Characterizing Sleep in Adolescents and Adults with Autism Spectrum Disorders.

We studied 28 adolescents/young adults with autism spectrum disorders (ASD) and 13 age/sex matched individuals of typical development (TD). Structured sleep histories, validated questionnaires, actigraphy (4 weeks), and salivary cortisol and melatonin (4 days each) were collected. Compared to those with TD, adolescents/young adults with ASD had longer sleep latencies and more difficulty going to bed and falling asleep. Morning cortisol, evening cortisol, and the morning-evening difference in cortisol did not differ by diagnosis (ASD vs. TD). Dim light melatonin onsets (DLMOs) averaged across participants were not different for the ASD and TD participants. Average participant scores indicated aspects of poor sleep hygiene in both groups. Insomnia in ASD is multifactorial and not solely related to physiological factors.

A randomized control trial of a psychosocial intervention for caregivers of allogeneic hematopoietic stem cell transplant patients: effects on distress.

Caregivers of patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCT) serve a pivotal role in patient care but experience high stress, anxiety and depression as a result. We theorized that stress management adapted for allo-HSCT caregivers would reduce distress compared with treatment as usual (TAU). Of 267 consecutive caregivers of allo-HSCT patients approached, 148 (mean=53.5 years, 75.7% female) were randomized to either psychosocial intervention (i=74) or TAU (n=74). Eight one-on-one stress management sessions delivered across the 100-day post-transplant period focused on understanding stress, changing role(s) as caregiver, cognitive behavioral stress management, pacing respiration and identifying social support. Primary outcomes included perceived stress (psychological) and salivary cortisol awakening response (CAR) (physiological). Randomized groups were not statistically different at baseline. Mixed models analysis of covariance (intent-to-treat) showed that intervention was associated with significantly lower caregiver stress 3 months post transplant (mean=20.0, 95% confidence interval (95% CI)=17.9-22.0) compared with TAU (mean=23.0, 95% CI=21.0-25.0) with an effect size (ES) of 0.39 (P=0.039). Secondary psychological outcomes, including depression and anxiety, were significantly reduced with ESs of 0.46 and 0.66, respectively. Caregiver CAR did not differ from non-caregiving controls at baseline and was unchanged by intervention. Despite significant caregiving burden, this psychosocial intervention significantly mitigated distress in allo-HSCT caregivers.

Multiple latent viruses reactivate in astronauts during Space Shuttle missions.

Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone were measured prospectively in 17 astronauts (16 male and 1 female) before, during, and after short-duration (12-16 days) Space Shuttle missions. Blood, urine, and saliva samples were collected during each of these phases. Antiviral antibodies and viral load (DNA) were measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). Three astronauts did not shed any virus in any of their samples collected before, during, or after flight. EBV was shed in the saliva in all of the remaining 14 astronauts during all 3 phases of flight. Seven of the 14 EBV-shedding subjects also shed VZV during and after the flight in their saliva samples, and 8 of 14 EBV-shedders also shed CMV in their urine samples before, during, and after flight. In 6 of 14 crewmembers, all 3 target viruses were shed during one or more flight phases. Both EBV and VZV DNA copies were elevated during the flight phase relative to preflight or post-flight levels. EBV DNA in peripheral blood was increased preflight relative to post-flight. Eighteen healthy controls were also included in the study. Approximately 2-5% of controls shed EBV while none shed VZV or CMV. Salivary cortisol measured preflight and during flight were elevated relative to post-flight. In contrast DHEA decreased during the flight phase relative to both preflight and post-flight. As a consequence, the molar ratio of the area under the diurnal curve of cortisol to DHEA with respect to ground (AUCg) increased significantly during flight. This ratio was unrelated to viral shedding. In summary, three herpes viruses can reactivate individually or in combination during spaceflight.

Environmental stress alters genetic regulation of novelty seeking in vervet monkeys.

Considerable attention has been paid to identifying genetic influences and gene-environment interactions that increase vulnerability to environmental stressors, with promising but inconsistent results. A nonhuman primate model is presented here that allows assessment of genetic influences in response to a stressful life event for a behavioural trait with relevance for psychopathology. Genetic and environmental influences on free-choice novelty seeking behaviour were assessed in a pedigreed colony of vervet monkeys before and after relocation from a low stress to a higher stress environment. Heritability of novelty seeking scores, and genetic correlations within and between environments were conducted using variance components analysis. The results showed that novelty seeking was markedly inhibited in the higher stress environment, with effects persisting across a 2-year period for adults but not for juveniles. There were significant genetic contributions to novelty seeking scores in each year (h(2) = 0.35-0.43), with high genetic correlations within each environment (rhoG > 0.80) and a lower genetic correlation (rhoG = 0.35, non-significant) between environments. There were also significant genetic contributions to individual change scores from before to after the move (h(2) = 0.48). These results indicate that genetic regulation of novelty seeking was modified by the level of environmental stress, and they support a role for gene-environment interactions in a behavioural trait with relevance for mental health.

Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic--pituitary--adrenal axis activity.

OBJECTIVE: The aim of this study was to determine whether salivary cortisol measured by a simple enzyme immunoassay (EIA) could be used as a surrogate for serum total cortisol in response to rapid changes and across a wide range of concentrations. DESIGN: Comparisons of matched salivary and serum samples in response to dynamic hypothalamic-pituitary-adrenal (HPA) axis testing. Subjects Healthy women (n=10; three taking oral oestrogens) and men (n=2), aged 23--65 years, were recruited from the community. Measurements Paired saliva and serum samples were obtained during three protocols: 10 min of exercise at 90% of maximal heart rate (n=8), intravenous administration of corticotrophin-releasing hormone (CRH; n=4), and dexamethasone suppression (n=7). Cortisol was measured in saliva using a commercial high-sensitivity EIA and total cortisol was measured in serum with a commercial radioimmunoassay (RIA). Results The time course of the salivary cortisol response to both the exercise and CRH tests paralleled that of total serum cortisol. Salivary cortisol demonstrated a significantly greater relative increase in response to the exercise and CRH stimuli (697+/- 826%vs. 209+/- 150%, P=0.04 saliva vs. serum). A disproportionately larger increase in free cortisol, compared with total, would be expected when the binding capacity of cortisol-binding globulin (CBG) is exceeded. In response to dexamethasone suppression, relative decreases in cortisol were not significantly different between the two media (-47+/- 56%vs.-84+/- 8%, P=0.13 saliva vs. serum). Although a significant linear correlation was found for all paired salivary and serum total cortisol samples (n=183 pairs, r=0.60, P<0.001), an exponential model provided a better fit (r=0.81, P<0.001). The linear correlations were strengthened when data from subjects on oral oestrogens (n=52 pairs, r=0.75, P < 0.001) were separated from those not taking oestrogens (n=131 pairs, r=0.67, P<0.001). Conclusions Salivary cortisol measured with a simple EIA can be used in place of serum total cortisol in physiological research protocols. Evidence that salivary measures represent the biologically active, free fraction of cortisol includes: (1) the greater relative increase in salivary cortisol in response to tests that raise the absolute cortisol concentration above the saturation point of CBG; (2) the strong exponential relationship between cortisol assessed in the two media; and (3) the improved linear correlations when subjects known to have increased CBG were analysed separately. Thus, an advantage of measuring salivary cortisol rather than total serum cortisol is that it eliminates the need to account for within-subject changes or between-subject differences in CBG.

Antidepressant treatment during social challenge prior to 1 year of age affects immune and endocrine responses in adult macaques.

Antidepressants are widely used in treating depression and other behavioral problems in children and adolescents. Little is known about the long-term effects of these agents, particularly on physiological systems. The effects of previous antidepressant treatment during a social challenge in 9-month-old rhesus monkeys (Macaca mulatta) on their adult immune and endocrine responses were studied. Prior to the social challenge, the monkeys were reared either by their mother or in a peer group. Monkeys were treated with either a serotonergic agonist (fluoxetine), a noradrenergic agonist (desipramine), or saline during social separation. Non-separated, saline-treated monkeys served as control monkeys. In order to evaluate immune effects of early antidepressant treatment, adult monkeys were immunized with a novel antigen, tetanus toxoid. Blood samples were collected prior to and at 4-5-day intervals for 28 days after immunization. Plasma total immunoglobulins (IgG and IgM), complement levels (C3 and C4), tetanus antibody titers, and cortisol were assessed. Antibody levels were lowest in monkeys treated with antidepressants regardless of specific drug treatment or early rearing condition. Drug-treated subjects had elevated plasma immunoglobulins and complement protein levels. Cortisol was also highest in drug-treated subjects. These results should be considered when prescribing commonly used antidepressants for treatment of childhood disorders.

A preliminary description of responses of free-ranging rhesus monkeys to brief capture experiences: behavior, endocrine, immune, and health relationships.

A cohort of free-ranging rhesus monkeys has been followed since birth in 1994 on the island of Cayo Santiago, Puerto Rico. At 3 years of age, subjects were trapped and blood samples were collected after capture and prior to release the following day. Blood samples were processed for natural cytotoxicity toward xenogeneic tumors, phenotyping, and plasma hormones. Intestinal parasites were determined from fresh stool samples collected during trapping. Data were also available from the previous year for antibody titers to latent viruses prevalent in this population. Behavioral traits of each monkey were characterized using a previously developed trait scale for rhesus monkeys. Natural cytotoxicity toward both K562 and Raji targets declined from capture until release the following day. Plasma cortisol rose and plasma prolactin and growth hormone fell during the period of captivity; a rise in insulin was significant. It was expected that individual differences in behavioral traits might predict immune and hormone levels at the time of capture or changes in these parameters during the capture period. Although behavioral adjectives tended to cluster along three orthogonal dimensions (Insecurity, Irritability, and Sociability), they bore no relationship to the physiological parameters collected acutely (in vitro immune and endocrine parameters). The individual difference markers of gender and maternal rank were not related to the magnitude of the observed changes in these in vitro parameters, either. However, an in vivo measure (CMV titer) was related to individual differences in Irritability. It was concluded that the magnitude of the stress associated with capture overwhelmed the individual difference effects.

Elevated cytotoxicity in combat veterans with long-term post-traumatic stress disorder: preliminary observations.

Resting immune [WBC and differential cell counts lymphocyte phenotyping (CD2, CD4, CD8, CD16, CD20, and CD56), and NK activity] and endocrine (cortisol, prolactin, growth hormone, and DHEA-SO4) parameters were measured in 10 male, Vietnam combat veterans diagnosed with long-term post-traumatic stress disorder (PTSD) and 9 control Vietnam combat veterans without a PTSD diagnosis but with a comparable history of alcohol abuse. Subjects completed a battery of psychological questionnaires. We report on preliminary observations of the relationship between PTSD and physiological and psychological parameters. With some important exceptions, PTSD patients did not differ from the age-matched control group with regard to hormone levels or lymphocyte phenotypes. However, NK activity was higher in the PTSD population than in the controls. Beck, Mississippi, and Combat Exposure scores were significantly elevated in the PTSD population. In contrast to previous observations in depressed populations, depression (indicated by elevated Beck scores), comorbid with PTSD, was associated with increased natural cytotoxicity.

Juvenile friends, behavior, and immune responses to separation in bonnet macaque infants.

Individual differences in the response to maternal separation in nonhuman primate infants have been attributed to (among other variables) presence or absence of processes that may model social support in humans. Alternative attachments to other members of the social group buffer the infant against a depressive response to maternal separation. This hypothesis was tested in a group of bonnet macaques by manipulating the presence or absence of alternative juvenile attachment figures (friends) during separation. Infants who retained such attachments showed fewer behavioral evidences of depression when separated from their mothers. These infants without friends also showed changes in lymphocyte activation by mitogens or natural cytotoxicity that were not evident in the infants with juvenile friends. Across all separated infants, natural cytotoxicity was positively correlated with juvenile affiliative behavior directed toward the infants during the separation. These results support the hypothesis that social support, available from alternative attachments, can modulate the response to loss, and can account for some of the individual differences seen in these responses.

Natural cytotoxicity toward K562 cells by macaque lymphocytes from infancy through puberty: effects of early social challenge.

The consequences of a single maternal separation experience followed by reunion at 6-7 months of age was studied in socially housed pigtail (Macaca nemestrina) and bonnet (Macaca radiata) macaques. At 15 months, these subjects were removed from their natal group and placed in same species social groups, consisting of other separated and matched control subjects. Some subjects were followed until they reached an average age of 4 years while remaining in this group. Blood samples were drawn to permit assessment of the ability of peripheral blood lymphocytes to lyse, in vitro, K562 cells. Maternal separation failed to affect lysis of K562 cells acutely, although lysis by matched control subjects appeared to be transiently reduced 2 h after removal of the adult female. A longer-term influence was noted such that lysis of targets in previously separated subjects was greater than that in matched controls. Lysis rose over time regardless of species or experimental condition. A striking internal consistency in the lysis was also noted. Lysis was highly intercorrelated (r's > .60, p < or = .005) within subjects across time during baseline, separation, and reunion phases of the acute social challenge. In addition, there was a tendency for this correlation to hold over the longitudinal phases. Lysis of K562 targets by macaque lymphocytes would appear to possess trait-like stability; however, the range of lysis may be modified by early experiences.

Morphine-induced alterations in antibody levels: receptor and immune mechanisms.

We have previously shown that an acute administration of morphine (10 mg/kg, i.v.) decreases IgG, but not IgM, antibody levels to antigen administered before morphine. Further, decreases in IgG were blocked by previous administration of naltrexone, indicating that receptor binding is critical to the decreased antibody levels. These studies investigated potential receptor and immune mechanisms for these effects. To investigate potential receptor mechanisms, the stereoselectivity and location of receptor binding was determined. The results of these experiments suggest morphine must bind stereoselectively to central sites to decrease antibody levels after antigen administration. To investigate potential immune mechanisms for these changes, antibody secreting cells (ASC) for keyhole limpet hemocyanin-specific IgG and IgM were enumerated. Morphine decreased ASC for IgG but increased ASC for IgM. Two pathways for the genetic switch from IgM to IgG production were investigated. One pathway requires interferon-gamma to stimulate IgM-secreting cells to switch to IgG2a-secreting cells. Another pathway requires interleukin-4 to stimulate IgM-secreting cells to switch to IgG1- secreting cells. IgG1 and IgG2a levels were measured to determine if these pathways were differentially affected and only IgG2a levels were decreased. Further, these decreases were accompanied by decreased IFN-gamma levels but not by altered numbers of splenocytes. These data indicate that morphine may alter the ability of ASC to switch from IgM to IgG2a production, possibly by reducing the availability of IFN-gamma.

A long-term increase in basal levels of corticosterone and a decrease in corticosteroid-binding globulin after acute stressor exposure.

Adrenal glucocorticoids play an important role in mediating many of the behavioral and physiological effects of exposure to stressors. Focus has been primarily on the acute stress-induced rise in glucocorticoids [corticosterone (CORT) in the rat]. There are reports, however, that exposure to chronic stressors can produce an increase in basal CORT and a decrease in corticotropin-binding globulin (CBG). These changes occur subsequent to the stress-induced rise in CORT. The following experiments examined whether exposure to an acute stressor (100 5-sec inescapable tail shocks; IS) could also produce long term changes in basal CORT and CBG. We report that a single session of IS results in an increase in basal total serum CORT that persists 48-96 h after IS termination. The increase is present only at the diurnal trough (morning). CBG levels ae also decreased for 24-48 h. The decrease is present at both the diurnal peak (evening) as well as the trough (morning). These changes result in an increase in the percent and amount of biologically active CORT (unbound or free). Thus, glucocorticoid-sensitive targets are exposed to high levels of free CORT for several days after IS termination. The long term increase in free CORT reported here may play an important role in mediating some of the effects produced by IS as well as those produced by other acute stressors.

Individual differences in macaques' responses to stressors based on social and physiological factors: implications for primate welfare and research outcomes.

Primates are used extensively in a variety of research settings. Federal regulations in the US mandate that caretakers provide for the 'psychological well-being of laboratory primates'. One of the difficulties in implementing this law has been both in the definition of psychological well-being and in the need to deal with each primate species and, in some cases, age or sex class, uniquely. Non-human primates exhibit distinct individual differences in their behavioural and physiological responses to experimental challenges and caretaking procedures. We have been investigating what factors can predict some of these individual differences, and have found that factors both intrinsic and extrinsic are significant. Extrinsic factors found to predict individual differences in response to stressors include the nature and prior experience with the challenge, the presence of familiar peers and availability of social support. Intrinsic factors include cognitive interpretations of the challenge and temperamental differences in reactivity. These studies highlight the importance of understanding the context and individual psychology of macaques in order to provide laboratory environments conducive to their welfare, and in order to understand the impact experimental and caretaking procedures are likely to have on the health and welfare of our subjects.

Stressed rats fail to expand the CD45RC+CD4+ (Th1-like) T cell subset in response to KLH: possible involvement of IFN-gamma.

Exposure to stressors effects various aspects of immune function, including the in vivo antibody response. We have previously reported that rats exposed to an acute session of inescapable tail shock (IS) show long-term reductions in anti-KLH (keyhole limpet hemocyanin) IgM and IgG. The mechanisms responsible for this suppression are currently unknown. Previous work has suggested changes in CD4+ T cells could be important. We report here that exposure to IS results in a reduction in Con A-stimulated IFN-gamma levels in mesenteric lymphocytes and splenocytes taken immediately after IS termination. In addition, IS exposure prevents the KLH-induced increase in the number of CD45RC+CD4+ T cells (Th1-like) in both the mesenteric lymph nodes and the spleen 4 days after immunization. The failure of KLH to expand the CD45RC+CD4+ subset could be due to the stress-induced reduction in IFN-gamma levels reported in cells taken at the time of immunization. Implications of these findings as a mechanism for the decrease in the in vivo antibody response previously reported is discussed.

Total cortisol, free cortisol, and growth hormone associated with brief social separation experiences in young macaques.

Many behavioral, immunological, and physiological consequences or brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched nonseparated controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables.

Stress-induced reduction in the rat mixed lymphocyte reaction is due to macrophages and not to changes in T cell phenotypes.

Exposure to aversive events or stressors modulates various aspects of immune function. We have previously reported that exposure to an acute stressor, inescapable tail shock (IS), resulted in a shift in T cell subpopulations in rat mesenteric lymph nodes but not in cervical lymph nodes (Fleshner et al. (1992) J. Neuroimmunol. 41, 131-142). The mesenteric CD4+/CD8+ ratio was increased immediately after exposure to IS and was due primarily to an increase in the percent of CD4+ cells. The present experiments were designed to determine the relationship between the IS-associated phenotypic shift and its significance in the function of CD4+ T cells. The function assessed was the in vitro proliferative response to alloantigens coded for by the Major Histocompatibility Complex (MHC). Using the mixed lymphocyte reaction (MLR), we report that exposure to IS resulted in a decrease in the MLR response of cells from both cervical and mesenteric lymph nodes. Depletion of macrophages (nylon wool adherent cells) eliminated the IS-induced reduction and co-culture of macrophages (irradiation-insensitive cells) from shocked rats produced the suppression. One interpretation of these data is that exposure to IS resulted in the activation of macrophages and the release of a suppressive factor which reduced the MLR response of peripheral lymph node lymphocytes.

Natural killer cell activity is reduced in association with oral contraceptive use.

Oral contraceptive (OC) use has been associated with increased incidence of a number of infections, but the mechanisms behind these changes is unclear. The present study compared OC users and nonusers in natural killer (NK) cell activity, NK phenotype, and illness frequency. Subjects were 55 female medical students (19 OC users, 36 nonusers). Three blood samples were obtained, 1 mo apart. Natural cytotoxicity was tested with a 51Cr assay. Self reports of illness symptoms during the previous week were collected at each blood sampling. NK phenotype number was assessed by flow cytometry. Oral contraceptive users had lower natural cytotoxicity and increased frequency of sneezing, gastrointestinal distress, runny nose, sore throat, coughing, and total illness symptoms, relative to nonusers. No differences were found between OC users and nonusers in NK phenotype number. These findings support the hypothesis that differences between users and nonusers in infection rates might be due to alterations in NK activity.

PIP: Oral contraceptive (OC) use has been associated with increased incidence of a number of infections, but the mechanisms behind such changes are unclear. Comparison of lymphocyte phenotypes between contraceptive users and nonusers found no difference in either the percentage or absolute numbers of any cell types. Other data, however, suggest differences in natural cytotoxic activity in contraceptive users. Natural cytotoxic activity in normal women has been found to vary with menstrual phase and estradiol levels, being lowest mid-cycle, shortly following peak estradiol levels. OC users, however, demonstrate no variation in either natural killer (NK) cell activity or estradiol levels over their cycles. The authors report their findings from a study comparing 19 OC users and 36 nonusers in NK cell activity, NK phenotype, and illness frequency. Three blood samples were obtained one month apart from each of the female medical student participants. Natural cytotoxicity was tested with a Cr assay, while NK phenotype number was assessed by flow cytometry. Relative to nonusers, OC users had lower natural cytotoxicity and increased frequency of sneezing, gastrointestinal distress, runny nose, sore throat, coughing, and total illness symptoms. No differences were found between OC users and nonusers in NK phenotype number. These findings support the hypothesis that differences between users and nonusers in infection rates may be due to alterations in NK activity.

Morphine-induced decreases in in vivo antibody responses.

Endogenous opioids have been shown to be released during acute stress and could play a role in immune modulation and activation of the hypothalamo-pituitary-adrenal axis. We investigated the ability of morphine sulfate to mimic stressor effects on decreases in in vivo antibody responses. Sprague-Dawley and Fischer 344 rats were given an intraperitoneal injection of an antigen, Keyhole limpet hemocyanin (KLH), followed by a single intravenous injection of either saline or varying doses of morphine sulfate. The corticosterone and anti-KLH IgG antibody responses to morphine were measured. A dose-dependent increase in corticosterone was observed. Significantly lower levels of anti-KLH IgG antibodies were observed in morphine-treated animals but these effects were strain and dose dependent. In Sprague-Dawley rats, 3 and 10 mg/kg doses of morphine decreased antibody levels while 1.5, 5, and 15 mg/kg did not change antibody responses. In Fischer 344 rats a dose of 5 mg/kg of morphine decreased antibody levels while 10 and 15 mg/kg did not change antibody responses. These results indicate that morphine can decrease antibody levels and that these decreases are not correlated with elevated levels of corticosterone. To determine if opioid binding is critical to these changes, animals received naltrexone prior to the administration of morphine. Naltrexone partially attenuated corticosterone levels, but completely blocked morphine-induced changes in immune function.

Intrinsic and extrinsic factors affect infant responses to maternal separation.

Responses of individuals to the loss of a primary attachment object may be quite variable. In humans, it has been suggested that only about 25% of bereavements result in substantial psychological or medical morbidity (Hamburg et al. 1975). In nonhuman primates, which are used to model responses to separation and loss, a similar estimate of about 25% has also been obtained (McKinney 1985). In addition, there are wide-ranging species differences in vulnerability with regard to the nature and severity of the response to maternal separation and/or loss. All of these findings suggest that there are important processes, intrinsic and/or extrinsic to the individual, that contribute to the probability that a loss will produce a major behavioral or physiological response. We have been systematically examining some of the factors that may account for a portion of this variability in two species of macaques (bonnet monkeys Macaca radiata; and pigtail monkeys, M. nemestrina).