The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes

Alexander T Ciota
Linda M Styer
Mark A Meola
Laura D Kramer
The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes
R E S E A R C H A R T I C L E Open Access
Background: Understanding the phenotypic consequences of interactions between arthropod-borne viruses (arboviruses) and their mosquito hosts has direct implications for predicting the evolution of these relationships and the potential for changes in epidemiological patterns. Although arboviruses are generally not highly pathogenic to mosquitoes, pathology has at times been noted. Here, in order to evaluate the potential costs of West Nile virus (WNV) infection and resistance in a primary WNV vector, and to assess the extent to which virusvector relationships are species-specific, we performed fitness studies with and without WNV exposure using a highly susceptible Culex pipiens mosquito colony. Specifically, we measured and compared survival, fecundity, and feeding rates in bloodfed mosquitoes that were (i) infected following WNV exposure (susceptible), (ii) uninfected following WNV exposure (resistant), or (iii) unexposed. Results: In contrast to our previous findings with a relatively resistant Cx. tarsalis colony, WNV infection did not alter fecundity or blood-feeding behaviour of Cx. pipiens, yet results do indicate that resistance to infection is associated with a fitness cost in terms of mosquito survival. Conclusions: The identification of species-specific differences provides an evolutionary explanation for variability in vector susceptibility to arboviruses and suggests that understanding the costs of infection and resistance are important factors in determining the potential competence of vector populations for arboviruses.

Background

A comprehensive understanding of the relationships between arthropod fitness and infection with arthropodborne viral pathogens (arboviruses) is vital to describing the ways in which selective pressures might drive the coevolution of vector and virus. With the ultimate evolutionary goal of pathogens being dispersal and transmission, and the probabilities of these generally increasing with increased vector fitness, evolution should tend to favor an innocuous relationship between arboviruses and their mosquito hosts [1]. Indeed, many studies have demonstrated that such a benign association often exists [2][3][4]. This is particularly the case with viruses which rely more extensively on vertical transmission for maintenance [5]. Despite this, evolution away from virulence is not always the rule if, as initially proposed in the trade-off hypothesis, increased virulence is itself coupled with increased transmission [6,7]. Specifically, increases in transmissibility are generally associated with increased replication, yet increased replication may be inherently coupled with increased virulence. Additional strategies such as infection causing increases in feeding rate as documented for malaria [8] and West Nile virus (WNV; [9]) may partially overcome the costs of virulence on pathogen transmission, yet ultimately pathogen evolution should move towards the balance between virulence and pathogen load which maximizes transmission.

On the host side, evolution should independently favor resistance and/or tolerance to pathogenic viruses, yet these immune defences are often associated with their own fitness costs, so predicting the way in which host-pathogen interactions will affect the evolution of host response requires a detailed understanding of the balance between the costs of infection and immunity in individual species.

West Nile virus is the most widespread arbovirus in the United States and is primarily vectored by Culex mosquitoes including Cx. pipiens in the northeast and north central, Cx. tarsalis in the west, and Cx. quinquefasciatus in the south and southwest USA [10,11]. Although all species have the capacity to support high levels of replication of WNV, vector competence differs among and within species [12][13][14]. Histopathology of WNV in colonized Cx. quinquefasciatus demonstrated that infection can be associated with significant tissue damage in salivary glands [15] and apoptosis of midgut cells [16]. Similar pathology has been noted in mosquito vectors during infection with other arboviruses including Semliki Forest virus [17] and Western equine encephalomyelitis virus [18]. Additionally, studies monitoring life history traits have also demonstrated that fitness costs in terms of both survival and fecundity are often associated with arbovirus infection in mosquitoes, particularly in the case of alphaviruses [19][20][21][22]. A previous study in our laboratory found that decreased fecundity but not survival was associated with infection of WNV in Cx. tarsalis mosquitoes [9]. Here, in order to evaluate the extent to which virus-vector relationships are species-specific, we performed similar studies in Cx. pipiens mosquitoes. Specifically, we measured and compared survival, fecundity and feeding rates in bloodfed mosquitoes that were (i) infected following WNV exposure (susceptible), (ii) uninfected following WNV exposure (resistant), or (iii) unexposed. We hypothesized that differences in the costs of both infection and resistance among mosquito species may partially explain differences in susceptibility in nature.

Results

WNV infection rates and viral titers

The titer of WNV in the serum of infected chicks fed upon by mosquitoes was 10 6.3 and 10 6.7 log 10 pfu/ml for replicates I and II, respectively. Infection rates were high for exposed groups in both replicates, with 36 of 42 fully-engorged mosquitoes infected in replicate I and 32 of 39 infected in replicate II. This translated to a combined infection rate of 84.0% and therefore relatively small numbers of resistant mosquitoes. As expected, WNV titers in mosquito bodies increased with time following infectious bloodmeal until approximately day 20, yet there was also substantial variation among titers which is not explained by time (Figure 1a). The proportions of infected mosquitoes with disseminated infection (i.e. WNV + legs) were 83.3% and 87.5% for replicates I and II, respectively. If mosquitoes that died prior to day 5 are omitted (due to low probability of dissemination), the combined proportion of disseminated infections among infected mosquitoes increases to 91.2%. WNV titer in legs was highly positively correlated with WNV body titer (r 2 = 0.73, p < 0.001), yet the threshold viral load required for WNV dissemination was 4.2 log 10 pfu/ mosquito body (Figure 1b).

Survival and Wing length

Cx. pipiens survival curves were generated and compared for susceptible, resistant and unexposed groups using both individual replicate and combined data. Survival was significantly decreased in resistant groups relative to both susceptible and unexposed groups in both replicates (p < 0.01, log-rank; Figure 2). Survival time was on average 9.7 days (replicate I) or 14.5 days (replicate II) less in resistant relative to susceptible mosquitoes. Although in both replicates, average, median, and maximum survival times were also higher in susceptible groups relative to unexposed groups (table 1), a difference in survival between these two groups was only significant for replicate I (p < 0.01, log-rank). In addition, significantly increased survival occurred for both susceptible and unexposed groups between replicates (table 1). The reason for the difference between replicates in not clear, although variations in colony fitness between generations are not uncommon.

Mean wing lengths between replicates and among groups were statistically similar for all individual comparisons (p > 0.05, t-test). Values for wing length ranged from 3.21 mm to 4.00 mm with a combined mean length of 3.70 mm (table 1).

Fecundity

The reproductive output of each group was monitored and compared throughout the study. Although the values for R 0 are substantially lower for the resistant group in both replicates, this can be wholly attributed to the decreased survival observed with this group (table 1). In fact, values for both r and T demonstrate that the rate of increase of the resistant population is similar or slightly greater than both other groups while surviving (table 1). In addition, although lacking in statistical significance due to low sample size, both smoothed m x and total % of females laying eggs were modestly larger for the resistant group when replicates are combined (Figure 3; chi-squared; p > 0.01). This difference cannot be attributed to WNV infection as all measures of egg output were similar among susceptible and unexposed groups (table 1; Figure 3). A gradual decline in reproductive output as noted by the number of eggs/raft was measured throughout the study ( Figure 4). An exception to this was seen with a spike in eggs in the resistant group at week 2, yet this data point represents just a single raft containing 218 eggs. Significant differences were measured in the egg hatch rates among groups ( Figure 5; chi-squared, p < 0.001). Again, there is no apparent indication that WNV infection is associated with a decreased egg hatch rate, as rates for the unexposed group were in fact significantly lower than both susceptible and resistant groups.

Bloodfeeding behaviour

The combined mean weekly feeding rate for all experimental mosquitoes was 31.65% and was statistically similar among groups (table 2; fisher's exact, p > 0.05). The percent of female Cx. pipiens not taking a subsequent bloodmeal following the initial feeding to enter the study was 28.4% (table 2). Although this again did not differ statistically among groups, it is notable that the lowest percentage of unfed females was observed in the resistant groups despite significantly decreased survival (table 2; fisher's exact, p > 0.05). Although a higher proportion of females fed, levels of engorgement were also on average lower in the resistant groups based on mean qualitative scores (table 2). Statistical significance was also not attained here when combined resistant groups were compared to both combined susceptible and unexposed groups (t-test, p > 0.05) yet this likely was due to the combination of small sample size and large deviations in the resistant group. In addition, since the statistical power was based solely on averaging qualitative observations, results do not rule out the potential for this difference to be biologically significant.

Discussion

Unlike the acute, lytic, and often pathogenic infections which are associated with arbovirus infection of vertebrates, infections of mosquito vectors are often persistent and generally thought to be largely benign [1]. Figure 2 Combined survival of individual groups of Cx. pipiens following initial bloodfeeding. Statistically significant differences in survival were measured between both WNV susceptible and unexposed groups relative to WNV resistant mosquitoes (log-rank, p < 0.001). While it is true that the primary vectors of arboviruses have the capacity to support high levels of virus replication, it is not always the case that mosquito hosts are free from fitness costs as a result of these infections [5].

In addition, recent studies demonstrate that mosquito immune responses to arbovirus infections are measurable and complex, suggesting that an evolutionary need to retain such a defence system likely exists [23].

Although the costs of immune defence have been documented with other insect-pathogen relationships [24][25][26][27], such costs generally remain uncharacterized for arbovirus-mosquito associations. Defining the costs of both defence and infection has direct implications for predicting the evolution of virus-vector interactions and, consequently, for forecasting the success of emerging vector-borne diseases. West Nile virus emergence and subsequent expansion in the U.S. has been driven by the availability of both highly susceptible avian hosts and highly competent mosquito vectors in the Culex genus [11,28]. While WNV strain has a significant impact on phenotype, studies have also clearly demonstrated that, although Cx. quinquefasciatus, Cx. pipiens, and Cx. tarsalis all have the capacity to maintain WNV activity, vector competence differs among and within each species [13,14]. Consistent with these findings, colonies of Cx. tarsalis and Cx. pipiens in our laboratory differ substantially in competence, with Cx. pipiens being more susceptible to WNV infection and Cx. tarsalis being more likely to transmit once infected [29]. Both susceptibility and tolerance to infection will directly affect vector competence and are inherently linked to the cost of arbovirus infection and/or immune defence in the mosquito. For this reason, it is reasonable to assume that differences in vector competence may be partially explained by the costs of infection or defence. A previous study in our laboratory with Cx. tarsalis demonstrated that infection with WNV resulted in decreased fecundity of mosquitoes [9]. In addition, this study demonstrated that a fitness cost was not associated with resistance to infection. Taken together, these results could partially explain the maintenance of a relatively   high number of WNV resistant Cx. tarsalis in this population. Here, we evaluated survival, fecundity and feeding rates in Cx. pipiens in groups which were (i) susceptible, (ii) resistant, or (iii) unexposed. Based on the assumption that the costs of resistance and infection can partially explain patterns of vector competence in nature, we hypothesized that the increased susceptibility of Cx. pipiens to WNV should be reflected in these costs.

Results clearly demonstrate that costs are indeed species-specific. Unlike Cx. tarsalis, there was no evidence of decreased fecundity with WNV infection of Cx. pipiens as measured by eggs/female (table 1), eggs/raft (table 1; Figure 4), or percent of females ovipositing ( Figure 4). In addition, hatch rate was in fact significantly higher in the infected group relative to the unexposed group (fisher's exact, p < 0.05; Figure 5). Although egg ovipositing females are likely to be mated, whether differences in hatch rate can truly be attributed to WNV infection cannot be fully assessed unless the reproductive status of individual females was known. In addition, hatch rates for resistant mosquitoes was significantly higher than both susceptible and unexposed groups (fisher's exact, p < 0.05; Figure 5). This difference can likely be attributed to the fact that eggs in primary rafts were more likely to be viable than those in subsequent rafts and that resistant individuals rarely survived long enough to produce more than a single egg raft (table 1, Figures 1 and 4). Overall, survival time was significantly decreased for resistant individuals as compared to both susceptible and unexposed groups (logrank, p < 0.01; Table 1; Figure 1). Combined median survival time for resistant groups was just 14 days, compared to 25.5 or 39.5 days for susceptible mosquitoes in replicates I and II, respectively (table 1, Figure 1). Although generational fitness differences in mosquito colonies are apparent here, both combined and individual survival data demonstrate significantly decreased survival for resistant mosquitoes relative to the more abundant susceptible population. In addition, although significant differences in survival were not identified between susceptible and unexposed groups, average, median and maximum survival time was lower in unexposed groups for both combined and individual replicate data (table 1; Figure 1). Since the unexposed group would be expected to be composed of a majority of susceptible mosquitoes together with a small percentage of less fit, resistant mosquitoes, this result is also consistent with the overall conclusion that WNV resistance is associated with decreased survival. Since the majority of mosquitoes were susceptible, sample sizes for resistant groups were small; but the fact that this difference in survival was observed in both replicates increases confidence in this finding. Differences in survival both among replicates and groups cannot be attributed to size, as wing sizes for all mosquito groups tested were statistically equivalent (table 1). The combined infection rate of 84.0% is comparable to previous studies with WNV and, as expected, was significantly higher than that measured with Cx. tarsalis using the same virus strain and experimental conditions (33.3%; fisher's exact, p < 0.0001; [9]). This confirms that these mosquitoes differ substantially in their capacity to maintain resistant individuals in the population and that these differences are consistent with the cost of resistance and infection Data represents combined values from two replicates. 1 Mean weekly feeding rates for surviving mosquitoes 2 Initial bloodmeal feeding was required for entry into the study. Percents represent individuals that did not take at least one subsequent bloodmeal. 3 Mean score +/-SD. Levels of engorgement were qualitatively scored as 0-4, with 0 representing no blood and 4 representing full engorgement. Figure 5 Combined percent of Cx. pipiens egg rafts hatched throughout the study. Significant differences were measured between groups (chi-squared, p < 0.001). now identified between these two species. Specifically, Cx. pipiens display no cost from infection yet a significant cost for resistance, which predicts that more susceptible individuals will be maintained in the population, while Cx. tarsalis display no cost for resistance yet a significant cost for infection, which predicts more resistant individuals will be maintained in the population. The bloodfeeding results are also consistent with these differences, as the increased feeding rate identified with WNV infection of Cx. tarsalis by Styer et al can be viewed either as compensatory effect selected by the virus to overcome the cost of infection in this host or, more likely, a host reaction to decreased fecundity associated with WNV infection. In either case, a species that does not accrue such costs from WNV infection should, in turn, not develop increased feeding rates with infection. Indeed, results here demonstrate that no differences in feeding rates exist among experimental groups of Cx. pipiens (table 2). What remains unclear is how well these colonized mosquitoes represent the variation within populations of Culex mosquitoes in nature. Clearly, genetic heterogeneity will be much greater in nature, and this will have significant implications for phenotypic variation in terms of susceptibility. In addition, the premise that population structure has been significantly impacted by infection status assumes that both prevalence of infection and time of co-evolution with WNV have been sufficient for such relationships to evolve. Although the co-evolutionary history of WNV and Culex mosquito populations in the U.S. spans back just over a decade, Cx. pipiens, unlike Cx. tarsalis, are an invasive species which likely have a more historic association with WNV [30]. This could potentially explain the increased tolerance to infection in Cx. pipiens identified here, yet these relationships are likely much more complex, as many populations of Cx. tarsalis are highly competent vectors and small variations in the genetic signature among Cx. pipiens can significantly alter susceptibility [14]. Alternatively, these relationships could be generic responses to a host of pathogens as Culex mosquitoes in the U.S. have had more historic relationships with other arboviruses including St. Louis encephalitis virus, a flavivirus which is closely related to WNV [31]. Future studies will help elucidate the specificity of the costs of infection and resistance.

In addition, it would be naïve to assume that all differences in arbovirus susceptibility and competence of individual mosquito species or populations can be explained exclusively by the costs of resistance and infection, as the capacity of an arbovirus to infect, replicate and disseminate in particular hosts will likely be largely due to virus-dependent molecular interactions beyond host immunity, such as the efficiency of recognition and entry, and replication in particular hosts. In addition, the relationship between temperature and vector competence is well established [32]. Despite this, results here clearly demonstrate that the costs of infection and resistance in vector populations are important factors to be considered when both predicting the way in which current vector-virus relationships might co-evolve and assessing the potential for arboviral emergence in previously naïve vector populations.

Conclusions

In contrast to our previous findings with a relatively resistant Cx. tarsalis colony, WNV infection did not alter fecundity or blood-feeding behaviour of Cx. pipiens, indicating that there is no cost associated with WNV infection in this population. Further studies will help clarify if additional WNV adaptation to a vector population could lead to increased virulence in these vectors and therefore constrain the evolution of more efficient strains. In addition, results clearly demonstrate a significant decrease in survival associated with WNV resistance under our experimental conditions. The identification of species-specific differences provides an evolutionary explanation for variability in susceptibility of mosquito vectors to arboviruses and suggests that understanding the costs of infection and resistance, together with the co-evolutionary history of vector and virus, are important considerations when evaluating the potential competence of vector populations for arboviruses.

Methods

Virus strains and testing

WNV used for experimentation was derived from WNV NY003356, isolated from an American crow in 2000 from Staten Island, NY [33] and prepared by three rounds of plaque purification and a single amplification on Vero cells (African green monkey kidney; ATCC CCL-81) as previously described [34]. Mosquito bodies and legs were separated and placed in individual tubes with 1 ml mosquito diluent [MD; 20% heat-inactivated FBS in Dulbecco's PBS plus 50 μg/ml penicillin/streptomycin, 50 μg/ml gentamicin, and 2.5 μg/ml Fungizone] plus one 5 mm metal bead (Daisy). Individual samples were thawed and homogenized for 30 seconds at 24 hz in a Mixer Mill MM301 (Retsch), and debris was pelleted by centrifugation at 6000 rcf for 5 minutes and screened or titrated by plaque assay in duplicate on Vero cells as previously described [35].

Mosquitoes

Cx. pipiens egg rafts were originally collected in Pennsylvania in 2004 (courtesy of M. Hutchinson) and subsequently colonized at the Arbovirus laboratory, Wadsworth Center. Mosquitoes were reared and maintained in 30.5 cm 3 cages in an environmental chamber at 27°C, 50-65% relative humidity with a photoperiod of 16:8 (light:dark) hours. 400 adult mosquitoes (200 male/ 200 female) to be used for experiments were collected upon emergence and held in mesh top 3.8 L paper cartons and provided cotton pads with 10% sucrose ad libitum. Mosquitoes were held for 4 days to allow for mating.

Chickens

Day-old, pathogen-free chickens (Gallus gallus) were obtained from Charles River (North Franklin, CT) and transferred to the Arbovirus laboratory BSL-3 animal facility in preparation for experimentation. Chickens were housed in metal cages with individual light sources and daily fresh food, water, and resting pads. Three dayold chickens were inoculated subcutaneously with either 10 3 pfu WNV in 100 ul animal diluent (endotoxin-free phosphate buffered saline [PBS] +1% fetal bovoine serum [FBS]) or 100 ul diluent alone, 3 days prior to mosquito feeding. All chicken work was approved by the Wadsworth Center Institutional Animal Care and Use Committee (IACUC 06-355).

Blood feeding

Mosquitoes were deprived of sucrose for 48 hours prior to feeding on chickens. Following starvation, female mosquitoes were removed from the large carton and distributed into two 0.6 L cups for experimental infections in the BSL-3 animal facility. A mock (control) or WNV (experimental) inoculated chicken was placed on top of the mesh of individual cups and carefully restrained manually while mosquitoes were given approximately 1 hour to feed. Following feeding, 100 ul of blood was drawn from the brachial vein of the chickens and transferred to serum separator tubes as previously described [36]. Chicken blood was processed as previously described and serum was saved at -80°C for subsequent plaque titration [9]. Mosquitoes were then anesthetized using CO 2 and fully-engorged mosquitoes were separated and housed individually in cups containing oviposition dishes with 15 mls of distilled water and with access to 10% sucrose.

Subsequent uninfected blood meals were offered to both control and experimental groups via hanging drops for two days each week of the study. Specifically, mosquitoes were again starved for 48 hrs and then offered 30 ul drops of defibrinated goose blood (Hema Resources) with 2.0% sucrose. Mosquitoes were monitored for one hour during these feedings and both numbers fed and levels of engorgement (1, small amount of blood in abdomen, no abdominal distention; 2, some distention, no pleural membrane observed; 3, significant abdominal blood, pleural membrane observed; 4, fully engorged, distended abdomen) were recorded.

Mosquito fitness

Survival, wing length, fecundity, and egg hatching were evaluated in this study. Mortality and egg production were monitored and recorded daily for all groups. Wings were removed from dead mosquitoes, individually mounted on slides with double-sided tape, and measured as previously described using a Zeiss microscope, Axiocam camera, and Axiovision software (Carl Zeiss; [9]). Mosquito bodies and legs were processed and tested as described above. Egg rafts were photographed under 50X magnification using a Nikon digital camera (Nikon) and individual eggs were counted using Photo Studio (ArcSoft).

Oviposition cups containing rafts were held for approximately 2 days at 27°C to allow for hatching and 1 st instar larvae were counted in order to calculate egg hatching rates.

Data analysis

Survival curves were generated and analyzed using GraphPad Prism software version 4.0. Comparisons of curves both among groups [(resistant (exposed and uninfected), susceptible (exposed and infected), and unexposed (fed on uninfected chicken)] and between replicates were done with a log-rank test, which is equivalent to the Mantel-Haenszel test. Resistance in this study is defined only as an inability to become infected under these experimental conditions and therefore does not necessarily imply resistance at higher WNV doses. Both survival and reproductive data were used to construct life history tables for each group in separate replicates. Specific calculations included survival (l x ), equivalent to the proportion of mosquitoes surviving to day x, and reproductive output (m x ), equivalent to the number of eggs produced on day x. Data for m x was smoothed by averaging an individual daily egg output with the egg output on both previous and subsequent days. Subsequent calculations for net reproductive rate (total eggs produced in an average females lifetime; R 0 = ∑ l x m x ), generation time (average age at which a females lays her eggs; T = ∑ l x m x x/R 0 ), and intrinsic rate of increase (instantaneous population growth rate; r = ln R 0 /T) [37,38] were performed. GraphPad 4.0 was used to construct contingency tables and perform subsequent Fisher's exact and Chi-squared tests for both female egg ovipositing and egg hatch rate among groups. Microsoft Excel was used to perform t-tests for comparisons of mean eggs/raft and levels of engorgement among groups.

Figure 1
1WNV titers in Cx. pipiens mosquitoes at the time of death. (a) Data points represent individual mosquitoes and the line represents the best-fit relationship between WNV body titer and time of death. (b) Relationship between WNV body titer and WNV leg titer in individual mosquitoes. The dotted line refers to the threshold body titer required for WNV dissemination (4.2 log 10 pfu) and the linear regression analysis was completed for all values above that threshold.

Figure 3
3Combined fecundity of individual groups of Cx. pipiens. The percent of females ovipositing refers to individuals producing at least one egg raft during the study and smoothed m x refers to the average daily reproductive output while surviving. No significant differences were measured in these statistics (fisher's exact, p > 0.05).

Figure 4
4Mean egg raft sizes produced by individual groups of Cx. pipiens during the study. No mosquitoes in the resistant group survived beyond week 3 of the study.

Table 1
1Summary statistics for Cx. pipiens fitness following feeding on a bloodmeal with (resistant, susceptible) or without (unexposed) WNV Resist. Suscept. Unexp. Resist. Suscept. Unexp.replicate 1
replicate 2

N
6
36
22
7
32
14

Wing (mm) 3.67
3.73
3.69
3.70
3.72
3.67

AST 1 (d)
16.8
26.5
20.9
19.7
38.2
34.2

MST 2 (d)
13.5
25.5
21.0
14.0
39.5
36.0
MaxST 3 (d)
35.0
59.0
45.0
41.0
68.0
57.0

Rafts/fem 4 0.46
1.39
1.32
0.86
1.25
1.29

Eggs/raft
174.0
162.1
151.7
152.2
144.5
140.1

R 0

5

37.1
94.6
79.1
65.2
87.1
88.2

T 6
14.2
15.0
16.8
13.4
21.2
23.6

r 7
0.25
0.30
0.26
0.31
0.21
0.19

1 average survival time
2 median survival time
3 maximum survival
4 mean total egg rafts produced per female
5 net reproduction rate
6 mean generation time
7 population growth rate

Table 2
2Summary of blood feeding behavior among study groupsweekly feeding
rate 1

total
unfed 2

engorgement
score 3

resistant
34.21%
23.1%
2.0 +/-1.3

susceptible
29.93%
28.8%
2.6 +/-1.3
unexposed
34.15%
29.7%
2.5 +/-1.1

Ciota et al. BMC Ecology 2011, 11:23 http://www.biomedcentral.com/1472-6785/11/23 Page 6 of 8
AcknowledgementsThe authors thank Claus Wilke for assistance with statistical analyses. The authors would like to acknowledge Pamela Chin for technical assistance. The authors thank the Wadsworth Center Media and Tissue Culture Facility for providing cells and media for this work. We also thank the BSL-3 animal facility staff at the Wadsworth Center, which was used in this study and is funded in part by the Northeast Biodefense Center's animal core on the NIH/NIAID award U54A17158. This work was supported partially by federal funds from the National Institute of Allergy and Infectious Disease, National Institutes of Health (contract number NO1-AI-25490) and, National Institutes of Health (grant number RO1-AI-077669).Authors' contributions
F M Burnet, D O White, Natural history of infectious diseases Cambridge. Cambridge University PressBurnet FM, White DO: Natural history of infectious diseases Cambridge, Cambridge University Press; 1972.

Lack of adverse effect of transovarially acquired La Crosse virus infection on the reproductive capacity of Aedes triseriatus (Diptera: Culicidae). L A Patrican, G R Defoliart, J Med Entomol. 22Patrican LA, DeFoliart GR: Lack of adverse effect of transovarially acquired La Crosse virus infection on the reproductive capacity of Aedes triseriatus (Diptera: Culicidae). J Med Entomol 1985, 22:604-611.

Impact of stressful conditions on the survival of Culex pipiens exposed to Rift Valley fever virus. D J Dohm, W S Romoser, M J Turell, K J Linthicum, J Am Mosq Control Assoc. 7Dohm DJ, Romoser WS, Turell MJ, Linthicum KJ: Impact of stressful conditions on the survival of Culex pipiens exposed to Rift Valley fever virus. J Am Mosq Control Assoc 1991, 7:621-623.

Blood feeding behavior of dengue-2 virus infected Aedes aegypti. J L Putnam, T W Scott, Am J Trop Med Hyg. 52Putnam JL, Scott TW: Blood feeding behavior of dengue-2 virus infected Aedes aegypti. Am J Trop Med Hyg 1995, 52:225-227.

Mode of transmission and the evolution of arbovirus virulence in mosquito vectors. L Lambrechts, T W Scott, Proc Biol Sci. 276Lambrechts L, Scott TW: Mode of transmission and the evolution of arbovirus virulence in mosquito vectors. Proc Biol Sci 2009, 276:1369-1378.

Coevolution of hosts and parasites. R M Anderson, R M May, Parasitology. 19822Anderson RM, May RM: Coevolution of hosts and parasites. Parasitology 1982, 85(Pt 2):411-426.

The evolution and maintenance of virulence in microparasites. B R Levin, Emerg Infect Dis. 2Levin BR: The evolution and maintenance of virulence in microparasites. Emerg Infect Dis 1996, 2:93-102.

Trade-offs, conflicts of interest and manipulation in Plasmodium-mosquito interactions. A Schwartz, J C Koella, Trends Parasitol. 17Schwartz A, Koella JC: Trade-offs, conflicts of interest and manipulation in Plasmodium-mosquito interactions. Trends Parasitol 2001, 17:189-194.

West Nile virus infection decreases fecundity of Culex tarsalis females. L M Styer, M A Meola, L D Kramer, J Med Entomol. 44Styer LM, Meola MA, Kramer LD: West Nile virus infection decreases fecundity of Culex tarsalis females. J Med Entomol 2007, 44:1074-1085.

K A Bernard, J G Maffei, S A Jones, E B Kauffman, G D Ebel, A P Dupuis, Ii, K A Ngo, D C Nicholas, D M Young, P Y Shi, V L Kulasekera, M Eidson, D J White, W B Stone, L D Kramer, Comparison of WNV infection in birds and mosquitoes in New York State in 2000. Emerg Inf Dis. 7Bernard KA, Maffei JG, Jones SA, Kauffman EB, Ebel GD, Dupuis AP II, Ngo KA, Nicholas DC, Young DM, Shi PY, Kulasekera VL, Eidson M, White DJ, Stone WB, Kramer LD: Comparison of WNV infection in birds and mosquitoes in New York State in 2000. Emerg Inf Dis 2001, 7:679-685.

West Nile virus: epidemiology and ecology in North America. N Komar, Adv Virus Res. 61Komar N: West Nile virus: epidemiology and ecology in North America. Adv Virus Res 2003, 61:185-234.

Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. W K Reisen, Y Fang, V M Martinez, J Med Entomol. 42Reisen WK, Fang Y, Martinez VM: Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J Med Entomol 2005, 42:367-375.

An update on the potential of north American mosquitoes (Diptera: Culicidae) to transmit West Nile Virus. M J Turell, D J Dohm, M R Sardelis, M L Oguinn, T G Andreadis, J A Blow, J Med Entomol. 42Turell MJ, Dohm DJ, Sardelis MR, Oguinn ML, Andreadis TG, Blow JA: An update on the potential of north American mosquitoes (Diptera: Culicidae) to transmit West Nile Virus. J Med Entomol 2005, 42:57-62.

Spatial and temporal variation in vector competence of Culex pipiens and Cx. restuans mosquitoes for West Nile virus. A M Kilpatrick, D M Fonseca, G D Ebel, M R Reddy, L D Kramer, Am J Trop Med Hyg. 83Kilpatrick AM, Fonseca DM, Ebel GD, Reddy MR, Kramer LD: Spatial and temporal variation in vector competence of Culex pipiens and Cx. restuans mosquitoes for West Nile virus. Am J Trop Med Hyg 2010, 83:607-613.

Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae). Y A Girard, V Popov, J Wen, V Han, S Higgs, J Med Entomol. 42Girard YA, Popov V, Wen J, Han V, Higgs S: Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae). J Med Entomol 2005, 42:429-444.

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection. R Vaidyanathan, T W Scott, Apoptosis. 11Vaidyanathan R, Scott TW: Apoptosis in mosquito midgut epithelia associated with West Nile virus infection. Apoptosis 2006, 11:1643-1651.

Cytopathic effect of Semliki Forest virus in the mosquito Aedes aegypti. C A Mims, M F Day, I D Marshall, Am J Trop Med Hyg. 15Mims CA, Day MF, Marshall ID: Cytopathic effect of Semliki Forest virus in the mosquito Aedes aegypti. Am J Trop Med Hyg 1966, 15:775-784.

Pathologic changes in the midgut of Culex tarsalis following infection with Western equine encephalomyelitis virus. S C Weaver, L H Lorenz, T W Scott, Am J Trop Med Hyg. 47Weaver SC, Lorenz LH, Scott TW: Pathologic changes in the midgut of Culex tarsalis following infection with Western equine encephalomyelitis virus. Am J Trop Med Hyg 1992, 47:691-701.

Reduction of Culiseta melanura fitness by eastern equine encephalomyelitis virus. T W Scott, L H Lorenz, Am J Trop Med Hyg. 59Scott TW, Lorenz LH: Reduction of Culiseta melanura fitness by eastern equine encephalomyelitis virus. Am J Trop Med Hyg 1998, 59:341-346.

Effect of eastern equine encephalomyelitis virus on the survival of Aedes albopictus, Anopheles quadrimaculatus, and Coquillettidia perturbans (Diptera: Culicidae). A C Moncayo, J D Edman, M J Turell, J Med Entomol. 37Moncayo AC, Edman JD, Turell MJ: Effect of eastern equine encephalomyelitis virus on the survival of Aedes albopictus, Anopheles quadrimaculatus, and Coquillettidia perturbans (Diptera: Culicidae). J Med Entomol 2000, 37:701-706.

Western equine encephalomyelitis virus infection affects the life table characteristics of Culex tarsalis (Diptera: Culicidae). F Mahmood, W K Reisen, R E Chiles, Y Fang, J Med Entomol. 41Mahmood F, Reisen WK, Chiles RE, Fang Y: Western equine encephalomyelitis virus infection affects the life table characteristics of Culex tarsalis (Diptera: Culicidae). J Med Entomol 2004, 41:982-986.

Exposure to chickungunya virus and adult longevity in Aedes aegypti (L.) and Aedes albopictus (Skuse). M H Reiskind, C J Westbrook, L P Lounibos, J Vector Evol. 35Reiskind MH, Westbrook CJ, Lounibos LP: Exposure to chickungunya virus and adult longevity in Aedes aegypti (L.) and Aedes albopictus (Skuse). J Vector Evol 2010, 35:61-68.

Advances in dissecting mosquito innate immune responses to arbovirus infection. R Fragkoudis, G Ttarzadeh-Yazdi, A A Nash, J K Fazakerley, A Kohl, J Gen Virol. 90Fragkoudis R, ttarzadeh-Yazdi G, Nash AA, Fazakerley JK, Kohl A: Advances in dissecting mosquito innate immune responses to arbovirus infection. J Gen Virol 2009, 90:2061-2072.

Costs of resistance in insect-parasite and insect-parasitoid interactions. A R Kraaijeveld, J Ferrari, H C Godfray, Parasitology. 125SupplKraaijeveld AR, Ferrari J, Godfray HC: Costs of resistance in insect-parasite and insect-parasitoid interactions. Parasitology 2002, 125(Suppl):S71-S82.

Resistance is costly: trade-offs between immunity, fecundity and survival in the pea aphid. D M Gwynn, A Callaghan, J Gorham, K F Walters, M D Fellowes, Proc Biol Sci. 272Gwynn DM, Callaghan A, Gorham J, Walters KF, Fellowes MD: Resistance is costly: trade-offs between immunity, fecundity and survival in the pea aphid. Proc Biol Sci 2005, 272:1803-1808.

The evolutionary costs of immunological maintenance and deployment. K A Mckean, C P Yourth, B P Lazzaro, A G Clark, BMC Evol Biol. 876McKean KA, Yourth CP, Lazzaro BP, Clark AG: The evolutionary costs of immunological maintenance and deployment. BMC Evol Biol 2008, 8:76.

Evaluating the costs of mosquito resistance to malaria parasites. H Hurd, P J Taylor, D Adams, A Underhill, P Eggleston, Evolution. 59Hurd H, Taylor PJ, Adams D, Underhill A, Eggleston P: Evaluating the costs of mosquito resistance to malaria parasites. Evolution 2005, 59:2560-2572.

West Nile virus activity in the United States. K A Bernard, L D Kramer, Viral Immunol. 14Bernard KA, Kramer LD: West Nile virus activity in the United States, 2001. Viral Immunol 2001, 14:319-338.

West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors. R M Moudy, B Zhang, P Y Shi, L D Kramer, Virology. 387Moudy RM, Zhang B, Shi PY, Kramer LD: West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors. Virology 2009, 387:222-228.

The Culex pipiens complex. P F Mattingly, L E Roseboom, K L Knight, H Laven, F H Drummond, S R Christophers, P G Shute, Trans R Entomol Soc London. 102Mattingly PF, Roseboom LE, Knight KL, Laven H, Drummond FH, Christophers SR, Shute PG: The Culex pipiens complex. Trans R Entomol Soc London 1951, 102:331-382.

T P Monath, Fx ; Heinz, B N Fields, D M Knipe, P M Howley, Philidelphia, Flaviviruses. In Fields Virology. Lippincott Williams and WilkinsMonath TP, Heinz FX: Flaviviruses. In Fields Virology. Edited by: Fields BN, Knipe DM, Howley PM. Philidelphia: Lippincott Williams and Wilkins; 1996:961-1034.

Temperature, viral genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes. A M Kilpatrick, M A Meola, R M Moudy, L D Kramer, PLoS Pathogens. 41000092Kilpatrick AM, Meola MA, Moudy RM, Kramer LD: Temperature, viral genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes. PLoS Pathogens 2008, 4:e1000092.

Partial genetic characterization of West Nile virus strains. G D Ebel, A P Dupuis, Ii, K A Ngo, D C Nicholas, E B Kauffman, S A Jones, D M Young, J G Maffei, P Y Shi, K A Bernard, L D Kramer, Emerg Infect Dis. 7Ebel GD, Dupuis AP II, Ngo KA, Nicholas DC, Kauffman EB, Jones SA, Young DM, Maffei JG, Shi PY, Bernard KA, Kramer LD: Partial genetic characterization of West Nile virus strains, New York State, 2000. Emerg Infect Dis 2001, 7:650-653.

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture. A T Ciota, A O Lovelace, K A Ngo, A N Le, J G Maffei, M A Franke, A F Payne, S A Jones, E B Kauffman, L D Kramer, Virology. 357Ciota AT, Lovelace AO, Ngo KA, Le AN, Maffei JG, Franke MA, Payne AF, Jones SA, Kauffman EB, Kramer LD: Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture. Virology 2007, 357:165-174.

Quantitation of flaviviruses by fluorescent focus assay. A F Payne, I Binduga-Gajewska, E B Kauffman, L D Kramer, J Virol Methods. 134Payne AF, Binduga-Gajewska I, Kauffman EB, Kramer LD: Quantitation of flaviviruses by fluorescent focus assay. J Virol Methods 2006, 134:183-187.

Characterization of mosquito-adapted West Nile virus. A T Ciota, A O Lovelace, Y Jia, L J Davis, D S Young, L D Kramer, J Gen Virol. 89Ciota AT, Lovelace AO, Jia Y, Davis LJ, Young DS, Kramer LD: Characterization of mosquito-adapted West Nile virus. J Gen Virol 2008, 89:1633-1642.

J R Carey, Applied Biodemography for Biologists with Special Emphasis on Insects. New YorkOxford University PressCarey JR: Applied Biodemography for Biologists with Special Emphasis on Insects New York, Oxford University Press; 1993.

. Re: Ecology Ricklefs, Newton, 10.1186/1472-6785-11-23Chiron PressRicklefs RE: Ecology Newton, Chiron Press; 1973. doi:10.1186/1472-6785-11-23

The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes. Ciota, BMC Ecology. 1123Cite this article asCite this article as: Ciota et al.: The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes. BMC Ecology 2011 11:23.

. Ciota, BMC Ecology. 1123Ciota et al. BMC Ecology 2011, 11:23 http://www.biomedcentral.com/1472-6785/11/23


The relationships between interoception and alexithymic trait. The Self-Awareness Questionnaire in healthy subjects
August 2015

Olga Pollatos
Glenn Carruthers
Neil Gerald Muggleton
Mariachiara Longarzo firstname.lastname@example.com
Dario Grossi firstname.lastname@example.com
Mariachiara Longarzo
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

Francesca D'olimpio
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

Angela Chiavazzo
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

Gabriella Santangelo
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

Hermitage Capodimonte
NapoliItaly

Luigi Trojano
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

Dario Grossi
Department of Psychology
Laboratory of Neuropsychology
Second University of Naples
CasertaItaly

University of Ulm
Germany

Macquarie University
Australia

Department of Psychology
Laboratory of Neuropsychology
National Central University
Taiwan

Second University of Naples
Viale Ellittico 3181100CasertaItaly

The relationships between interoception and alexithymic trait. The Self-Awareness Questionnaire in healthy subjects

Frontiers in Psychology | www.frontiersin.org
11149August 201510.3389/fpsyg.2015.01149Received: 23 December 2014 Accepted: 23 July 2015ORIGINAL RESEARCH Edited by: Reviewed by: *Correspondence: Specialty section: This article was submitted to Consciousness Research, a section of the journal Frontiers in Psychology Citation: Longarzo M, D'Olimpio F, Chiavazzo A, Santangelo G, Trojano L and Grossi D (2015) The relationships between interoception and alexithymic trait. The Self-Awareness Questionnaire in healthy subjects. Front. Psychol. 6:1149.interoceptive awarenessemotionalexithymiahypochondriasishealthinsular cortex
Interoception is the basic process enabling evaluation of one's own bodily states. Several previous studies suggested that altered interoception might be related to disorders in the ability to perceive and express emotions, i.e., alexithymia, and to defects in perceiving and describing one's own health status, i.e., hypochondriasis. The main aim of the present study was to investigate the relationships between alexithymic trait and interoceptive abilities evaluated by the "Self-Awareness Questionnaire" (SAQ), a novel self-report tool for assessing interoceptive awareness. Two hundred and fifty healthy subjects completed the SAQ, the Toronto Alexithymia Scale-20 items (TAS-20), and a questionnaire to assess hypochondriasis, the Illness Attitude Scale (IAS). The SAQ showed a two-factor structure, with good internal consistency (Cronbach's alpha = 0.88). We observed significant direct correlations between SAQ, TAS-20 and two of its subscales, and the IAS. Regression analysis confirmed that the difficulty in identifying and expressing emotions is significantly related with awareness for one's own interoceptive feelings and with a tendency to misinterpret and amplify bodily sensations. From a clinical point of view, the assessment of interoceptive awareness by the SAQ could be pivotal in evaluating several psychopathological conditions, such as the somatoform disorders.

Introduction

Interoception is the basic process collecting information coming from one's own body, such as heartbeat, hunger, thirst, breathing, or visceral sensations. Such information is less distinct than that provided by "exteroceptive" somatosensory systems (e.g., touch, or skin temperature), but contributes to maintain homeostasis (Craig, 2003), and allows the brain to build a sense of one's own physical condition and to answer questions such as "how do you feel now?" (Craig, 2009).

The relationships between interoception and emotion are still under debate. Classic and modern theories of emotion maintain that bodily states contribute to or are essential for emotional experience (James, 1884;Lange, 1885;Bard, 1928;Cannon, 1932;Lane and Schwartz, 1987). Several studies using heartbeat perception tasks demonstrated a positive association between interoception and intensity of emotional experience (e.g., Schandry, 1981;Jones, 1994;Wiens et al., 2000;Barrett et al., 2004;Critchley et al., 2004;Wiens, 2005;Herbert et al., 2007;Pollatos et al., 2007a,b). Thus, according to Craig (2004Craig ( , 2009, individual differences in emotional awareness may be directly related to individual differences in interoception. Similarly, Pollatos et al. (2005) and Dunn et al. (2010) reported that individuals with high sensitivity to interoceptive signals showed higher arousal in response to emotional visual stimuli.

The close relationships between interoception and emotional awareness have recently been supported by a neurofunctional study. Zaki et al. (2012) demonstrated that the anterior insular cortex, which is considered essential for both interoception monitoring and emotional processing (Craig, 2004(Craig, , 2009Critchley et al., 2004;Pollatos et al., 2007a), was activated during tasks in which healthy subjects were required to monitor their own heartbeat or to assess their own emotional experience after viewing videos of people recounting emotional stories. On this basis, the anterior insular cortex has been regarded as "a convergence zone" of interoceptive and emotional awareness (Zaki et al., 2012; see also Lamm and Singer, 2010). Enhanced activation in a network including the anterior insular cortex, the anterior cingulate cortex, ventromedial prefrontal cortex and somatosensory cortex has been reported in subjects with high interoceptive awareness (Critchley et al., 2004).

Emotional awareness and processing of interoceptive signals are thought to be impaired in alexithymia (Lane et al., 1998(Lane et al., , 2000Craig, 2004), defined by Sifneos (1973) as a reduced ability to identify and describe one's own emotions in patients with psychosomatic diseases. Alexithymia has been hypothesized to be associated with altered activation and morphology in the anterior cingulate cortex and the anterior insular cortex (Lane et al., 1998(Lane et al., , 2000Berthoz et al., 2002;Kano et al., 2003;Borsci et al., 2009). Consistent with such findings, Herbert et al. (2011) demonstrated that the ability to process interoceptive signals (assessed on a heartbeat perception task) is negatively associated with all aspects of alexithymia.

Available studies on the relationships between alexithymia and interoception, however, did not take into account the distinct facets of this complex construct. In this respect, Terasawa et al. (2013) underscored that tasks assessing heartbeat detection (e.g., Schandry, 1981) provide a measure of interoceptive "sensitivity, " i.e., of the accuracy in objective tests of bodily sensations, but are not suitable to investigate other aspects of interoception, such as interoceptive "sensibility" and interoceptive "awareness." Interoceptive "sensibility" would express one's own tendency to be focused on internal states, whereas interoceptive "awareness" (IA) would imply cognitive appreciation of interoceptive sensations, and the ability of expressing bodily feelings. Interoceptive "sensibility" and IA are best addressed by means of self-report questionnaires (Terasawa et al., 2013). The distinction of the three aspects of interoception can help explaining the reason why studies using self-report questionnaires or "objective" heartbeat detection tasks provide divergent findings about the relationships between interoception and alexithymic trait. In fact, contrary to findings reported by Herbert et al. (2011), Ernst et al. (2014 observed a positive correlation between IA (assessed by a self-report tool, the Body Perception Questionnaire; Porges, 1993) and alexithymic trait in a small sample of healthy subjects in a study on neurotransmitter concentration in the insula and in the anterior cingulate cortex.

A positive correlation between IA and alexithymic trait could be consistent with the view that close attention toward interoceptive sensations could hamper interpretation of one's own emotional feelings (Biondi, 1991). Accordingly, in their review Kano and Fukudo (2013) suggested that persons with alexithymia tend to over-report physical symptoms and interpret even low-intensity emotion-related sensations as signs of illness. This hypothesis is consistent with the results of a PET study showing stronger awareness of visceral sensations in alexithymics, with greater activity in the posterior insula and in the rostral anterior cingulate cortex (Kano et al., 2007). Moreover, Tominaga et al. (2014) found a strong correlation between alexithymia (assessed by Toronto Alexithymia Scale-20 items; Bagby et al., 1994) and hypochondriasis and somatoform disorders (assessed by Somatosensory Amplification Scale; Barsky et al., 1988;Nakao and Barsky, 2007). Taken together, such observations would show a significant direct correlation between measures of IA and alexithymic trait, when self-report assessment tools are used.

The aim of the present study was to investigate the hypothesis of a direct association between IA and alexithymic trait in a large sample of healthy subjects. In a systematic review of the available self-report measures assessing body-awareness, Mehling et al. (2009) concluded that no instruments assess all aspects of interoception, but mainly explore anxiety or emotions, without sufficient details on physical sensations and with a few items on body awareness. For these reasons, Mehling et al. (2012) developed the Multidimensional Assessment of Interoceptive Awareness (MAIA) to evaluate dimensions of body awareness such as quality of body sensations, attention regulation for body sensations, emotional awareness for physiological signs of emotion or tendency to evaluate one's own body as safe and trustworthy. However, to the best of our knowledge, there is no tool evaluating perception of a wide range of bodily sensations. For this purpose, we devised a new self-report measure of IA to be used in a large non-clinical sample. This questionnaire, termed "Self-Awareness Questionnaire" (SAQ), is short and easy to administer and refers to commonly felt bodily sensations. This novel self-report tool is based on the "How do you feel questionnaire" (Grossi et al., 2014), which allowed to demonstrate that poor interoceptive awareness is associated with insular damage in stroke patients, and includes further items derived from other available questionnaires (e.g., Barsky et al., 1988;Porges, 1993).

Materials and Methods

Participants

We recruited 250 healthy subjects among students and academic staff of the Departments of Psychology and Political Science at the Second University of Naples. To be included in the present study, subjects had to meet the following criteria: (1) lack of current or past history of alcohol or drug abuse, (2) lack of current or past history of major psychiatric diseases, (3) lack of history of brain injury, stroke, or any other major clinical condition, (4) lack of past or current use of psychoactive medications. The eligibility criteria were assessed by means of a brief semi-structured clinical interview.

All individuals were naïve to the scopes and purposes of the study and gave their written informed consent to participate without any reward.

Materials and Methods

All participants completed three questionnaires assessing: (i) IA, (ii) the ability to identify and describe emotions, and (iii) attitudes associated with hypochondriasis. To assess IA, and to specifically investigate how and how frequently subjects feel signals arising from their own body, we used an extended version of the "How do you feel questionnaire" (Grossi et al., 2014). The questionnaire included 35 items (Appendix 1) to be rated on a 5-point Likert scale (0 = never; 1 = sometimes; 2 = often; 3 = very often; 4 = always). The total score ranges 0-140, with higher scores meaning higher IA. In a preliminary study on an independent sample of 50 healthy students, we required participants to rate whether each item was clearly comprehensible and whether it assessed common physical sensations. All items were considered simple to comprehend, and suitable to address bodily sensations; all 35 items were thus included in the SAQ.

To assess the ability to identify and describe emotions, we used the Toronto Alexithymia Scale-20 items (TAS-20; Bagby et al., 1994), the most widely used self-report tool to assess the Alexithymia construct. The 20 items explore three factors reflecting the main aspects of the alexithymia: difficulty in identifying feelings; difficulty in describing feelings; externally oriented thinking. Each item has to be rated on a 5-point Likert scale (from 1 = "completely agree" to 5 = "strongly disagree"). The total score ranges 20-100, with higher scores indicating higher levels of alexithymia. The Italian version of TAS-20 has been demonstrated to show good test-retest reliability (0.86) and adequate internal consistency (Cronbach's alpha: 0.75) in a wide sample of healthy adults and of medical and psychiatric outpatients (Bressi et al., 1996).

The Illness Attitude Scale (IAS; Kellner, 1987) investigates attitude, fear and beliefs associated with hypochondriac behavior, and includes 27 items rated on a 5-point Likert scale (from 0 = "no" to 4 = "most of the time"). The total score ranges 0-108, with higher scores indicating more severe hypochondriac symptoms. The IAS is a reliable instrument, distinguishing hypochondriac patients from psychiatric patients and healthy individuals (Kellner, 1987). The IAS has been translated in several languages and its psychometric properties are well established (Sirri et al., 2008); an Italian version of the scale has been used in studies on clinical samples (e.g., Fava et al., 2000), but its psychometric properties have not been assessed specifically.

The study was approved by the Local Ethics Committees.

Statistical Analysis

Quality of data for SAQ questionnaire was evaluated by computing percentage of missing or invalid items; a percentage <5% is considered as an index of acceptable data quality. Moreover, data quality was assessed using mean, median, skewness, kurtosis and extent of ceiling and floor effects. Floor and ceiling effects <15% were defined as optimal (Cronbach, 1951). Internal consistency of the SAQ and of its dimensions was evaluated by Cronbach's alpha (McHorney and Tarlov, 1995); a value ≥0.70 was considered acceptable (Scientific Advisory Committee of the Medical Outcomes Trust, 2002). Scaling assumptions referring to the correct grouping of items were checked using alpha correction if item is removed (item is removed if the alpha value increases). Furthermore, we run an Exploratory Factor Analysis, by Principal Axis Factor analysis (PAF), to explore the latent structure of the scale and to perform data reduction; we extracted the components explaining an amount of variance greater than a single item do (i.e., with Eigenvalues > 1; Kaiser, 1960). We computed factor loadings after oblimin rotation, allowing factors to correlate.

Thirdly, we assessed correlations of SAQ with TAS-20 (total and subscales scores for both measures) and IAS total score by computing Pearson's correlation coefficients (Bonferroni correction for multiple comparisons was adopted to reduce type-I errors).

Last, to investigate whether IA explained a significant portion of variance of alexithymia, a regression analysis was performed on TAS total score, using the SAQ total score as a predictor. The specific contribution of SAQ in predicting TAS scores was also assessed by means of hierarchical multiple regression analysis performed on TAS total scores, in which we first entered demographic data (age and gender), then IAS total score, and last SAQ total score as independent variables.

Results

Two hundred and fifty healthy subjects (175 females, 75 males) participated in the present study. Table 1 shows the participants' demographic features and data about the psychometric variables.

Data collected for each item of the SAQ were computable and there was no missing value. There was no floor or ceiling effect; skewness was 0.78 and kurtosis was 0.30.

Descriptive analyses on normality distribution and communality indexes from PAF, suggested removing 7 of the 35 items (Items: 1 , 2, 5, 8, 20, 22, 29). The mean score on the resulting 28-item SAQ "awareness index" (max value = 112) for the present sample was 23.33 (SD = 11.67). PAF extracted three factors with an eigenvalue higher than 1. The screen test indicated a two-factor solution, which accounted for about 29% of the total variance. Factor loadings obtained after oblimin rotation are shown in Table 2.  For each item the highest factor loading is printed in bold.

The first factor (F1) was the most relevant (eigenvalue after rotation = 5.81), and mainly (but not exclusively) included items related to visceral sensations (items 3, 6, 7, 12, 13, 17, 19, 21, 23, 26, 27, 30, 32, 33, 35). The second factor (F2, eigenvalue = 4.50) mainly included items referring to somatosensory sensations (items 4, 9, 10, 11, 14, 15, 16, 18, 24, 25, 28, 30, 31, 34). The SAQ awareness index significantly correlated with both F1 (r = 0.89, p < 0.01) and F2 factors (r = 0.86, p < 0.01); the two factors significantly correlated with each other (r = 0.55, p < 0.01). The SAQ awareness index and its two factors showed good internal consistency (F1: alpha = 0.85; F2: alpha = 0.81; total alpha = 0.88). Correlation analyses ( Table 3) revealed that the SAQ awareness index (and its two factors F1 and F2) significantly correlated with the TAS-20 total score and its two subscales "difficulty identifying feelings" and "difficulty describing feelings, " but not with the TAS-20 subscale assessing "externally oriented thinking." In addition, the SAQ awareness index and its factors F1 and F2 significantly correlated with the IAS total score. All such correlations were positive, meaning that high scores on the SAQ and on its factors were associated to high scores on the questionnaires assessing two specific facets of alexithymia and hypochondriasis. By the same token, high IAS total scores were associated with high scores on TAS-20 and with high scores on two components of TAS-20, "difficulty identifying feelings" and "difficulty describing feelings."

The results from the regression analysis showed that Interoception was a moderately significant predictor of Alexithymia, explaining 13% of the variance (beta = 0.37, p < 0.001). The results from the hierarchical regression models showed that: age and gender were not significant predictors of total TAS score [step# 1: R2 = 0.002; F (2, 247) = 1.28; p = 0.28; beta for age = 0.08, beta for gender = 0.06]; hypochondria was a significant predictor of alexithymia [step# 2: R2 = 0.12; F (3, 246) = 11.96; p < 0.001; beta = 0.35]; IA, as evaluated by the SAQ, was still a significant predictor of TAS scores accounting for a further 8% of the variance [step# 3: R2 = 0.20; F (4, 245) = 16.21; p < 0.001; beta for gender = 0.18, beta for IAS = 0.21, beta for SAQ = 0.33].

Discussion

The aim of the present study was to investigate the relationships between bodily awareness, i.e., "interoceptive awareness" after Terasawa et al. (2013), and the ability to identify and to describe emotions. For this purpose we used a self-report questionnaire (SAQ) specifically assessing how and how frequently participants felt signals from their own body. The SAQ demonstrated a good internal consistency; items clustered into two factors, the first mainly related to visceral feelings, and the second mainly related to somatosensory feelings.

In exploring the relationships between IA and emotion processing, we observed significant positive relationships of SAQ awareness index and of its two factors with TAS-20. In particular, the two subscales of the TAS-20 investigating "difficulty in identifying feelings" and "difficulty in describing feelings" showed strong positive relationships with the SAQ awareness index and its two factors. It is important to underscore that the correlation of IA with alexithymic trait was positive in the present study, consistent with findings reported by Ernst et al. (2014), who observed that a high IA, as assessed by a self-report index, correlated with high alexithymic trait. Although studies assessing interoceptive "sensitivity" (e.g., via the heartbeat detection task) reported an inverse correlation (Herbert et al., 2011), our and Ernst et al.'s findings clearly support the idea that IA is directly correlated with alexithymic trait.

The association between hypochondriasis and interoception might suggest that high IA is related to a strong concern for one's own bodily sensations. Along these lines, Salkovskis and Warwick (1986) maintained that cognitive processes concerning body, health and illness might increase attention toward bodily signals. Barsky et al. (1990) subsumed this hypothesis in the Somatosensory Amplification model, according to which hypochondriac subjects tend to focus on their somatic sensations and experience them as intense and disturbing. This group of subjects are hypervigilant about their own body, concentrate on their own physical sensations and consider them as dangerous. The significant positive correlations among the SAQ awareness index (and its two factors), TAS-20 scores and IAS total score suggest that focusing one's own attention on bodily feelings can hamper interpretation of one's own emotional feelings, and of bodily sensations that are direct expressions of such feelings. In other words, these results might be compatible with the idea that IA, emotion processing and hypochondriasis trait are different facets of a condition in which individuals focus on their own body signals and do not recognize the emotional nature of such signals. These individuals might be prone to interpret their physiological modifications as a cue that something life threatening is going on. Increased attention to one's own somatic signals might lead to a corresponding inattention to one's own emotions. These findings seem to confirm the interconnection between these conditions. However, from the present study we cannot infer the causal directionality of such correlations, and it remains entirely plausible that high scores of TAS-20 factors may contribute to the development of somatoform disorders through attention, amplification and misinterpretation of somatic sensations with emotional arousal (Kano and Fukudo, 2013).

It is important to take into account that our conclusions are based on observations gathered from a sample of relatively young and educated healthy adults, and this might limit generalization of our findings. However, most available scales, including the Italian version of the TAS-20 used in this study, have been developed and tested on similar samples. Moreover, in our study we did not assess validity and reliability of the SAQ questionnaire and did not investigate its relationships with objective measures of interoception (e.g., the heartbeat detection task). We also acknowledge that the inclusion of clinical samples would have added further informative values to the present study, allowing ascertaining whether the same relationships among IA, alexithymia and hypochondriasis also apply in individuals with somatoform disorders. These potential limitations might trigger further extensive investigation, in which the different aspects of interoception are assessed concurrently, and levels of anxiety and of depressive symptoms are controlled, since they could modulate alexithymia, and particularly the "difficulty identifying emotion" and "difficulty describing emotion" factors of TAS-20 (Tominaga et al., 2014).

Despite the above limitations, our study demonstrated relevant interactions between IA, alexithymia and hypochondriasis. IA is necessary for maintaining homeostasis, but excessive attention to the body can interfere with the ability to interpret bodily signals correctly. From a clinical point of view, the assessment of IA could be pivotal in evaluating several psychopathological conditions. Misinterpretation of physical sensations, rather than physical sensations in themselves, can determine concerns in individuals with a high level of attention to their own body; this could result in a vicious circle involving metacognition and evaluation of one's own thinking and symptoms (Marcus et al., 2007). These observations suggest that IA could contribute in maintaining such syndromes. The SAQ might reveal useful in comprehending the role of IA in clinical samples of patients with somatoform disorders.

TABLE 1 |
1Participants' demographic and psychometric variables.Mean
Standard deviation
Range

Age (years)
27.9
9.4
18-57

Education (years)
14.3
2.3
5-18

IAS
33.6
15.4
4-81

TAS-20
34.0
9.8
9-69

SAQ
27.4
13.5
2-78

TABLE 2 |
2Factor analysis of SAQ.Items
F1
F2

3. I feel my heart beat in my ears
0.41
0.21

4. I feel very hot in comparison to others
−0.16
0.62

6. I feel pain extremely
0.32
0.02

7. I feel my stomach tightening
0.49
0.18

9. I feel a sudden hunger pang
0.01
0.47

10. I feel my back ache
0.10
0.32

11. I feel pins and needles
0.19
0.32

12. I feel that I can't get enough air into my
lungs

0.72
−0.09

13. I have an extra-strong heartbeat
0.60
0.13

14. I feel full and bloated after eating
0.21
0.43

15. I have the sudden urge to urinate
0.17
0.36

16. I feel as if I am on fire
0.23
0.51

17. I feel a burning sensation in my stomach
0.32
0.13

18. I feel a pain in my stomach
0.24
0.30

19. I feel very cold in comparison to others
0.64
−0.08

21. I feel like I have to throw up
0.44
0.17

23. I feel chilled
0.60
−0.10

24. I feel my legs heavy
0.25
0.41

25. I feel my throat dry
0.21
0.47

26. I have heavy feeling in my chest
0.69
−0.06

27. I feel my heart thudding
0.71
−0.10

28. I feel sudden thirst pangs
−0.10
0.65

30. I feel breathless without engaging in any
type of exertion and effort

0.32
0.31

31. I feel my ears burning
0.14
0.30

32. I feel a lump in my throat
0.63
−0.05

33. I feel faint
0.37
0.07

34. I feel my palms sweaty
−0.14
0.51

35. I have difficulty in swallowing
0.30
0.07

TABLE 3 |
3Correlation analyses between SAQ with IAS and TAS-20.Bonferroni corrected p-values: *p < 0.05; **p < 0.01.TAS-20
IAS

Difficulty identifying
Difficulty describing
Externally oriented
Total score
Total score

feelings
feelings
thinking

SAQ -Awareness index
0.47**
0.29**
−0.02
0.34**
0.32**

SAQ -F1 factor
0.40**
0.20*
−0.08
0.25**
0.31**

SAQ -F2 factor
0.45**
0.33**
0.04
0.37**
0.26**

IAS -Total score
0.36**
0.27**
0.10
0.33**
-

Frontiers in Psychology | www.frontiersin.org
August 2015 | Volume 6 | Article 1149
AppendixSELF-AWARENESS QUESTIONNAIRE (Original Italian version)1. Mi capita che quando qualcuno tossisce, viene da tossire anche a me.(When somebody coughs, I feel like coughing too) 2 never 2 sometimes 2 often 2 very often 2 always 2. Mi capita di sentire molto fastidio anche per una piccola ferita (I am excessively bothered even by a small wound) 2 never 2 sometimes 2 often 2 very often 2 always 3. Mi capita di sentire il mio battito cardiaco pulsare nelle orecchie (I feel my heart beat in my ears) 2 never 2 sometimes 2 often 2 very often 2 always 4. Mi capita di sentire eccessivamente caldo rispetto agli altri (I feel very hot in comparison to others) 2 never 2 sometimes 2 often 2 very often 2 always 5. Mi capita di sentire la testa vuota (I feel my head empty) 2 never 2 sometimes 2 often 2 very often 2 always 6. Mi capita di sentire eccessivamente il dolore (I feel pain excessively) 2 never 2 sometimes 2 often 2 very often 2 always 7. Mi capita di sentire una stretta allo stomaco (I feel my stomach tightening) 2 never 2 sometimes 2 often 2 very often 2 always 8. Mi capita di sentire un pizzicore alla gola (I feel a tickle in my throat) 2 never 2 sometimes 2 often 2 very often 2 always 9. Mi capita di sentire un improvviso stimolo della fame (I feel a sudden hunger pang) 2 never 2 sometimes 2 often 2 very often 2 always 10. Mi capita di sentire mal di schiena (I feel my back ache) 2 never 2 sometimes 2 often 2 very often 2 always 11. Mi capita di sentire formicolii (I feel pins and needles) 2 never 2 sometimes 2 often 2 very often 2 always 12. Mi capita di sentire che mi manca l'aria (I feel that I can't get enough air into my lungs) 2 never 2 sometimes 2 often 2 very often 2 always 13. Mi capita di avere il batticuore (I have an extra-strong heartbeat) 2 never 2 sometimes 2 often 2 very often 2 always 14. Mi capita di sentirmi gonfio dopo un pasto (I happen full and bloated after eating) 2 never 2 sometimes 2 often 2 very often 2 always 15. Mi capita di sentire un improvviso stimolo della pipì (I have a sudden urge to urinate) 2 never 2 sometimes 2 often 2 very often 2 always 16. Mi capita di sentirmi infuocare (I feel as if I am on fire) 2 never 2 sometimes 2 often 2 very often 2 always 17. Mi capita di sentire bruciore di stomaco (I feel a burning sensation in my stomach) 2 never 2 sometimes 2 often 2 very often 2 always 18. Mi capita di sentire mal di pancia (I feel a pain in my stomach) 2 never 2 sometimes 2 often 2 very often 2 always 19. Mi capita di sentire eccessivamente freddo rispetto agli altri (I feel very cold in comparison to others) 2 never 2 sometimes 2 often 2 very often 2 always 20. Mi capita di sentire prurito (I feel itchy) 2 never 2 sometimes 2 often 2 very often 2 always 21. Mi capita di sentire che devo vomitare (I feel as if I have to throw up) 2 never 2 sometimes 2 often 2 very often 2 always 22. Mi capita di sentire un improvviso stimolo della cacca (I happen a sudden urge to defecate) 2 never 2 sometimes 2 often 2 very often 2 always 23. Mi capita di sentirmi agghiacciare (I feel chilled) 2 never 2 sometimes 2 often 2 very often 2 always 24. Mi capita di sentire le gambe pesanti (I feel my legs are heavy) 2 never 2 sometimes 2 often 2 very often 2 always 25. Mi capita di sentire la gola secca (I feel my throat dry) 2 never 2 sometimes 2 often 2 very often 2 always 26. Mi capita di sentire un peso al petto (I have a heavy feeling in my chest) 2 never 2 sometimes 2 often 2 very often 2 always 27. Mi capita di sentire un tonfo al cuore (I feel my heart thudding) 2 never 2 sometimes 2 often 2 very often 2 always 28. Mi capita di sentire un improvviso stimolo della sete (I feel sudden thirst pangs) 2 never 2 sometimes 2 often 2 very often 2 always 29. Mi capita di sentire un dolore che si sposta da una parte all'altra del corpo (I feel a pain that seems to migrate around the body) 2 never 2 sometimes 2 often 2 very often 2 always 30. Mi capita di sentire l'affanno senza aver fatto alcuno sforzo (I feel breathless without engaging in any type of exertion or effort) 2 never 2 sometimes 2 often 2 very often 2 always 31. Mi capita di sentire le orecchie bollenti (I feel my ears burning) 2 never 2 sometimes 2 often 2 very often 2 always 32. Mi capita di sentire un nodo alla gola (I feel a lump in my throat) 2 never 2 sometimes 2 often 2 very often 2 always 33. Mi capita di sentirmi svenire (I feel faint) 2 never 2 sometimes 2 often 2 very often 2 always 34. Mi capita di sentire le mani sudate (I feel my palms sweaty) 2 never 2 sometimes 2 often 2 very often 2 always 35. Mi capita di sentire difficoltà ad ingoiare (I have difficulty swallowing) 2 never 2 sometimes 2 often 2 very often 2 always ISTRUZIONI. Per cortesia legga attentamente ogni domanda e scelga la risposta che meglio descrive quanto spesso avverte ogni sensazione. Indichi una sola risposta. Non ci sono risposte corrette o errate.(Please read each item carefully and tick the one box that best describes how often you feel each sensation. Tick one only. There are no right or wrong answers.)
The twenty-item Toronto Alexithymia Scale-I. Item selection and cross-validation of the factor structure. R M Bagby, J D Parker, G J Taylor, 10.1016/0022-3999(94J. Psychosom. Res. 38Bagby, R. M., Parker, J. D., and Taylor, G. J. (1994). The twenty-item Toronto Alexithymia Scale-I. Item selection and cross-validation of the factor structure. J. Psychosom. Res. 38, 23-32. doi: 10.1016/0022-3999(94)90005-1

A diencephalic mechanism for the expression of rage with special reference to the sympathetic nervous system. P Bard, Am. J. Physiol. 84Bard, P. (1928). A diencephalic mechanism for the expression of rage with special reference to the sympathetic nervous system. Am. J. Physiol. 84, 490-515.

Interoceptive sensitivity and self-reports of emotional experience. L F Barrett, K S Quigley, E Bliss-Moreau, K R Aronson, 10.1037/0022-35188.8.131.524J. Pers. Soc. Psychol. 87Barrett, L. F., Quigley, K. S., Bliss-Moreau, E., and Aronson, K. R. (2004). Interoceptive sensitivity and self-reports of emotional experience. J. Pers. Soc. Psychol. 87, 684-697. doi: 10.1037/0022-35184.108.40.2064

The amplification of somatic symptoms. A J Barsky, J D Goodson, R S Lane, P D Cleary, 10.1097/00006842-198809000-00007Psychosom. Med. 50Barsky, A. J., Goodson, J. D., Lane, R. S., and Cleary, P. D. (1988). The amplification of somatic symptoms. Psychosom. Med. 50, 510-519. doi: 10.1097/00006842- 198809000-00007

The somatosensory amplification scale and its relationship to hypochondriasis. A J Barsky, G Wyshak, G L Klerman, 10.1016/0022-3956(90)90004-AJ. Psychiatr. Res. 24Barsky, A. J., Wyshak, G., and Klerman, G. L. (1990). The somatosensory amplification scale and its relationship to hypochondriasis. J. Psychiatr. Res. 24, 323-334. doi: 10.1016/0022-3956(90)90004-A

Effect of impaired recognition and expression of emotions on fronto-cingulate cortices: an fMRI study of men with alexithymia. S M Berthoz, E Artiges, P F Van Der Moortele, J Poline, S M Consoli, J L Martinot, 10.1176/appi.ajp.159.6.961Am. J. Psychiatry. 159Berthoz, S. M., Artiges, E., Van der Moortele, P. F., Poline, J., Consoli, S. M., and Martinot, J. L. (2002). Effect of impaired recognition and expression of emotions on fronto-cingulate cortices: an fMRI study of men with alexithymia. Am. J. Psychiatry 159, 961-967. doi: 10.1176/appi.ajp.159. 6.961

La Psicosomatica Nella Pratica Clinica. M Biondi, Pensiero Scientifico EditoreRomaBiondi, M. (1991). La Psicosomatica Nella Pratica Clinica. Roma: Pensiero Scientifico Editore.

Alexithymia in healthy women: a brain morphology study. G Borsci, M Boccardi, R Rossi, G Rossi, J Perez, M Bonetti, 10.1016/j.jad.2008.07.013J. Affect. Disord. 114Borsci, G., Boccardi, M., Rossi, R., Rossi, G., Perez, J., Bonetti, M., et al. (2009). Alexithymia in healthy women: a brain morphology study. J. Affect. Disord. 114, 208-215. doi: 10.1016/j.jad.2008.07.013

Cross validation of the factor structure of the 20-items Toronto Alexithymia Scale: an Italian multicenter study. C Bressi, G Taylor, J Parker, S Bressi, V Brambilla, E Aguglia, 10.1016/S0022-3999(96)00228-0J. Psychosom. Res. 41Bressi, C., Taylor, G., Parker, J., Bressi, S., Brambilla, V., Aguglia, E., et al. (1996). Cross validation of the factor structure of the 20-items Toronto Alexithymia Scale: an Italian multicenter study. J. Psychosom. Res. 41, 551-559. doi: 10.1016/S0022-3999(96)00228-0

The Wisdom of the Body. W B Cannon, W.W. Norton and CoNew York, NYCannon, W. B. (1932). The Wisdom of the Body. New York, NY: W.W. Norton and Co.

Interoception: the sense of the physiological condition of the body. A D Craig, 10.1016/S0959-4388(03)00090-4Curr. Opin. Neurobiol. 13Craig, A. D. (2003). Interoception: the sense of the physiological condition of the body. Curr. Opin. Neurobiol. 13, 500-505. doi: 10.1016/S0959-4388(03)00090-4

Human feelings: why are some more aware than others?. A D Craig, 10.1016/j.tics.2004.04.004Trends Cogn. Sci. 8Craig, A. D. (2004). Human feelings: why are some more aware than others? Trends Cogn. Sci. 8, 39-241. doi: 10.1016/j.tics.2004.04.004

How do you feel now? The anterior insula and human awareness. A D Craig, 10.1038/nrn2555Nat. Rev. Neurosci. 10Craig, A. D. (2009). How do you feel now? The anterior insula and human awareness. Nat. Rev. Neurosci. 10, 59-70. doi: 10.1038/nrn2555

Neural system supporting interoceptive awareness. H D Critchley, S Wiens, P Rothstein, A Ohlman, R J Dolan, 10.1038/nn1176Nat. Neurosci. 7Critchley, H. D., Wiens, S., Rothstein, P., Ohlman, A., and Dolan, R. J. (2004). Neural system supporting interoceptive awareness. Nat. Neurosci. 7, 189-195. doi: 10.1038/nn1176

Coefficient alpha and the internal structure of tests. L J Cronbach, 10.1007/BF02310555Psychometrika. 16Cronbach, L. J. (1951). Coefficient alpha and the internal structure of tests. Psychometrika 16, 297-334. doi: 10.1007/BF02310555

Listening to your heart: how interoception shapes emotion experience and intuitive decision making. B D Dunn, H C Galton, R Morgan, D Evans, C Oliver, M Meyer, 10.1177/0956797610389191Psychol. Sci. 21Dunn, B. D., Galton, H. C., Morgan, R., Evans, D., Oliver, C., Meyer, M., et al. (2010). Listening to your heart: how interoception shapes emotion experience and intuitive decision making. Psychol. Sci. 21, 1835-1844. doi: 10.1177/0956797610389191

The association of interocpetive awareness and alexithymia with neurotransmitter concentrations in insula and anterior cingulate. J Ernst, H Böker, J Hättenschwiler, D Schüpbach, G Northoff, E Seifritz, 10.1093/scan/nst058Soc. Cogn. Affect. Neurosci. 9Ernst, J., Böker, H., Hättenschwiler, J., Schüpbach, D., Northoff, G., Seifritz, E., et al. (2014). The association of interocpetive awareness and alexithymia with neurotransmitter concentrations in insula and anterior cingulate. Soc. Cogn. Affect. Neurosci. 9, 857-863. doi: 10.1093/scan/nst058

Explanatory therapy in hypochondriasis. G A Fava, S Grandi, C Rafanelli, S Fabbri, M Cazzaro, 10.4088/JCP.v61n0414J. Clin. Psychiatry. 61Fava, G. A., Grandi, S., Rafanelli, C., Fabbri, S., and Cazzaro, M. (2000). Explanatory therapy in hypochondriasis. J. Clin. Psychiatry 61, 317-322. doi: 10.4088/JCP.v61n0414

The brain network for self-feeling: a symptom-lesion mapping study. D Grossi, A Di Vita, L Palermo, U Sabatini, L Trojano, C Guariglia, 10.1016/j.neuropsychologia.2014.08.004Neuropsychologia. 63Grossi, D., Di Vita, A., Palermo, L., Sabatini, U., Trojano, L., and Guariglia, C. (2014). The brain network for self-feeling: a symptom-lesion mapping study. Neuropsychologia 63, 92-98. doi: 10.1016/j.neuropsychologia.2014. 08.004

On the relationship between interoceptive awareness and alexithymia: is interoceptive awareness related to emotional awareness?. B M Herbert, C Herbert, Pollatos , O , 10.1111/j.1467-6494.2011.00717.xJ. Pers. 79Herbert, B. M., Herbert, C., and Pollatos, O. (2011). On the relationship between interoceptive awareness and alexithymia: is interoceptive awareness related to emotional awareness? J. Pers. 79, 1149-1175. doi: 10.1111/j.1467- 6494.2011.00717.x

Interoceptive sensitivity and emotion processing: an EEG study. B M Herbert, O Pollatos, R Schandry, 10.1016/j.ijpsycho.2007.04.007Int. J. Psychophysiol. 65Herbert, B. M., Pollatos, O., and Schandry, R. (2007). Interoceptive sensitivity and emotion processing: an EEG study. Int. J. Psychophysiol. 65, 214-227. doi: 10.1016/j.ijpsycho.2007.04.007

What is an emotion? Mind IX. W James, 10.1093/mind/os-IX.34.188James, W. (1884). What is an emotion? Mind IX, 188-205. doi: 10.1093/mind/os- IX.34.188

Perception of visceral sensation: a review of recent findings, methodologies, and future directions. G E Jones, Advance Psychophysiology. J. R. Jennings and P. K. AcklesLondonJessica Kingsley publisher5Jones, G. E. (1994). "Perception of visceral sensation: a review of recent findings, methodologies, and future directions, " in Advance Psychophysiology, Vol. 5, eds J. R. Jennings and P. K. Ackles (London: Jessica Kingsley publisher), 55-192.

The application of electronic computers to factor analysis. H F Kaiser, 10.1177/001316446002000116Educ. Psychol. Meas. 20Kaiser, H. F. (1960). The application of electronic computers to factor analysis. Educ. Psychol. Meas. 20, 141-151. doi: 10.1177/001316446002000116

The alexithymic brain: the neural pathways linking alexithymia to physical disorders. M Kano, S Fukudo, 10.1186/1751-0759-7-1Biopsychosoc. Med. 71Kano, M., and Fukudo, S. (2013). The alexithymic brain: the neural pathways linking alexithymia to physical disorders. Biopsychosoc. Med. 7:1. doi: 10.1186/1751-0759-7-1

Specific brain processing of facial expressions in people with alexithymia: an H2 150-PET study. M Kano, S Fukudo, J Gyoba, M Kamachi, M Tagawa, H Mochizuki, 10.1093/brain/awg131Brain. 126Pt 6Kano, M., Fukudo, S., Gyoba, J., Kamachi, M., Tagawa, M., Mochizuki, H., et al. (2003). Specific brain processing of facial expressions in people with alexithymia: an H2 150-PET study. Brain 126(Pt 6), 1474-1484. doi: 10.1093/brain/awg131

Correlation between alexithymia and hypersensitivity to visceral stimulation in human. M Kano, T Hamaguchi, M Itoh, K Yanai, S Fukudo, 10.1016/j.pain.2007.01.032Pain. 132Kano, M., Hamaguchi, T., Itoh, M., Yanai, K., and Fukudo, S. (2007). Correlation between alexithymia and hypersensitivity to visceral stimulation in human. Pain 132, 252-263. doi: 10.1016/j.pain.2007.01.032

Abridged Manual of the Illness Attitude Scale (Mimeographed). R Kellner, Kellner, R. (1987). Abridged Manual of the Illness Attitude Scale (Mimeographed).

. Albuquerque , NM: Department of Psychiatry, University of New MexicoAlbuquerque, NM: Department of Psychiatry, University of New Mexico.

The role of anterior insular cortex in social emotions. C Lamm, T Singer, 10.1007/s00429-010-0251-3Brain Struct. Funct. 214Lamm, C., and Singer, T. (2010). The role of anterior insular cortex in social emotions. Brain Struct. Funct. 214, 579-591. doi: 10.1007/s00429-010- 0251-3

Neural correlates of levels of emotional awareness: evidence of an interaction between emotion and attention in the anterior cingulated cortex. R D Lane, E M Reimann, B Axelrod, L S Yun, A Holmes, G E Schwartz, 10.1162/089892998562924J. Cogn. Neurosci. 10Lane, R. D., Reimann, E. M., Axelrod, B., Yun, L. S., Holmes, A., and Schwartz, G. E. (1998). Neural correlates of levels of emotional awareness: evidence of an interaction between emotion and attention in the anterior cingulated cortex. J. Cogn. Neurosci. 10, 525-535. doi: 10.1162/089892998562924

Levels of emotional awareness: a cognitivedevelopmental theory and its application to psychopathology. R D Lane, G E Schwartz, Am. J. Psychiatry. 144Lane, R. D., and Schwartz, G. E. (1987). Levels of emotional awareness: a cognitive- developmental theory and its application to psychopathology. Am. J. Psychiatry 144, 133-143.

Pervasive emotion recognition deficit common to alexithymia and the repressive coping style. R D Lane, L Sechrest, R Riedel, D E Shapiro, A W Kaszniak, 10.1097/00006842-200007000-00007Psychosom. Med. 62Lane, R. D., Sechrest, L., Riedel, R., Shapiro, D. E., and Kaszniak, A. W. (2000). Pervasive emotion recognition deficit common to alexithymia and the repressive coping style. Psychosom. Med. 62, 492-501. doi: 10.1097/00006842- 200007000-00007

The mechanism of the emotions. C G Lange, The Classical Psychologists. B. RandBoston, MA: Houghton MifflinLange, C. G. (1885). "The mechanism of the emotions, " in The Classical Psychologists, ed B. Rand (Boston, MA: Houghton Mifflin), 672-684.

Cognitive and perceptual variables in hypochondriasis and health anxiety: a systematic review. D K Marcus, J R Gurley, M M Marchi, C Bauer, 10.1016/j.cpr.2006.09.003Clin. Psychol. Rev. 27Marcus, D. K., Gurley, J. R., Marchi, M. M., and Bauer, C. (2007). Cognitive and perceptual variables in hypochondriasis and health anxiety: a systematic review. Clin. Psychol. Rev. 27, 127-139. doi: 10.1016/j.cpr.2006.09.003

Individual-patient-monitoring in clinical practice: are available health surveys adequate? Qual. C A Mchorney, A R Tarlov, 10.1007/BF01593882Life Res. 4McHorney, C. A., and Tarlov, A. R. (1995). Individual-patient-monitoring in clinical practice: are available health surveys adequate? Qual. Life Res. 4, 293-307. doi: 10.1007/BF01593882

Body awareness: construct and selfreport measures. W E Mehling, V Gopisetty, J Daubenmier, C J Price, F M Hecht, A Stewart, 10.1371/journal.pone.0005614PLoS ONE. 45614Mehling, W. E., Gopisetty, V., Daubenmier, J., Price, C. J., Hecht, F. M., and Stewart, A. (2009). Body awareness: construct and self- report measures. PLoS ONE 4:e5614. doi: 10.1371/journal.pone. 0005614

The Multidimensional Assessment of Interoceptive Awareness (MAIA). W E Mehling, C Price, J J Daubenmier, M Acree, E Bartmess, A Stewart, 10.1371/journal.pone.0048230PLoS ONE. 748230Mehling, W. E., Price, C., Daubenmier, J. J., Acree, M., Bartmess, E., and Stewart, A. (2012). The Multidimensional Assessment of Interoceptive Awareness (MAIA). PLoS ONE 7:e48230. doi: 10.1371/journal.pone.0048230

Clinical application of somatosensory amplification in psychosomatic medicine. M Nakao, A J Barsky, 10.1186/1751-0759-1-17Biopsychosoc. Med. 117Nakao, M., and Barsky, A. J. (2007). Clinical application of somatosensory amplification in psychosomatic medicine. Biopsychosoc. Med. 1:17. doi: 10.1186/1751-0759-1-17

Neural systems connecting interoceptive awareness and feelings. O Pollatos, K Gramann, R Schandry, 10.1002/hbm.20258Hum. Brain Mapp. 28Pollatos, O., Gramann, K., and Schandry, R. (2007a). Neural systems connecting interoceptive awareness and feelings. Hum. Brain Mapp. 28, 9-18. doi: 10.1002/hbm.20258

Heart rate response after emotional presentation is modulated by interoceptive awareness. O Pollatos, B M Herbert, E Matthias, R Schandry, 10.1016/j.ijpsycho.2006.09.003Int. J. Psychophysiol. 63Pollatos, O., Herbert, B. M., Matthias, E., and Schandry, R. (2007b). Heart rate response after emotional presentation is modulated by interoceptive awareness. Int. J. Psychophysiol. 63, 117-124. doi: 10.1016/j.ijpsycho.2006.09.003

Brain structures involved in interoceptive awareness and cardioafferent signal processing: a dipole source localization study. O Pollatos, W Kirsch, R Schandry, 10.1002/hbm.20121Hum. Brain Mapp. 26Pollatos, O., Kirsch, W., and Schandry, R. (2005). Brain structures involved in interoceptive awareness and cardioafferent signal processing: a dipole source localization study. Hum. Brain Mapp. 26, 54-64. doi: 10.1002/hbm.20121

Body Perception Questionnaire. Laboratory of Developmental Assessment. S Porges, University of MarylandPorges, S. (1993). Body Perception Questionnaire. Laboratory of Developmental Assessment, University of Maryland. Available online at: http://stephenporges. com/index.php/publicationss/21-body-perception-questionnaires

Morbid preoccupation, health anxiety and reassurance: a cognitive-behavioral approach to hypochondriasis. P M Salkovskis, H Warwick, 10.1016/0005-7967(86)90041-0Behav. Res. Ther. 24Salkovskis, P. M., and Warwick, H. (1986). Morbid preoccupation, health anxiety and reassurance: a cognitive-behavioral approach to hypochondriasis. Behav. Res. Ther. 24, 597-602. doi: 10.1016/0005-7967(86)90041-0

Assessing health status and quality-of-life instruments: attributes and review criteria. R Schandry, 10.1111/j.1469-8986.1981.tb02486.xdoi: 10.1023/A:1015291021312Psychophysiology. 18Qual. Life Res.Schandry, R. (1981). Heart beat perception and emotional experience. Psychophysiology 18, 483-488. doi: 10.1111/j.1469-8986.1981.tb02486.x Scientific Advisory Committee of the Medical Outcomes Trust. (2002). Assessing health status and quality-of-life instruments: attributes and review criteria. Qual. Life Res. 11, 193-205. doi: 10.1023/A:1015291021312

The prevalence of "alexithymic" characteristic mechanisms in psychosomatic patients. P E Sifneos, 10.1159/000286529Psychother. Psychosom. 22Sifneos, P. E. (1973). The prevalence of "alexithymic" characteristic mechanisms in psychosomatic patients. Psychother. Psychosom. 22, 255-262. doi: 10.1159/000286529

The illness attitude scale. A clinicmetric index for assessing hypochondriacal fears and beliefs. R Sirri, S Grandi, G A Fava, 10.1159/000151387Psychother. Psychosom. 77Sirri, R., Grandi, S., and Fava, G. A. (2008). The illness attitude scale. A clinicmetric index for assessing hypochondriacal fears and beliefs. Psychother. Psychosom. 77, 337-350. doi: 10.1159/000151387

Anterior insular cortex mediates bodily sensibility and social anxiety. Y Terasawa, M Shibata, Y Moriguchi, S Umeda, 10.1093/scan/nss108Soc. Cogn. Affect. Neurosci. 8Terasawa, Y., Shibata, M., Moriguchi, Y., and Umeda, S. (2013). Anterior insular cortex mediates bodily sensibility and social anxiety. Soc. Cogn. Affect. Neurosci. 8, 259-266. doi: 10.1093/scan/nss108

Relationship between alexithymia and coping strategies in patients with somatoform disorders. T Tominaga, H Choi, Y Nagoshi, Y Wada, K Fukui, 10.2147/NDT.S55956Neuropsychiatr. Dis. Treat. 10Tominaga, T., Choi, H., Nagoshi, Y., Wada, Y., and Fukui, K. (2014). Relationship between alexithymia and coping strategies in patients with somatoform disorders. Neuropsychiatr. Dis. Treat. 10, 55-62. doi: 10.2147/NDT.S55956

Interoception in emotional experience. S Wiens, 10.1097/01.wco.0000168079.92106.99Curr. Opin. Neurol. 18Wiens, S. (2005). Interoception in emotional experience. Curr. Opin. Neurol. 18, 442-447. doi: 10.1097/01.wco.0000168079.92106.99

Heartbeat detection and the experience of emotions. S Wiens, E S Mezzacappa, E Katkin, 10.1080/026999300378905Cogn. Emot. 14Wiens, S., Mezzacappa, E. S., and Katkin, E. (2000). Heartbeat detection and the experience of emotions. Cogn. Emot. 14, 417-427. doi: 10.1080/026999300378905

Overlapping activity in anterior insula during interoception and emotional experience. J Zaki, J I Davis, K N Ochsner, 10.1016/j.neuroimage.2012.05.012Neuroimage. 62Zaki, J., Davis, J. I., and Ochsner, K. N. (2012). Overlapping activity in anterior insula during interoception and emotional experience. Neuroimage 62, 493-499. doi: 10.1016/j.neuroimage.2012.05.012