example of plastic stopper

intact refer to Appendix A picture b). Colony set-up with clipboard ramp (b).

(a) (b)

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Appendix C – Ethograms

Ethogram 1: Behaviors scored during In-Colony and breeding pair recordings (The Biology of

the Naked Mole Rat, 1991)

Aggressive Behaviors

Bite and Drag NMR bites another’s tail or leg and drags it a

certain distance.

Open Mouth Gaping Stand-off between two NMRs with their

mouths open.

Incisor Fencing Two NMRs interlock teeth and attempt to flip

each other over.

General Aggression Any sort of biting, scratching, aggressive

lunging behaviors.

Muzzle Fencing Two NMRs collide muzzles and begin

pushing each other back and forth.

Shoving NMR pushes another using its muzzle.

Sex Affiliative Behaviors

Genital Sniffing NMR sniffs another in the genital area.

Genital Nuzzling Two NMRs sniff and nuzzle each other’s

genitalia at the same time.

Mounting NMR (generally male) mounts another NMR

(generally female).

Lordosis NMR (generally female) arches back in

typical lordosis position.

“P” Interaction Sexual interaction between two NMRs where

one animal performs self-nuzzling while the

other performs genital sniffing.

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Non-Sex Affiliative Behaviors

Huddling NMR joins a group huddle where numerous

colony members are resting together in a pile,

typically in the nest area.

Face Sniffing NMR sniffs the face and head region of

another.

Body Sniffing NMR sniffs the body region of another.

Typically, body is considered from neck to

haunches.

Tetany NMR becomes still and takes on a submissive

pose to allow another NMR to investigate.

Accommodation NMR makes space or leaves to make space

for another.

Working Behaviors

Digging NMR digs bedding with forepaws or kicks

away bedding with hind paws.

Climbing NMR stands on its hind legs and attempts to

climb up the cage wall.

Gnawing NMR uses its teeth to chew or gnaw away at a

cage wall, plastic tube, or bedding.

Maintenance of Environment NMR transports or changes setting of paper

bedding within or away from the nest area.

47

In-Transit Behaviors

Active NMR walks around the cage without

performing other behaviors.

Tunnel Guarding/Travel NMR peers into or walks across tubing.

Inactive Behaviors

Inactive NMR stays still.

Grooming NMR grooms itself (but not genitalia).

Self-Nuzzling NMR specifically grooms its genital area.

Feeding

Food interaction NMR either eats or transports food

throughout the cage.

Pup Care

Pup Care NMR interacts with, including licking and

sniffing, colony pups.