Infection of male rats with Toxoplasma gondii results in enhanced delay aversion and neural changes in the nucleus accumbens core

Rats infected with the protozoan parasite Toxoplasma gondii exhibit reduced avoidance of predator odours. This behavioural change is likely to increase transmission of the parasite from rats to cats. Here, we show that infection with T. gondii increases the propensity of the infected rats to make more impulsive choices, manifested as delay aversion in an intertemporal choice task. Concomitantly, T. gondii infection causes reduction in dopamine content and neuronal spine density of the nucleus accumbens core, but not of the nucleus accumbens shell. These results are consistent with a role of the nucleus accumbens dopaminergic system in mediation of choice impulsivity and goal-directed behaviours. Our observations suggest that T. gondii infection in rats causes a syndromic shift in related behavioural constructs of innate aversion and making foraging decisions.     

Necrosis and necroptosis in germ cell depletion from mammalian ovary

The maximum number of germ cells is present during the fetal life in mammals. Follicular atresia results in rapid depletion of germ cells from the cohort of the ovary. At the time of puberty, only a few hundred (<1%) germ cells are either culminated into oocytes or further get eliminated during the reproductive life. Although apoptosis plays a major role, necrosis as well as necroptosis, might also be involved in germ cell elimination from the mammalian ovary. Both necrosis and necroptosis show similar morphological features and are characterized by an increase in cell volume, cell membrane permeabilization, and rupture that lead to cellular demise. Necroptosis is initiated by tumor necrosis factor and operated through receptor interacting protein kinase as well as mixed lineage kinase domain-like protein. The acetylcholinesterase, cytokines, starvation, and oxidative stress play important roles in necroptosis-mediated granulosa cell death. The granulosa cell necroptosis directly or indirectly induces susceptibility toward necroptotic or apoptotic cell death in oocytes. Indeed, prevention of necrosis and necroptosis pathways using their specific inhibitors could enhance growth/differentiation factor-9 expression, improve survivability as well as the meiotic competency of oocytes, and prevent decline of reproductive potential in several mammalian species and early onset of menopause in women. This study updates the information and focuses on the possible involvement of necrosis and necroptosis in germ cell depletion from the mammalian ovary.     

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.     

Adrenocorticotropin stimulation of cyclic adenosine 3′,5′-monophosphate formation in isolated rat adrenal cells the role of membrane sialic acid

Neuraminidase treatment of isolated rat adrenal cells inhibited the cyclic AMP response to adrenocorticotropin stimulation. Greater percent inhibition was observed with the lower doses of adrenocorticotropin. Although the maximum amount of cyclic AMP produced by the neuraminidase-treated cells was similar to that obtained with untreated cells, the concentration of adrenocorticotropin required to produce it was greatly increased. The increase in adrenocorticotropin concentration to produce half-maximum amounts of cyclic AMP was 2-fold. Neuraminidase treatment caused a dose-related depletion of sialic acid from the cells with about 60% of the total sialic acid being released by 20 munits/ml of the enzyme. Stimulation of cyclic AMP formation by NaF in a particulate fraction obtained from homogenates of cells was unchanged after treatment of the cells with neuraminidase. The data implicate a role for sialic acid in the early events in the action of adrenocorticotropin on the cell membrane. Sialic acid may be involved in the interaction of the adrenocorticotropin molecule with the receptor or it may have a role in transmission of the signal arising from adrenocorticotropin-receptor interaction to the catalytic unit of adenyl cyclase.     

Predator cat odors activate sexual arousal pathways in brains of Toxoplasma gondii infected rats

Cat odors induce rapid, innate and stereotyped defensive behaviors in rats at first exposure, a presumed response to the evolutionary pressures of predation. Bizarrely, rats infected with the brain parasite Toxoplasma gondii approach the cat odors they typically avoid. Since the protozoan Toxoplasma requires the cat to sexually reproduce, this change in host behavior is thought to be a remarkable example of a parasite manipulating a mammalian host for its own benefit. Toxoplasma does not influence host response to non-feline predator odor nor does it alter behavior on olfactory, social, fear or anxiety tests, arguing for specific manipulation in the processing of cat odor. We report that Toxoplasma infection alters neural activity in limbic brain areas necessary for innate defensive behavior in response to cat odor. Moreover, Toxoplasma increases activity in nearby limbic regions of sexual attraction when the rat is exposed to cat urine, compelling evidence that Toxoplasma overwhelms the innate fear response by causing, in its stead, a type of sexual attraction to the normally aversive cat odor.     

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p?=?0.003) and 54% (p?=?0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p?=?0.001), MYOG (94%, p?=?0.000), and MYF5 (36%, p?=?0.000), cell cycle regulator p21 (62%, p?=?0.000), MSTN receptor ACVR2B (23%, p?=?0.061), MSTN antagonist follistatin (81%, p?=?0.000), and downstream signaling mediators SMAD2 (20%, p?=?0.060) and SMAD3 (49%, p?=?0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p?=?0.354) and 79% compared to untransduced myoblasts (p?=?0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p?=?0.000)- and sh2 (1.71-fold; p?=?0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.     

α2u-globulins mediate manipulation of host attractiveness in Toxoplasma gondii–Rattus novergicus association

Uninfected female rats (Rattus novergicus) exhibit greater attraction to the males infected with protozoan parasite Toxoplasma gondii. This phenomenon is contrary to the aversion towards infected males observed in multitude of other host–parasite associations. In this report, we describe a proximate mechanism for this anomaly. We demonstrate that T. gondii infection enhances hepatic production and urinary excretion of α2u-globulins in rats. We further demonstrate that α2u-globulins are sufficient to recapitulate male sexual attractiveness akin to effects of the infection. This manipulation possibly results in greater horizontal transmission of this parasite between the infected male and the uninfected female. It supports the notion that in some evolutionary niches parasites can alter host sexual signaling, likely leading to an increased rate of sexual transmission.Upon infection, Toxoplasma gondii invades immune-privileged organs of male rats like brain and testes. Ensuing chronic infection causes change in the behavior of the infected rats, namely: (a) loss of aversion to cat odors (Berdoy et al., 2000; Vyas and Sapolsky, 2010) and (b) gain of enhanced sexual attractiveness (Dass et al., 2011; Vyas, 2013). These changes plausibly lead to greater transmission of the parasite by trophic route to the cat intestine and by sexual route to female rats, although incontrovertible evidence of greater transmission in field conditions is not yet available (Worth et al., 2013). Correlative suggestions have also been made about increased attractiveness of infected human males and sexual transmission in humans (Hodkova et al., 2007; Flegr et al., 2014), although definitive evidence is presently unavailable. The gain in attractiveness post infection is particularly interesting because parasitism is generally observed to reduce sexual attractiveness of the host across varied phylogenies (Hamilton and Zuk, 1982; Folstad and Karter, 1992; Kavaliers et al., 2005). In this report, we investigate the proximate mechanism of this paradoxical effect.We first determined preference of uninfected estrus females for urine marks from control and infected males in an approach–approach conflict assay (detailed methods are described in Supplementary Information). Females spent greater time in bisect containing urine from the infected males (8/11 females, control < infected). For each second spent in the vicinity of urine from control males, receptive females spent 2.12 s with the infected male urine (paired t-test: |t₁₀|=2.78, P=0.019). Subsequently, female preference for low- and high-molecular weight fractions of the urine was separately quantified (LMW and HMW, respectively; cutoff=3 kDa). Volatiles bound to HMW fraction were displaced by a competitive molecule, menadione (Papes et al., 2010). Volatiles in HMW fractions before and after menadione displacement were compared with vehicle using direct analysis in real-time mass spectrometry (DART-MS). This analysis indicated the absence of any bound volatiles in displaced samples used for behavioral measurements. Analysis of variance (ANOVA) revealed significant interaction between infection status and the fractions (F_(1,30)=8.23, P=0.007). Females exhibited comparable preference to the LMW fraction of control and infected males (Figure 1a; control: 387±47 s, infected: 398±35 s; paired t-test: |t₁₅|=0.22, P>0.8; statistical power=0.054). In contrast, HMW fraction of the infected male was significantly preferred over that of control males (Figure 1a; control: 282±20 s, infected: 457±26 s; |t₁₅|=4.84, P<0.001). HMW fraction of infected animals exhibited greater attractiveness even when grafted on control LMW fraction (Figure 1b; control: 317±25 s, infected: 493±34 s; |t₁₁|=3.09, P=0.01). The majority of urinary proteins in the HMW fraction were determined to be α2u-globulins through mass spectrometric analysis (LOC259246 and LOC298109). Proteins homologous to rat α2u-globulins are also involved in chemical communication in mice (Hurst, 2009; Papes et al., 2010; Vasudevan and Vyas, 2013), including sexual signaling (Roberts et al., 2010).Open in a separate windowFigure 1The greater attractiveness of T. gondii-infected males was communicated by HMW, and not LMW, fraction of the male urine. Estrus females exhibited greater attraction to HMW fraction of the urine obtained from the infected males (a, black dots). N=16 females. ANOVA: P=0.514 for fractions; P=0.018 for infection status; P=0.007 for interaction. Abscissa and ordinate depict time spent in bisect containing urine from infected or control animals, respectively (in second, trial duration=1200 s). Solid circles depict data obtained from individual females. Gray diagonal line depicts chance (abscissa=ordinate). The attraction was not recapitulated by LMW fraction (<3 kDa) of the urine (a, gray dots). HMW obtained from infected animals was attractive to females even when combined with LMW of control urine (b). N=12 females. Mass spectroscopic analysis revealed major component of HMW fraction to be α2u-globulins.Infected animals contained greater amounts of α2u-globulin mRNA and protein in liver (Figures 2a and b). Congruently, urine from infected animals contained greater amounts of α2u-globulins (Figure 2b, 138% increase; P=0.011). In both liver and urine, the 25th percentile of α2u-globulin quantity in infected animals surpassed the 75th percentile of respective controls. α2u-globulins levels in preputial glands did not significantly differ between control and infected males (P=0.37).Open in a separate windowFigure 2Toxoplasma gondii infection increased α2u-globulins production in liver and excretion in urine. Infection enhanced α2u-globulins mRNA abundance in the liver (a). Ordinate depicts PCR cycles needed to reach a threshold when using α2u-globulins primers minus when using GAPDH primers (a housekeeping gene). Box plot depicts median, 25th percentile and 75th percentile. *P<0.05; exact Mann–Whitney test. N =7. Liver and urine from infected animals contained greater amounts of α2u-globulin protein. N=6 control, 8 infected (liver); N=7 (urine) (b). Ordinate depicts densitometric intensity, normalized to intensity of a pooled sample run in the same gel. Same pooled samples were used in all gels of the experiment. Creatinine-adjusted urine samples were used. Both groups exhibited comparable filtration rates of urine from the kidney (creatinine content; P>0.9) and a comparable tendency to place urine marks in a novel arena (P>0.9). *P<0.05 and **P<0.01; exact Mann–Whitney test. N=7. Renatured α2u-globulins postdialysis were sufficient to recapitulate greater attractiveness (c). Females exhibited attraction to FPLC-purified HMW fraction containing α2u-globulins, even after its denaturation in 3 m guanidinium chloride, subsequent dialysis and then renaturation in buffered physiological saline to remove all bound volatiles. N=12 females.Volatile substances in urine are known to bind α2u-globulins. The observation that HMW fraction from urine of infected animals was able to retain greater attractiveness even after menadione displacement supports the notion that α2u-globulins rather than volatiles bound to it were the active ingredients. We conducted a further experiment to test the possibility of residual volatiles still being bound to α2u-globulins in amounts undetectable by DART-MS. The fast protein liquid chromatography (FPLC)-purified α2u-globulins fraction was denatured, dialyzed and transferred to physiological buffer for renaturation. Dialysis with excess buffer was used in an intervening part of the cycle to remove any residual volatiles. Renaturation was confirmed by circular dichroism and tryptophan imaging. This procedure failed to rescue female attractiveness. Females exhibited robust preference to dialyzed α2u-globulins (Figure 2c; 10/12 females; |t₁₁|=4.09, P=0.002). Moreover, females exhibited clear preference for a greater concentration of FPLC-purified α2u-globulins in two-choice preference task comparing low and high doses (5 μg μl⁻¹: 334±41 s, 1.66 μg μl⁻¹: 208±24 s; paired t-test: |t₁₀|=2.78, P=0.014). These observations suggest that α2u-globulins can signal attractiveness without necessity of volatiles and in a dose-dependent manner. This is consistent with ability of HMW urinary fraction from the infected animals to evoke greater attraction, coupled with greater production of α2u-globulins postinfection. In other words, T. gondii infection increases urinary excretion of α2u-globulins, and greater α2u-globulins in the infected urine is sufficient to signal greater attractiveness. Greater α2u-globulins production in the infected rats is congruent with the observations that these proteins require testosterone for synthesis and that T. gondii infection increases testosterone production in male rats (Kulkarni et al., 1985; Lim et al., 2013).Many models of sexual selection posit that male sexual advertisement is an ‘honest'' proxy of an ability to fight infections. This honesty is thought to arrive because resources used for sexual advertising produce a handicap in ability to fight parasites or pathogens (Hamilton and Zuk, 1982; Wingfield et al., 1997). In other words, sexual signals are expensive to produce or maintain, thus allowing only fit males to engage in the advertisement. Many parasites do exploit host sexual signals. More frequently, this exploitation takes the form of either eavesdropping on sexual signals to find a new host or to inter-species mimicry of sexual signals by parasites to attract a potential host (Zuk and Kolluru, 1998; Haynes and Yeargan, 1999; Zuk et al., 2006). Parasites in these cases do not influence host advertisement per se. In this backdrop, rat–T. gondii association provides additional plausibility of the parasites changing magnitude of host sexual advertisement.Parasites are known to affect the behavior of their hosts (Hughes et al., 2012). This observation is frequently employed to argue that natural selection acts on genes and not necessarily individuals. In this narrative, the body of the host becomes an extended phenotype of the parasite (Dawkins, 1999; Hunter, 2009) and the behavioral changes correlate with increased transmission of the parasite. T. gondii has earlier been shown to increase sexual attractiveness of the infected males, resulting in greater sexual transmission of the parasite (Dass et al., 2011). Current observations present a molecular mechanism for T. gondii-induced extended phenotype.