Adult F1 Research Articles

Pre-reproductive stress in adolescent female rats alters oocyte microRNA expression and offspring phenotypes: pharmacological interventions and putative mechanisms

Pre-reproductive stress (PRS) to adolescent female rats alters anxiogenic behavior in first (F1)- and second-generation (F2) offspring and increases mRNA expression of corticotropin-releasing factor receptor type 1 (Crhr1) in oocytes and in neonate offspring brain. Here, we ask whether the expression of Crhr1 and Crhr1-targeting microRNA is altered in brain, blood, and oocytes of exposed females and in the brain of their neonate and adult F1 and F2 offspring. In addition, we inquire whether maternal post-stress drug treatment reverses PRS-induced abnormalities in offspring. We find that PRS induces a selective increase in Crhr1-targeting mir-34a and mir-34c in blood and oocytes, while non-Crhr1 microRNA molecules remain unaltered. PRS induces similar microRNA changes in prefrontal cortex of F1 and F2 neonates. In adult animals, cortical Crhr1, but not mir-34, expression is affected by both maternal and direct stress exposure. Post-PRS fluoxetine (FLX) treatment increases pup mortality, and both FLX and the Crhr1 antagonist NBI 27914 reverse some of the effects of PRS and also have independent effects on F1 behavior and gene expression. PRS also alters behavior as well as gene and miRNA expression patterns in paternally derived F2 offspring, producing effects that are different from those previously found in maternally derived F2 offspring. These findings extend current knowledge on inter- and trans-generational transfer of stress effects, point to microRNA changes in stress-exposed oocytes as a potential mechanism, and highlight the consequences of post-stress pharmacological interventions in adolescence.

The Postnatal Offspring of Finasteride-Treated Male Rats Shows Hyperglycaemia, Elevated Hepatic Glycogen Storage and Altered GLUT2, IR, and AR Expression in the Liver

Background: A growing body of data indicates that the physiology of the liver is sex-hormone dependent, with some types of liver failure occurring more frequently in males, and some in females. In males, in physiological conditions, testosterone acts via androgen receptors (AR) to increase insulin receptor (IR) expression and glycogen synthesis, and to decrease glucose uptake controlled by liver-specific glucose transporter 2 (GLUT-2). Our previous study indicated that this mechanism may be impaired by finasteride, a popular drug used in urology and dermatology, inhibiting 5α-reductase 2, which converts testosterone (T) into dihydrotestosterone (DHT). Our research has also shown that the offspring of rats exposed to finasteride have an altered T–DHT ratio and show changes in their testes and epididymides. Therefore, the goal of this study was to assess whether the administration of finasteride had an trans-generational effect on (i) GLUT-2 dependent accumulation of glycogen in the liver, (ii) IR and AR expression in the hepatocytes of male rat offspring, (iii) a relation between serum T and DHT levels and the expression of GLUT2, IR, and AR mRNAs, (iv) a serum glucose level and it correlation with GLUT-2 mRNA. Methods: The study was conducted on the liver (an androgen-dependent organ) from 7, 14, 21, 28, and 90-day old Wistar male rats (F1:Fin) born by females fertilized by finasteride-treated rats. The control group was the offspring (F1:Control) of untreated Wistar parents. In the histological sections of liver the Periodic Acid Schiff (PAS) staining (to visualize glycogen) and IHC (to detect GLUT-2, IR, and AR) were performed. The liver homogenates were used in qRT-PCR to assess GLUT2, IR, and AR mRNA expression. The percentage of PAS-positive glycogen areas were correlated with the immunoexpression of GLUT-2, serum levels of T and DHT were correlated with GLUT-2, IR, and AR transcript levels, and serum glucose concentration was correlated with the age of animals and with the GLUT-2 mRNA by Spearman’s rank correlation coefficients. Results: In each age group of F1:Fin rats, the accumulation of glycogen was elevated but did not correlate with changes in GLUT-2 expression. The levels of GLUT-2, IR, and AR transcripts and their immunoreactivity statistically significantly decreased in F1:Fin animals. In F1:Fin rats the serum levels of T and DHT negatively correlated with androgen receptor mRNA. The animals from F1:Fin group have statistically elevated level of glucose. Additionally, in adult F1:Fin rats, steatosis was observed in the liver (see Appendix A). Conclusions: It seems that treating male adult rats with finasteride causes changes in the carbohydrate metabolism in the liver of their offspring. This can lead to improper hepatic energy homeostasis or even hyperglycaemia, insulin resistance, as well as some symptoms of metabolic syndrome and liver steatosis.

Developmental Programming: Physiological Impacts of Prenatal Melatonin Administration on Reproductive Capacity and Serum Triiodothyronine of Adult Female Offspring Rat Born to Moms Exposed to Bisphenol A During Pregnancy.

Gestational bisphenol A (BPA) exposure induced multiple programmed diseases in the adult offsprings. Thus, this study targeted exploring the physiological impacts of melatonin (MEL) as a reprogramming strategy against in utero BPA exposure on reproductive capacity of adult F1 female rat offspring. Forty adult pregnant albino female rats were divided equally into 5 groups (n = 8): group I (control), group II (low-dose BPA; 25μg BPA/kgB.w.t.), group III (low-dose BPA + 10mg MEL/kgB.w.t.), group IV (high-dose BPA; 250μg/kgB.w.t.), and group V (high-dose BPA + MEL). Treatments were given daily by subcutaneous (s/c) injection from the fourth day of pregnancy until full term. After delivery, female offspring were selected, and on postnatal day 60, adult offspring were examined for estrus regularity and then were sacrificed at estrus to collect blood and tissue samples. Findings clarified that in utero BPA exposure (both doses) increased significantly (P < 0.05) the ovarian weights and the serum levels of estrogen but decreased that of triiodothyronine (T3) compared to control groups. Significant increasing of serum malondialdehyde (MDA) and decreasing of total antioxidant capacity (TAC) were also detected. Both doses of BPA disturbed remarkably the estrus cycles and caused marked aberrations in ovarian and uterine tissues. Interestingly, prenatal MEL co-treatment with BPA mitigated significantly all of these degenerative changes. Thus, this study first demonstrated that prenatal MEL therapy could be used as a potent reprogramming intervention against BPA-induced reproductive disorders in the adult F1 female rat offspring.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish

BackgroundCongenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown.MethodsHistopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated.ResultsWhile TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, l-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by l-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by l-carnitine treatment.ConclusionsThese results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Developmental cannabidiol exposure increases anxiety and modifies genome-wide brain DNA methylation in adult female mice

BackgroundUse of cannabidiol (CBD), the primary non-psychoactive compound found in cannabis, has recently risen dramatically, while relatively little is known about the underlying molecular mechanisms of its effects. Previous work indicates that direct CBD exposure strongly impacts the brain, with anxiolytic, antidepressant, antipsychotic, and other effects being observed in animal and human studies. The epigenome, particularly DNA methylation, is responsive to environmental input and can direct persistent patterns of gene regulation impacting phenotype. Epigenetic perturbation is particularly impactful during embryogenesis, when exogenous exposures can disrupt critical resetting of epigenetic marks and impart phenotypic effects lasting into adulthood. The impact of prenatal CBD exposure has not been evaluated; however, studies using the psychomimetic cannabinoid Δ9-tetrahydrocannabinol (THC) have identified detrimental effects on psychological outcomes in developmentally exposed adult offspring. We hypothesized that developmental CBD exposure would have similar negative effects on behavior mediated in part by the epigenome. Nulliparous female wild-type Agouti viable yellow (Avy) mice were exposed to 20 mg/kg CBD or vehicle daily from two weeks prior to mating through gestation and lactation. Coat color shifts, a readout of DNA methylation at the Agouti locus in this strain, were measured in F1 Avy/a offspring. Young adult F1 a/a offspring were then subjected to tests of working spatial memory and anxiety/compulsive behavior. Reduced-representation bisulfite sequencing was performed on both F0 and F1 cerebral cortex and F1 hippocampus to identify genome-wide changes in DNA methylation for direct and developmental exposure, respectively.ResultsF1 offspring exposed to CBD during development exhibited increased anxiety and improved memory behavior in a sex-specific manner. Further, while no significant coat color shift was observed in Avy/a offspring, thousands of differentially methylated loci (DMLs) were identified in both brain regions with functional enrichment for neurogenesis, substance use phenotypes, and other psychologically relevant terms.ConclusionsThese findings demonstrate for the first time that despite positive effects of direct exposure, developmental CBD is associated with mixed behavioral outcomes and perturbation of the brain epigenome.

Insights into factors sustaining persistence of high malaria transmission in forested areas of sub-Saharan Africa: the case of Mvoua, South Cameroon

BackgroundIn Mvoua, a village situated in a forested area of Cameroon, recent studies have reported high prevalence of Plasmodium falciparum infection among the population. In order to understand factors that can sustain such a high malaria transmission, we investigated the biology of Anopheles vectors and its susceptibility to insecticides, as well as long-lasting insecticidal net (LLIN) coverage, use and bio-efficacy.MethodsA longitudinal entomological survey was conducted from July 2018 to April 2019. Adult mosquitoes were collected using the human landing catch (HLC) method and identified using morphological and molecular techniques. Anopheles gambiae (s.l.) larvae were sampled from several stagnant water pools throughout the village and reared to generate F1 adults. The presence of P. falciparum circumsporozoite antigen was detected in the heads and thoraces of mosquitoes collected as adults using an enzyme-linked immunosorbent assay. The insecticide susceptibility status of the local An. gambiae (s.l.) F1 population to the pyrethroid insecticides deltamethrin 0.5% and permethrin 0.75% was determined using World Health Organization-tube bioassays, while the frequency of the knockdown resistance (kdr) mutation was determined by PCR. Coverage, use and physical integrity of LLINs were assessed in households, then cone assays were used to test for their bio-efficacy on both the reference insecticide-susceptible Kisumu strain and on field F1 An. gambiae (s.l.)ResultsIn total, 110 Anopheles mosquitoes were collected, of which 59.1% were identified as Anopheles funestus (s.l.), 38.18% as An. gambiae (s.l.) and 2.72% as An. ziemanii. Anopheles funestus was the most abundant species except in the long rainy season, when An. gambiae (s.l.) predominated (65.8%). In the dry seasons, vectors were principally endophagous (76% of those collected indoors) while they tended to be exophagous (66% of those collected outdoors) in rainy seasons. High Plasmodium infection was observed in An. gambiae (s.l.) and An. funestus, with a circumsporozoitic rate of 14.29 and 10.77%, respectively. Anopheles gambiae (s.l.) was highly resistant to pyrethroid insecticides (mortality rates: 32% for permethrin and 5% for deltamethrin) and harbored the kdr-L1014F mutation at a high frequency (89.74%). Of the 80 households surveyed, only 47.69% had achieved universal coverage with LLNs. Around 70% of the LLINs sampled were in poor physical condition, with a proportionate hole index > 300. Of the ten LLNs tested, eight were effective against the An. gambiae reference insecticide-susceptible Kisumu strain, showing mortality rate of > 80%, while none of these LLINs were efficient against local An. gamabie (s.l.) populations (mortality rates < 11.5%).ConclusionA combination of elevated P. falciparum infection in Anopheles vector populations, insufficient coverage and loss of effectiveness of LLINs due to physical degradation, as well as high resistance to pyrethroid insecticides is responsible for the persistence of high malaria transmission in forested rural area of Mvoua, Cameroon.Graphical

Sexual dimorphism of the extraorbital lacrimal glands in SF-1 knockout mice

Sexual dimorphism (SD) represents all the differences between males and females of the same species. SD of the murine lacrimal gland and the major effect of testosterone on its formation are well documented. Steroidogenic factor-1 (SF-1, NR5a1) is a nuclear receptor essential for the fetal development of steroid hormones producing organs and SF-1 knockout mice (Sf-1 KO) are therefore born without gonads and adrenal glands. The aim of this study was to investigate whether SD in lacrimal glands is present in the absence of exposure to sex hormones during development. Lacrimal glands from adult Sf-1 KO male and female mice without hormonal exposure, and from males that were treated with testosterone propionate (TP) prior to sacrifice, were examined. After sacrifice, glandular tissue was processed using standard histological procedures. Paraffin sections were analysed by stereology and immunostained against the androgen receptor (AR). Our results showed that there were no statistically significant differences in the mean volumes of acini, connective tissue or ductal system between males, females, and males on TP. The same pertains to the mean length of the ducts in all three groups. In the absence of sex hormones, sex chromosomes proved to be insufficient in inducing sexual dimorphism in LG. However, nuclei of the acinar cells in males on TP were positive for AR, whereas in males without TP no expression of AR was detected. Administration of TP induced the expression of AR in the nuclei of acinar cells of males but did not affect the morphology of LG. We conclude that SD in the lacrimal gland is not present in Sf-1 KO mice and this suggests that sex hormones have a major role in the development of SD in the lacrimal gland.

Effect of Tasmanian blue gum (Eucalyptus globulus Labill.) leaf extract on cowpea weevil (Callosobruchus maculatus [Fabricius, 1775], Coleoptera: Chrysomelidae)

A laboratory study was carried out to examine the efficacy of solvent extract of Eucalyptus globulus leaves in reducing post-harvest losses caused by cowpea weevil (Callosobruchus maculatus) on cowpea seed variety RSH 256. 450 g of E. globulus leaf powder were put in 1000 ml flask and 450 ml of n-hexane was used as the solvent. The extract was tested at dosages of 50, 100 and 150 µl 50 g-1 of cowpea grains on the weevil in stored cowpea. The different rates of treatment recorded significant differences (p &lt; 0.05) in causing adult mortality compared to the untreated control. The different rates of treatment also recorded significant differences (p &lt; 0.05) in emergence of F1 adults of each treatment compared to the control. The percentage masst loss and grain damage were also suppressed as a result of treatment with the plant material compared to the untreated control. However, among the treatments 150 µl 150 g-1 cowpea recorded the highest adult mortality rate and lowest emergence while control had the lowest mortality rate and the highest emergence of the insect. The rates of application were indicative of bioactive characteristics of the plant extract.

Recovery of Prenatal Baicalein Exposure Perturbed Reproduction by Postnatal Exposure of Testosterone in Male Mice.

Baicalein (BC), a flavonoid, which lacks the qualities of reproductive health and shows adverse effects, is tested in this study. Inseminated mice were injected with 30, 60, and 90 mg BC/Kg body weight on gestation days 11, 13, 15, and 17. The F1 BC-exposed males of each dosage were divided into six groups. First three groups (n = 6 from each BC dosage) were used for assessment of reproductive performance, the others (n = 4 from each BC dosage) were administered with testosterone 4.16 mg/kg body weight on postnatal days 21, 31, and 41. The reproductive health of adult F1 males at the age of 55 and 60 was tested. Prenatal BC exposure showed reduced fertility after cohabitation with control females. The BC exposure significantly reduced the body weight, tissue indices, and sperm parameters (motility, count, viability, and daily sperm count) and altered the sperm membrane in a hypoosmotic swelling test. A downward trend was observed in testicular steroidogenic marker enzymes (3β- and 17β-steroid dehydrogenases) and serum testosterone, whereas increase in serum titers of FSH and LH along with altered the testicular histology. Conversely, testosterone (4.16 mg/kg body weight) partially recovered reduced male reproductive health by BC. BC impaired male reproductive health due to low levels of testosterone is reverted by external testosterone is evidenced in this study.

Prenatal exposure to an environmentally relevant phthalate mixture accelerates biomarkers of reproductive aging in a multiple and transgenerational manner in female mice

Phthalates are known endocrine-disrupting chemicals that are found in many consumer products. Our laboratory previously developed a relevant phthalate mixture consisting of six phthalates and found that it disrupted female fertility in mice. However, it is unknown if prenatal exposure to phthalate mixtures can accelerate reproductive aging and if this occurs in multiple generations. Thus, we tested the hypothesis that prenatal exposure to a mixture of phthalates accelerates biomarkers of reproductive aging in multiple generations of female mice. Pregnant CD-1 mice were orally dosed with vehicle control or a phthalate mixture (20 μg/kg/day-500 mg/kg/day) daily from gestational day 10 to birth. Adult F1 females born to these dams were used to create the F2 and F3 generations by mating them with unexposed males. At 13 months, estrous cyclicity was monitored and ovaries and sera were collected for analysis. In the F1 generation, the mixture decreased testosterone and inhibin B levels, but increased follicle-stimulating hormone and luteinizing hormone levels compared to control. In the F2 generation, the phthalate mixture decreased the percent of antral follicles and testosterone hormone levels compared to control. In the F3 generation, prenatal exposure to the phthalate mixture increased ovarian weight, increased the time in metestrus/diestrus, altered follicle numbers, and decreased the levels of luteinizing hormone compared to control. Collectively, these data suggest that prenatal exposure to a phthalate mixture may accelerate several biomarkers of reproductive aging in a multi- and transgenerational manner in female mice.

Pre‐release evaluation of Heikertingerella sp. as a potential biocontrol agent for Tecoma stans in South Africa

AbstractNative to Central America, Tecoma stans (L.) Juss ex Kunth var. stans (Bignoniaceae) is a small tree that is invasive in South Africa and neighbouring countries. The plant was targeted for biological control in South Africa in 2003, with two insect agents released and established so far. The root‐feeding flea beetle, Heikertingerella sp. (Coleoptera: Galerucinae: Alticini), was imported from Mexico as an additional biocontrol agent and its biology and host specificity was assessed under quarantine conditions. The beetle displayed a generation time (i.e. from adult to adult) of 49 to 67 days, ensuring four annual generations under laboratory conditions. The beetle's larval and adult stages inflicted high levels of damage on the root system and the leaves of T. stans, respectively. No‐choice tests with 40 test‐plant species revealed adult feeding on only two non‐target species, Tecoma × alata and T. capensis (Thunb.) Spach, with feeding four times higher on T. stans. Larvae developed to adulthood on T. stans only. Multi‐choice tests involving the three Tecoma species confirmed these trends, demonstrating that Heikertingerella sp. is host specific. Since T. × alata is a hybrid of T. stans with invasive tendencies, any unlikely attacks by Heikertingerella sp. would be inconsequential in South Africa. The native T. capensis, which suffered little leaf damage and produced no F1 adults, is also at minimal risk of attack. We conclude that Heikertingerella sp. is a suitable biocontrol agent for T. stans and that permission for its release in South Africa be sought.

Vertical transmission of zika virus in Aedes albopictus

BackgroundZika virus (ZIKV) is an arthropod-borne flavivirus transmitted by Aedes mosquitoes. Aedes albopictus is an important vector of ZIKV worldwide. To date, most experiments have focused on the vertical transmission of ZIKV in Ae. aegypti, while studies on Ae. albopictus are very limited. To explore vertical transmission in Ae. albopictus, a series of laboratory studies were carried out.Methodology/Principal findingsIn this study, Ae. albopictus were blood-fed with ZIKV-infectious blood, and the ovaries and offspring viral infection rates were analyzed by reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). ZIKV was detected in the ovaries and oviposited eggs in two gonotrophic cycles. The minimum filial egg infection rates in two gonotrophic cycles were 2.06% and 0.69%, and the effective population transmission rate was 1.87%. The hatching, pupation, and emergence rates of infected offspring were not significantly different from those of uninfected offspring, indicating that ZIKV did not prevent the offspring from completing the growth and development process. ZIKV was detected in three of thirteen C57BL/6 suckling mice bitten by ZIKV-positive F1 females, and the viremia persisted for at least seven days.Conclusions/SignificanceZIKV can be vertically transmitted in Ae. albopictus via transovarial transmission. The vertical transmission rates in F1 eggs and adults were 2.06% and 1.87%, respectively. Even though the vertical transmission rates were low, the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to suckling mice through bloodsucking. This is the first experimental evidence of offspring with vertically transmitted ZIKV initiating new horizontal transmission. The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases.

Paternal exposure to di-n-butyl-phthalate induced developmental toxicity in zebrafish (Danio rerio).

Dibutyl phthalate (DBP) is an environmental endocrine disruptor detected in water, soil, and other environmental media frequently. Growing concerns regarding DBP exposure focus on toxicity to male reproduction. Reports about the developmental toxicity of paternal DBP exposure are rare. In this study, we investigated the developmental toxicity of paternal exposure to DBP on offspring in zebrafish. Adult male zebrafish with normal reproductive function were exposed to 0.2, 0.6, 1.8 mg/L of DBP or acetone solvent control for 30 days, and then mated with females. Thirty embryos per group were randomly selected to be observed, and malformations were recorded and photographed. The mating and observations were repeated three times, for a total of 90 embryos per group. The results showed that the percentage of malformations, such as edema and a bent trunk, was increased in the 0.6 and 1.8 mg/L DBP exposure groups, the heart rate and spontaneous contraction decreased in the 0.6 and 1.8 mg/L DBP exposure groups and migration of primordial germ cells was disrupted in some F1 embryos in all DBP exposure group after paternal exposure. The axial skeleton was affected in some F1 adults in the 1.8 mg/L DBP exposure group. Our findings demonstrate the developmental toxicity of paternal DBP exposure in zebrafish.

Chronic toxicity of technical atrazine to the fathead minnow (Pimephales promelas) during a full life-cycle exposure and an evaluation of the consistency of responses

Fathead minnows (Pimephales promelas) were continuously exposed to the herbicide atrazine (0.15, 0.25, 0.46, 0.99, and 2.0 mg a.i./L, plus dilution water and solvent controls) for a complete life cycle (274 days). Concentrations of atrazine up to 2.0 mg a.i./L did not significantly reduce hatching success, larval survival at 30 or 60 days post-hatch, or reproduction (eggs/spawn, total eggs, spawns/female, or eggs/female) in the F0 generation. However, at 60 days of exposure, total length and total survival to study completion were significantly reduced in ≥0.46 mg a.i./L and ≥ 0.99 mg a.i./L treatments, respectively. In the F1 generation, hatchability of embryos at ≥0.25 mg a.i./L (range 74–82%) was significantly less than that of pooled control organisms (86%). Following 30 days' post-hatch exposure, F1 survival was not significantly different from pooled control for any treatment. Finally, tissues representing major life stages had bioconcentration factors ranging from 3.7× (F1 embryos, <24 h) to 8.5× (F0 adults), indicating little to no evidence of bioconcentration. We developed a series of questions to assess the consistency of observed responses in order to place the data in context with the wider available and relevant literature (e.g., Observed between studies? Observed between species? Observed at lower levels of biological organization?). The analysis for consistency supports the conclusion that atrazine does not pose a significant chronic risk to freshwater fish in terms of growth, reproduction, or survivorship at concentrations of up to at least 100 μg/L.

Interactive effects of extreme temperature and a widespread coastal metal contaminant reduce the fitness of a common tropical copepod across generations

Tropical coastal areas are increasingly exposed to temperature extremes from marine heatwaves and contaminants from anthropogenic activities. The interactive effects of these environmental changes on marine life are understudied. We investigated the direct and cross-generational effects of copper (Cu) on F0 and F1 generations of the common tropical copepod Pseudodiaptomus annandalei under extreme temperatures (30 and 34 °C). In F0, Cu exposure reduced survival and nauplii production; these patterns were more pronounced at 34 °C and in females. F0 Copepods produced more faecal pellets at 34 °C than 30 °C, indicating a higher energetic demand. In F1, the number of F1 adults was lower in CuF0 and at 34 °C. Cu-exposed F0 produced larger adult F1, while exposure to 34 °C resulted in smaller adult F1. Our results show that tropical copepods are highly vulnerable to the interactive effects of contaminants and extreme temperatures.

Sex-specific transgenerational effects of morphine exposure on reward and affective behaviors

Current estimates indicate that millions of people in the United States abuse opioid drugs, which may also affect their offspring. To determine whether parental exposure to morphine alters reward and affective behaviors in subsequent generations we exposed male and female C57BL/6NTac mice to morphine (75 mg) or placebo pellets for 4 weeks. Naïve mice were used as mating partners to create subsequent generations (F1 and F2). Adult male and female F1 and F2 mice were tested in the morphine conditioned place preference paradigm (CPP), marble burying (MB), acoustic startle response (ASR), and open field tests (OFT). Paternal morphine exposure resulted in significantly attenuated preference scores amongst F1 male offspring, but significantly higher preference scores amongst F1 female offspring at the lowest CPP dose tested (5 mg/kg). In contrast, maternal exposure to morphine did not affect morphine reward in the F1 generation; however, the F2 male offspring of morphine-exposed F0 females displayed significantly higher CPP preference scores. Preference scores in F2 females were not affected by F0 male or female morphine exposure. Sex-specific alterations in affective behaviors were observed only in the offspring of F0 males exposed to morphine with F1 males spending less time in the center of the open field and F1 females spending more time in the center of the open field. One generation later, affective behaviors were no longer altered in F2 males but F2 females from the F0 male morphine exposure buried more marbles in the MB test. In summary, early exposure to morphine in males and females causes lineage-specific inheritance of reward and affective behaviors.

Vector competence of Rhipicephalus sanguineus sensu stricto for Anaplasma platys

Anaplasma platys is a Gram-negative, obligate intracellular bacteria that causes canine infectious cyclic thrombocytopenia in dogs. The brown dog tick Rhipicephalus sanguineus sensu lato is presumed to be the vector of A. platys based on the overlap in distribution of R. sanguineus and A. platys infections, detection of A. platys DNA in both flat and engorged field-collected R. sanguineus, and the fact that dogs are primary hosts of both brown dog ticks and A. platys. However, the only study evaluating the vector competence of R. sanguineus for A. platys under controlled laboratory conditions reported an apparent inability of ticks to acquire or maintain the pathogen. In 2016, engorged female Rhipicephalus sanguineus sensu stricto ticks were collected off dogs to start a laboratory tick colony. After one generation of colony maintenance on tick-naïve and pathogen-free New Zealand White rabbits, a rabbit used to feed F1 adults seroconverted to Anaplasma phagocytophilum antigen. PCR and subsequent DNA sequencing identified the presence of A. platys in both the adult ticks fed on this rabbit and their resulting F2 progenies. Retrospective testing of all previous (P and F1) life stages of this colony demonstrated that the infection originated from one field-collected A. platys-infected female whose progeny was propagated in the laboratory and produced the PCR-positive F1 adults. Over the following (F2-F4) generations, the prevalence of A. platys in this colony reached 90–100 % indicating highly efficient transovarial and horizontal transmission, as well as transstadial maintenance, of this pathogen by R. sanguineus s.s.

Establishment and quantitative measure of Stomoxys calcitrans (Linnaeus, 1758) colony production in Rio de Janeiro, Brazil.

Functional insect vector colonies are essential for the study of their biology, evolution, behavior as well as control strategies of these organisms that transmit pathogens of medical and veterinary importance. In addition to the establishment, improvement and adaptation of pre-established protocols in the maintenance of a colony is important, as it may result in higher production of insects. Stomoxys calcitrans or stable fly causes important economic losses in production of beef and milk, besides mechanically transmitting a great variety of pathogens to cattle. In order to generate flies under laboratory conditions, a colony of S. calcitrans was established at our laboratory considering local environment geographical characteristics. Adults collected in UFRRJ campus were kept in the laboratory and maintained with daily feeding on anticoagulant-containing bovine blood offered in disposable sanitary napkins. Immature forms were maintained on diets rich in organic matter. The colony temperature and relative humidity were daily monitored as well as the development of insects. The monthly measured biological parameters included the development of eggs into pupae, of pupae into F1 adults and the comparison of the number of flies collected in the field with the number of F1 flies emerged in the laboratory. The data presented here was obtained from March to November 2019. In that period, the average per month of flies collected in the field was 604.2, of generated eggs was 2694.3, of generated pupae was 349.5 and of generated F1 flies in the laboratory was 205.9. Fluctuations of abiotic and biological factors contributed to an uneven production of flies in the colony throughout the months. This is the first report with numerical quantification of S. calcitrans flies production in a laboratory in Brazil. Some methodologies used in the colony shall be reevaluated and modified with the goal of obtaining higher numbers of flies generated in the laboratory over time.

Glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis programming mediated hepatic lipid-metabolic in offspring caused by prenatal ethanol exposure

Prenatal ethanol exposure (PEE) could increase offspring’s susceptibility to adult liver lipid-metabolism diseases. This study aimed to confirm intrauterine programming mechanism of glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis for liver dysfunction in offspring rats induced by PEE. The results showed that levels of hepatic IGF1, lipid metabolism-related enzymes (e.g. FASN and HMGCR) and serum phenotype (TG, TCH, HDL-C, and LDL-C) were low in fetal rats of PEE but high in adult offspring except for HDL-C, meanwhile, hepatic H3K9ac and expression levels of IGF1 were low in fetal rats but high in adult offspring. Furthermore, levels of serum corticosterone and hepatic glucocorticoid-activation system (mainly including expression of 11β-HSD1, GR, and C/EBPα as well as 11β-HSD1/11β-HSD2 ratio) were high in fetal rats of PEE but low or unchanged in adult offspring. The adult F2 generation of PEE maintained the same GC-IGF1 axis programming alteration as the F1 generation despite gender differences. In vitro, cortisol was proved to activate hepatocyte glucocorticoid-activation system and decrease H3K9ac and expression levels of IGF1 by GR. Therefore, PEE has a long-term effect on the offspring’s liver functional development, which may be mainly related to the epigenetic programming alteration of the GC-IGF1 axis mediated by the glucocorticoid-activation system.

695-P: Grand-Maternal Exercise Improves Glucose Tolerance in Second Generation Offspring

Maternal obesity and diet are critical risk factors for development of obesity and diabetes in both first (F1) and second (F2) generation offspring. Maternal exercise in mice improves F1 metabolic health and prevents the detrimental effects of maternal high-fat-diet (HFD) on F1 offspring. Whether the beneficial effects of maternal exercise can be transmitted to the F2 generation is unknown. Here, C57BL/6 females were fed a chow or HFD and subdivided into cages with (Exercise) or without (Sedentary) running wheels for 2wks before breeding and during ∼3wks gestation. Male F1 offspring were sedentary and chow-fed, and at 8 wks of age were bred with age-matched females from untreated parents. F2 offspring were studied based on grand-maternal treatment: chow sedentary; chow trained; HFD sedentary; HFD trained (n= ∼10 litters/group; 348 total mice). F2 offspring were sedentary and chow-fed and studied up to 52 wks of age. For F2 males, grand-maternal exercise significantly improved glucose tolerance at 24, 36 and 52 wks and decreased insulin concentrations at 52 wks. Grand-maternal exercise also resulted in F2 males with lower body weights, decreased fat mass and increased lean mass at 52wks. Grand-maternal HFD decreased glucose uptake in skeletal muscles and adipose tissue in F2 males, but there was no effect of grand-maternal exercise, suggesting that the mechanism for improved glucose tolerance in F2 males does not involve glucose uptake in these tissues. For female F2, grand-maternal exercise had no effect on insulin concentrations, body weights or tissue glucose uptake. However, grand-maternal exercise significantly improved glucose tolerance in female F2 at 36 and 52 wks, and decreased fat mass and increased lean mass. Maternal exercise has beneficial effects on the metabolic health of adult F2 male and female offspring. These novel findings suggest that maternal exercise can prevent obesity and diabetes in multiple generations and may help reduce the incidence of these diseases worldwide. Disclosure A.B.A. Wagner: None. J. Kusuyama: None. P. Nigro: None. N.S. Makarewicz: None. M.F. Hirshman: Stock/Shareholder; Self; Abbott Laboratories, AbbVie Inc., Amgen, Colgate-Palmolive, Eli Lilly and Company, Medtronic. L.J. Goodyear: None. Funding American Diabetes Association (1-17-PMF-009 to A.B.A.W.); National Institutes of Health (R01DK101043)