James M. Harper
Professor
Department of Biological Sciences
Sam Houston State University
Comparative Physiology Research Lab
Research Projects
An overview research projects conducted in the lab over the last several years is provided below. These includes both ongoing and past research projects by undergraduate majors in biology, chemistry and animal science, as well as MS students.
Biology of Aging & Life History Evolution in Speckled Cockroaches
Aging and Physiological Function
Known-age adults are housed individually and maintained on puppy chow for the duration of their lives. Newly molted adults are identified by the appearance of wings and white body color. For the survival study shown all roaches were maintained at 22 - 24 degrees C on an approximately 12:12 light-dark cycle.
Hemimetabolous Development
Drosophila melanogaster, the most widely used insect model in aging research is a holometabolous insect species. That is, it undergoes a complete metamorphosis and has a developmental program that is quote different from many other organisms, including vertebrates. On the other hand, cockroaches are hemimetabolous insects emerge as nymphs that are similar to adults. Development is characterized by the passage through a series of nymphal stages (called instars) that exhibit a progressive increase in size until the final molt into an adult which involves reproductive maturation. The nymphs are free-living and can survive on the same diet as the adults. This is akin to vertebrate development and allows for the easy manipulation of early life events, such as diet, to study its effect on physiology.
Life History Evolution
When the legs of an insect lose contact with a substrate, a reflexive righting program (i.e. the righting response) is initiated to restore dorsoventral orientation. This reflex is critical for survival and is seen in many species of invertebrates as well as vertebrates. Here we found that larger individuals were slower to right themselves in both males (open symbols) and females (closed symbols) as a function of body length (left), body mass (center) or body mass index (right). In addition, the reproductive history of the individuals changed the nature of the relationship. For mated individuals (A - C) females were significantly slower than males to right themselves. For virgins (D - F) males were slower. Because larger individuals of both sexes are more fecund, this may represent an life history trade-off in this species. The mechanistic basis for this effect remains unknown.
Life History Evolution
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At its simplest, life history theory postulates that organismal fitness is due to a series of physiological and behavioral trade-offs that result in a negative correlation among individual life history traits. Ultimately, this is the consequence of devoting resources toward one process, such as growth or reproduction, at the expense of other processes, such as maintenance. For example, increased fecundity is often associated with reduced longevity. Life-history trade-offs have been well-documented within multiple species of both vertebrates and invertebrates which typically involve reduced growth, developmental delays and/or reduced survival in conjunction with a reciprocal increase in reproductive success or vice versa.
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The following article is a nice overview:
The Quarterly Review of Biology 89(4): 285-318
Body size at the time of death correlates with longevity on speckled cockroaches. As illustrated in the top figure, there is a positive relationship between body mass and longevity in that larger individuals were longer-lived. This relationship was true for measures of total body length and body width (pronotum) as well. In all cases, this relationship was statistically significant and was reproducible among cohorts of cockroaches that underwent their final larval molt in each of three years (2016, n = 21; 2017, n = 40; 2018, n = 33). The data for males and females are combined as there is no significant effect of sex on longevity in this species (data not shown). The bottom figure illustrates Kaplan-Meier survival curves for cockroaches divided into tertiles based on their body mass (WtScore). More specifically, individuals in tertile 1 were at least 1 SD smaller than the mean body mass pooled across all three years while those in tertile 3 were at least 1 SD larger than the mean. The difference in median longevity among the tertiles is statistically significant by log-rank test. The bottom figure illustrates the mean (+/- SEM) body mass (in grams) of the individual tertiles across each of the three cohorts. There is no significant difference among years, although there is a statistically significant difference among tertiles by two-way ANOVA. This relationship is unusual in that larger individuals within a species are typically shorter-lived than smaller individuals in mammals, as well as mutant strains of D. melanogaster. The mechanistic basis for this relationship is currently unknown but may involve differential activity of insulin-signaling pathways.
Food availability alters stress resistance in speckled cockroaches. Specifically, individual cockroaches had access to either: (1) an unlimited food (puppy chow; ad libitum or AL) or (2) limited food availability via the manipulation of the number of feeding stations, as well as the provision of the diet itself. In one instance, individual cockroaches had access to chow at each of two feeding stations that were refilled only when all chow had been consumed (2T). For the second condition, chow was provided at a single feeding station which was refilled only when completely empty as above (1T). This feeding regimen was maintained for over 300 days prior to the initiation of this study; test subjects are 3rd or 4th generation individuals continuously exposed to these conditions. Interestingly, cockroaches from the 2T condition (red line) were more resistant to the lethal effect of both heat (45 C) and acute exposure to CdCl2 (@ 50 mM) relative to the AL and 1T populations. Additional stressors currently being tested include the metabolic poison potassium cyanide (KCN), a potent inhibitor of the electron transport chain, acetaminophen, a well known inducer of lipid peroxidation, and bacterial LPS. Preliminary data assessing KCN toxicity has suggested that cockroaches from the 1T population are extremely sensitive to its effects. Mechanistic studies will aim to assess whether there is differential activation of heat shock protein mediated activity, differences in antioxidant enzyme activities and the quantification of specific oxidation products such as protein carbonyls among the populations.
Future Work
I am currently seeking students interested in working on the effect of life history parameters and/or aging on indices of behavior and cognition, carbohydrate metabolism, immune function, and neuromuscular function in speckled cockroaches.
Potential experimental interventions include dietary manipulation beyond varying food availability (e.g. diet composition, meal predictability, et cetera), the imposition of forced exercise/differential foraging effort or the treatment with pharmacological agents associated with increased longevity (e.g. metformin, rapamycin).
In addition, speckled cockroaches have become a popular model for toxicological research and are an ideal candidate for followup studies to those listed in the section below. In particular, we have begun to examine the effect of plasticizer exposure on stress resistance, body composition and reproductive parameters. See the opportunities page for details about research opportunities.
Cellular Toxicology
Prolonged exposure (14 days) to serum-free media leads to stress-induced premature senescence (SIPS) in NIH-3T3 cells (see figure to left) as indicated by the percent cells positive for β-galactosidase staining (see inset). The addition of dimethylsulfoxide (DMSO) to serum-free media exacerbated this effect; although it was not significant. Moreover, there was no additive effect of the plasticizing agent bisphenol-A (BPA) on the induction of SIPS. The mechanism(s) responsible for these effects remain unknown as well as the effect of other plasticizing agents.
Plasticizing agents alter the inflammatory response of RAW 264.7 cells. A 24 hr exposure to dibutyl phthalate (DBP) alone and especially in comjunction with BPA prior to challenge with the toll-like receptor ligand LPS significantly reduces the secretion of the pro-inflammatory cytokines IL-6 and TNF-alpha. Values are mean (+/- SEM) level of IL-6 and TNF-alpha present in the cell culture media at 8 - 48 hours after LPS exposure. Asterisks indicate a significant different relative to untreated controls. TLRs are important mediators of innate immune function in vertebrates. Future work will determine whether this is a TLR-4 specific effect or whether it involves other TLR-mediated signaling pathways using additional TLR agonists.
Dibutyl phthalate (DBP) induces mortality in the mouse macrophage cell line RAW 264.7 in a dose dependent manner regardless of the duration of exposure. Open bars indicates cell viability after 12 hours of exposure; closed bars indicate viability after a 24 hour exposure (n = 5). Cell viability was determined using the exclusionary dye Trypan Blue. Values are the mean (+/- SEM) proportion of dead cells for each of the treatment*time combinations. The mechanism of cell death remains unknown (e.g. apoptosis, necrosis, necroptosis).
A small pilot study suggested that the most popular commercially available vaping solution causes cell death in NIH-3T3 cells in a dose dependent manner as indicated by the level of lactate dehydrogenase (LDH) activity in the media due to cell lysis. Vaping solution at the indicated dose (in mM) was added directly to the media and sampled at 24, 72 or 120 hours after the initial exposure. The values presented are from a single experiment.
Physiological Correlates of Human Behaviors
I am a member of a large interdisciplinary collaboration involving faculty from biology, psychology, criminal justice and forensics whose focus is the elucidation of salivary markers associated with specific behaviors such as aggression, as well as genetic markers such as single nucleotide polymorphisms (SNPs) in target genes. In addition, we are examining markers of stress and inflammation in select populations of individuals (e.g. children of alcoholic parents) and will soon examine the effect of an intervention on psychological and physiological outcomes in populations with significant health disparities (pending funding).
Other research
I have a long-standing interest in developing non- or minimally invasive measures of physiological function in free-living populations of rodents. Prior work utilized fecal extracts proved to be a reliable indicator of adrenal function but this approach is limited to using relatively stable compounds such as steroids or thyroid hormones. I am particularly interested in developing urine-based assays to expand the physiological repertoire available for analysis.