George A Brusch
email: bruschg@gmail.com

Dale F Denardo

Olivier Lourdais

Reproductive state and water deprivation increase plasma corticosterone in a capital breeder

Keywords: Hydration, Squamate, Reproductive investment, Antaresia childreni, Osmotic stress

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The endogenous secretion of glucocorticoids is one of the main mechanisms by which animals modulate their physiological and behavioral responses to unpredictable and predictable stimuli [START_REF] Romero | Tempests, poxes, predators, and people: Stress in wild animals and how they cope[END_REF]. The main glucocorticoid in rodents and non-mammalian terrestrial vertebrates (i.e., birds, amphibians, and reptiles) is corticosterone (CORT) [START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF], and it can affect growth [START_REF] Busch | Impacts of frequent, acute pulses of corticosterone on condition and behavior of Gambel's whitecrowned sparrow (Zonotrichia leucophrys gambelii)[END_REF][START_REF] Midwood | Does cortisol manipulation influence outmigration behaviour, survival and growth of sea trout? A field test of carryover effects in wild fish[END_REF], survival [START_REF] O'connor | Seasonal carryover effects following the administration of cortisol to a wild teleost fish[END_REF][START_REF] Romero | The reactive scope model -A new model integrating homeostasis, allostasis, and stress[END_REF], and reproductive success [START_REF] Robert | The effects of maternal corticosterone levels on offspring behavior in fast-and slow-growth garter snakes (Thamnophis elegans)[END_REF][START_REF] Wingfield | Reproduction and resistance to stress: When and how[END_REF]. There is an impressive body of work examining the elevation in CORT in response to unpredictable stressors and noxious stimuli (i.e., allostatic response, see [START_REF] Romero | The reactive scope model -A new model integrating homeostasis, allostasis, and stress[END_REF][START_REF] Vera | Expanding the actions of cortisol and corticosterone in wild vertebrates: A necessary step to overcome the emerging challenges[END_REF]. However, baseline CORT levels are also important to consider because they may reflect the homeostatic demands faced by the organism [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes[END_REF][START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF]. CORT is important for regulating shifts in metabolism and behavior [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes[END_REF] and it oscillates to meet demands associated with shifts in life-history stages [START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF]. For example, baseline CORT levels are typically related to energy balance during reproduction and can either increase to support direct allocation to reproduction (i.e., energy model hypothesis) or decrease to minimize maintenance requirements (Bonier et al., 2009a,b).

However, important variations exist among and even within species in the directionality in which CORT levels respond (i.e., increase, decrease, or no change) to endogenous demands and predictable exogenous constraints [START_REF] Dickens | A consensus endocrine profile for chronically stressed wild animals does not exist[END_REF]. For example, individuals can have dramatically different CORT responses to annual, expected events such as decreased temperature during winter (increase - [START_REF] Sheriff | Mountain-top and valley-bottom experiences: the stress axis as an integrator of environmental variability in arctic ground squirrel populations[END_REF]decrease -Xu and Hu, 2017;no change -Khonmee et al., 2016) or seasonal precipitation (increase - [START_REF] Strier | Seasonal and social correlates of fecal testosterone and cortisol levels in wild male muriquis (Brachyteles arachnoides)[END_REF]decrease -Schoof et al., 2016;no change -Wilkening et al., 2016). These variable responses suggest that changes in CORT levels are extremely context dependent [START_REF] De Bruijn | The role of glucocorticoids in the vertebrate response to weather[END_REF][START_REF] Graham | Are invasive species stressful? The glucocorticoid profile of native lizards exposed to invasive fire ants depends on the context[END_REF], emphasizing the importance of considering factors such as life-history stage, body condition, and environmental conditions when investigating the role of CORT within an organism. Despite these variable patterns, CORT is critical to survival, maintenance, and reproduction [START_REF] Darlington | Corticosterone, but not glucose, treatment enables fasted adrenalectomized rats to survive moderate hemorrhage[END_REF], and CORT levels are modulated to adjust energy demands [START_REF] Dallman | Feast and famine: Critical role of glucocorticoids with insulin in daily energy flow[END_REF][START_REF] Remage-Healey | Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird[END_REF] and maintain homeostatic balance [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes[END_REF][START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF].

While energy is critical, water is another vital resource [START_REF] Kleiner | Water: An essential but overlooked nutrient[END_REF], and CORT has been shown to have important mineralocorticoid roles involved with water balance in rodents [START_REF] Agarwal | General overview of mineralocorticoid hormone action[END_REF][START_REF] Bentley | Endocrines and osmoregulation[END_REF][START_REF] Liu | Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rats[END_REF][START_REF] Thunhorst | Glucocorticoids increase salt appetite by promoting water and sodium excretion[END_REF]. While more limited in number, several studies have examined the relationship between CORT and water balance in non-rodent taxa. For example, a link between CORT levels and hydration state has been demonstrated in water-constrained environments [START_REF] Bradshaw | Homeostasis in Desert Reptiles[END_REF]. However, high variability in CORT levels suggests the response is context dependent. For example, recent studies found that baseline CORT levels are not affected by dehydration in animals with low metabolic demands (non-reproductive snakes and lizards, [START_REF] Dupoué | Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children's python (Antaresia childreni)[END_REF][START_REF] Moeller | Dehydration enhances multiple physiological defense mechanisms in a desert lizard, Heloderma suspectum[END_REF]. However, in animals with higher metabolic requirements and greater water deficits (e.g., pelagic sea birds), the opposite has been documented [START_REF] Brischoux | Marine lifestyle is associated with higher baseline corticosterone levels in birds[END_REF]. At least two studies have considered the additive challenges of reproduction and water deprivation on CORT levels (Dauphin-Villemant and Xavier, 1986;[START_REF] Dupoué | Water deprivation increases maternal corticosterone levels and enhances offspring growth in the snake Vipera aspis[END_REF]. In the aspic viper (Vipera aspis), CORT has been found to be closely related to the energy demands of pregnancy [START_REF] Lorioux | Are glucocorticoids good indicators of pregnancy constraints in a capital breeder?[END_REF]. However, [START_REF] Dupoué | Water deprivation increases maternal corticosterone levels and enhances offspring growth in the snake Vipera aspis[END_REF] found that changes in CORT levels were further increased when pregnant females faced water deprivation. In these studies, the study animals were able to thermoregulate, possibly resulting in differing selected body temperatures and thermal depression (see [START_REF] Ladyman | The influence of dehydration on the thermal preferences of the Western tiger snake, Notechis scutatus[END_REF] that, in turn, could have influenced the CORT response.

To further explore the influence that reproduction and water balance have on plasma CORT levels, we used Children's pythons (Antaresia childreni) in a two-by-two experiment where reproductive status and hydric states varied but body temperature was controlled to avoid confounding thermal effects on CORT levels. This capital-breeding, oviparous squamate faces considerable physiological and performance costs associated with reproduction, especially when considering that the energetic [START_REF] Lourdais | Structural and performance costs of reproduction in a pure capital breeder, the children's python Antaresia childreni[END_REF] and hydric [START_REF] Brusch | Reproduction alters hydration state but does not impact the positive effects of dehydration on innate immune function in children's pythons (Antaresia childreni)[END_REF] demands of reproduction must be met when energy and water resources are limited in the environment [START_REF] Taylor | Rainfall in the wet-dry tropics: Extreme events at Darwin and similarities between years during the period 1870-1983 inclusive[END_REF]. We tested the hypothesis that plasma CORT concentration is influenced by both reproductive status and hydric state. We predicted that (1) reproduction will increase CORT; (2) water deprivation, leading to dehydration, will increase CORT; and (3) the combined challenges from reproduction and water deprivation will have an additive effect on CORT.

Material and methods

Study species

Children's pythons inhabit the wet-dry tropics of northern Australia [START_REF] Wilson | A Complete Guide to Reptiles of Australia[END_REF] where they experience substantial, natural fluctuations in water availability during the Austral dry-season, which frequently lasts 3-4 months (May-August, [START_REF] Taylor | Rainfall in the wet-dry tropics: Extreme events at Darwin and similarities between years during the period 1870-1983 inclusive[END_REF]. Egg development and oviposition typically occurs towards the end of the dry season so that eggs hatch at the start of the wet season. As capital breeders, females accumulate energy resources over a long duration (typically in excess of a year) and utilize the resultant lipid reserves to support the reproductive effort from vitellogenesis through egg brooding [START_REF] Stephens | Capital breeding and income breeding: their meaning, measurement, and worth[END_REF].

However, a lack of free-standing water during the period of egg development results in considerable challenges to female water balance [START_REF] Brusch | Reproduction alters hydration state but does not impact the positive effects of dehydration on innate immune function in children's pythons (Antaresia childreni)[END_REF]. Coupled with a lack of available water to support themselves, reproductive females face additional water requirements associated with egg development [START_REF] Deeming | Post-hatching phenotypic effects of incubation in reptiles[END_REF][START_REF] Lourdais | Embryonic water uptake during pregnancy is stage-and fecundity-dependent in the snake Vipera aspis[END_REF]. In the weeks just prior to oviposition, in the absence of free-standing water in the environment, females must transfer substantial amounts of body water to their eggs [START_REF] Stahlschmidt | The use of ultrasonography to assess reproductive investment and output in pythons[END_REF] and this can dramatically challenge female water balance [START_REF] Brusch | Reproduction alters hydration state but does not impact the positive effects of dehydration on innate immune function in children's pythons (Antaresia childreni)[END_REF].

Experimental design

All work was conducted under the oversight of the Arizona State University Institutional Animal Care and Use Committee (protocol # 17-1532R). Snakes used for this study were part of a longterm colony at Arizona State University, AZ, USA, and were housed individually in 91 × 71 × 46 cm cages (Freedom Breeder, Turlock, CA, USA). Snakes were deprived of food from the onset of overwintering until oviposition (~5 months) because they typically do not eat when reproductively active. To stimulate the reproductive cycle, snakes were over-wintered for six weeks from mid-December through January (light-dark cycles of 6:18 h and a daily temperature cycle of 20:15°C that changed in conjunction with the light cycle). During the 6-hr light phase a supplemental sub-surface heat source was provided under one end of each cage to allow for thermoregulation. After overwintering, room temperature was increased to a constant 31.5°C, which is the preferred temperature of gravid Children's pythons [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): a test of the preadaptation hypothesis for maternal thermophily in snakes[END_REF], with a 12:12 h light-dark cycle and no supplemental heat provided. Females were held at a constant temperature without the opportunity to thermoregulate to prevent temperature-based difference resulting from differential thermoregulation between treatment groups.

Females were randomly assigned to either the reproductive (n = 19) or non-reproductive groups (n = 16). Females in the reproductive group were exposed to a different male every 2-3 days, resulting in each reproductive female being sequentially housed with 6-8 males during this 18-day breeding period. Each non-reproductive female was yoked to a reproductive female of similar body mass so that the timing of its treatments, measurements, and sampling coincided with that of the reproductive female to which it was yoked. At ovulation (based on a periovulation shed, [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): a test of the preadaptation hypothesis for maternal thermophily in snakes[END_REF], half of the reproductive females, and their yoked partners, were deprived of water until the reproductive female laid her eggs (22±1 d). Deprivation of water during this time leads to ecologically relevant levels of dehydration [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF].

Treatment groups were: gravid with water provided ad libitum (number of females, mean mass ± SEM, and plasma osmolality at the end of the experiment ± SEM: n = 9, 464±28 g, 324±5 mOsm kg -1 ), non-reproductive with water provided ad libitum (n = 8, 414±18 g, 297±4 mOsm kg -1 ), gravid with no water provided (n = 10, 424±41 g, 354±3 mOsm kg -1 ), and non-reproductive with no water provided (n = 8, 423±33 g, 340±4 mOsm kg -1 ).

Blood sampling and analyses

We used heparinized 1 ml syringes with a 25 gauge X 1.6 cm (5/8 in) needle to collect a 0.8 ml blood sample via cardiocentesis. Blood samples were collected from females once the reproductive female of the yoked partners reached late vitellogenesis (20 mm follicles determined by ultrasonographic examination, Sonosite MicroMazz, Bothell, WA, USA) and again when it reached late gravidity (twenty days after periovulation ecdysis and 1-4 days prior to oviposition). Total time for capture, restraint, and blood collection was typically less than three minutes and did not exceed seven minutes, which is less than the time required to detect measurable CORT concentration changes in squamates [START_REF] Romero | Corticosterone levels predict survival probabilities of Galapagos marine iguanas during El Nino events[END_REF]). To control for potential circadian fluctuations in CORT concentrations, all blood samples were collected between 0900-1100 hr. We immediately centrifuged the blood samples at 3000 rpm for three minutes to separate plasma from blood cells. We aliquoted plasma (~50 µl) into separate vials that were then frozen at -80°C. From the collected blood samples, we measured plasma CORT concentration as well as plasma osmolality (based on triplicate analysis using a vapor pressure osmometer, #5600, Wescor Inc., Logan, UT, USA) and circulating concentrations of glucose as a metric of circulating energy resources (blood glucose meter, #EG220546, Medline Industries, Northfield, IL, USA).

Plasma CORT concentrations (ng ml -1 ) were determined at the Centre d'Etudes Biologiques de Chizé (CEBC) following a well-established radioimmunoassay protocol [START_REF] Lormée | Sex-specific patterns in baseline corticosterone and body condition changes in breeding Red-footed Boobies Sula sula[END_REF]. The sample dilution curve in assay buffer was parallel to the standard curve, suggesting that the assay is specific for A. childreni with limited interference. We used a polyclonal anti-CORT antibody produced in rabbit whole antiserum (C 8784, Sigma-Aldrich, St Louis, MO, USA). Cross-reactions of the anti-CORT antibody were: 11-deoxycorticosterone 20%, progesterone 15.7%, 20α-hydroxyprogesterone 8.8%, testosterone 7.9%, 20βhydroxyprogesterone 5.2%, cortisol 4.5%, aldosterone 4.4%, cortisone 3.2%, androstenedione 2.6%, 17-hydroxyprogesterone 1.8%, 5α-dehydrotestosterone 1.4%, androsterone <0.1%, estrone <0.1%, and estriol <0.1%. The percent retrievable fraction of CORT after diethyl ether extraction was greater than 95% and all samples were run in duplicate (intra-assay variation: 8.05%, interassay variation: 9.79%).

Statistical Analyses

We performed all statistical analyses in R, version 3.5.0 (R Development Core Team 2018). We checked to ensure the data met the assumptions for parametric testing and used transformations where necessary. First, we examined the effect of reproductive status and water treatment on CORT values using repeated measures analysis of variance (rmANOVA). We tested for threeway interactions and used treatment (water or no water), status (reproductive or nonreproductive), and sampling period (late-vitellogenesis and late-gravidity bleeds) as fixed effects, and individual as a random effect. We included parameters addressing potential size effects by using a body condition index (BCI, standardized residuals from a linear regression using mass and SVL). However, this variable was removed from the final model as a result of stepwise removal using ΔAIC and model weights [START_REF] Arnold | Uninformative parameters and model selection using akaike's information criterion[END_REF][START_REF] Zuur | A protocol for data exploration to avoid common statistical problems[END_REF]. We used an ANOVA to examine changes in CORT values between females in each of the four groups (reproductive or non-reproductive, with or without water from late-vitellogenesis to late-gravidity). We used a post-hoc Tukey's HSD test to determine which of the groups were significantly different within and between time.

We then used linear regressions comparing the profiles among individuals to explore the relationships between CORT and osmolality or glucose across both sampling periods. We first compared the relationship by grouping all females together, regardless of treatment or status, after which we separately explored the effects of treatment, status, and the combination of the two. We next used variance partitioning methods described by Anderson and Gribble (1998) to decompose our full response into orthogonal subsets to examine how treatment (water or no water) or status (reproductive or non-reproductive) affected the relationship between CORT and osmolality or glucose and performed similar linear regressions. Using methods described by [START_REF] Nakagawa | A general and simple method for obtaining R2 from generalized linear mixed-effects models[END_REF], we calculated a general measure of R 2 , marginal R 2 (R 2 GLMM), from our models to estimate the variance explained by CORT and treatment, status, or a combination of the two. We used the packages "nlme" and "multcomp" [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF][START_REF] Pinheiro | nlme: Linear and nonlinear mixed effects models[END_REF]) for rmANOVA's, "CAR" [START_REF] Fox | An {R} companion to applied regression[END_REF] for linear regressions, "MuMIn" [START_REF] Barton | MuMIn: Multi-model inference[END_REF] for estimates of marginal R 2 , and "agricolae" (de Mendiburu, 2017) for post-hoc tests. Significance was set at α = 0.05.

Results

Variation in CORT levels

We found significant main effects of sampling (F1,31 = 25.08, p < 0.001), status (F1,31 = 26.69, p < 0.001), and treatment (F1,31 = 4.41, p = 0.044). We also found significant sampling-byreproductive status (F1,31 = 8.29, p = 0.007) and sampling-by-water treatment (F1,31 = 5.32, p = 0.028) interactions (Table 1, Fig. 1A,B), but no three-way interaction (F1,31 = 1.19, p = 0.283). A Tukey's HSD post-hoc test revealed that, while reproductive females initially had higher CORT (mean ± SEM; 16.77 ± 1.67 ng mL -1 ) compared to non-reproductive females (5.52 ± 1.46 ng mL - 1 ), the difference was more pronounced at the second sampling period (42.28 ± 6.72 ng mL -1 and 11.75 ± 4.84 ng mL -1 , respectively; Fig 1A). At the start of the experiment, CORT values were comparable in females with (11.08 ± 2.16 ng mL -1 ) and without (12.15 ± 2.06 ng mL -1 ) water.

However, water-deprived females had much higher CORT (36.56 ± 7.74 ng mL -1 ) at the time of the second sampling period, compared to those who had water provided (19.61 ± 3.59 ng mL -1 ;

Fig 1B).

When examining changes in CORT, we found significant differences among groups (F3,31 = 493.19, p = 0.006). Specifically, a Tukey's HSD post-hoc revealed that water deprived reproductive females had significantly greater (all p < 0.05) changes in CORT (36.0 ± 6.29 ng mL -1 ) when compared to other females (13.9 ± 6.66 ng mL -1 , 10.0 ± 6.84 ng mL -1 and 2.5 ± 1.15 ng mL -1 for reproductive with water, non-reproductive without water and non-reproductive with water, respectively). CORT change was not significantly different between these three groups (all p < 0.05).

Correlates of CORT levels

We found a significant positive relationship between CORT and plasma osmolality (F1,34 = 34.20, p < 0.001, R 2 GLMM = 0.331) when all females and sampling sessions were grouped together. When we isolated the effects of treatment and status, we found a significant positive relationship between CORT and osmolality in reproductive females with (F1,8 = 8.06, p = 0.022, R 2 GLMM = 0.321; Fig. 2A) and without access to water from late vitellogenesis to late gravidity (F1,9 = 22.05, p = 0.001, R 2 GLMM = 0.537; Fig. 2B), and in non-reproductive females without access to water during a similar period (F1,7 = 6.77, p = 0.035, R 2 GLMM = 0.311; Fig. 2C). We did not detect a significant relationship in non-reproductive females with access to water (p > 0.05; Fig. 2D).

When evaluating CORT and blood glucose, we did not detect a significant relationship (p > 0.05) when all females were grouped together. However, when we isolated the effects of treatment and status, we found a significant positive relationship between CORT and blood glucose in reproductive females, both those with (F1,8 = 8.45, p = 0.019, R 2 GLMM = 0.332; Fig. 3A) and those without access to water during gravidity (F1,9 = 7.41, p = 0.024, R 2 GLMM = 0.266; Fig. 3B). We did not detect a significant relationship in either of the non-reproductive groups (p > 0.05; Fig. 3C,D).

Discussion

Throughout an organism's lifetime, glucocorticoids play a fundamental role in responding to fluctuations in internal, biotic, and abiotic conditions [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes[END_REF][START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF].

CORT is widely recognized as an important hormone that mediates energy balance [START_REF] Landys | Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes[END_REF][START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF][START_REF] Sapolsky | How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions[END_REF]. For example, growth [START_REF] Belden | Corticosterone and growth in pacific treefrog (Hyla regilla) tadpoles[END_REF][START_REF] Cadby | Are increased concentrations of maternal corticosterone adaptive to offspring? A test using a placentotrophic lizard[END_REF][START_REF] Hayward | Maternal corticosterone is transferred to avian yolk and may alter offspring growth and adult phenotype[END_REF], reproduction [START_REF] Angelier | How does corticosterone affect parental behaviour and reproductive success? A study of prolactin in black-legged kittiwakes[END_REF][START_REF] Love | Evidence for baseline glucocorticoids as mediators of reproductive investment in a wild bird[END_REF][START_REF] Moore | Stress, reproduction, and adrenocortical modulation in amphibians and reptiles[END_REF], and migration [START_REF] Cease | Corticosterone and the transition from courtship behavior to dispersal in male red-sided garter snakes (Thamnophis sirtalis parietalis)[END_REF][START_REF] Eikenaar | Corticosterone, food intake and refueling in a long-distance migrant[END_REF] are, in part, regulated through the dynamic actions of CORT. A large body of literature on rodents has also recognized mineralocorticoid-like effects of CORT [START_REF] Bartter | The different effects of aldosterone-like steroids and hydrocortisone-like steroids on urinary secretion of potassium and acid[END_REF][START_REF] Bidet | Na+-H+ exchanger in proximal cells isolated from kidney. II. Short-term regulation by glucocorticoids[END_REF][START_REF] Schultz | A century of (epithelial) transport physiology: from vitalism to molecular cloning[END_REF]. The potential role of CORT in regulating salt and water balance has attracted far less attention in other taxa [START_REF] Vera | Expanding the actions of cortisol and corticosterone in wild vertebrates: A necessary step to overcome the emerging challenges[END_REF]. Our results provide evidence that increased CORT is contextually linked to both reproduction and plasma osmolality in an ectotherm.

At the time of the first blood sampling, all females had access to water and the reproductive females were in late vitellogenesis, which is the period of peak energetic investment in oviparous taxa [START_REF] Stephens | Capital breeding and income breeding: their meaning, measurement, and worth[END_REF]. The reproductive females had higher CORT compared to their non-reproductive yoked partners (Fig. 1A), and this is not surprising when considering the classic, energy-centric view of CORT [START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF]. As capital breeders, Children's pythons rely on internal reserves to fuel reproductive investment during vitellogenesis and increased CORT allows females to mobilize internal resources to fund the creation of energy-rich vitellogenin [START_REF] Jaatinen | Context dependency of baseline glucocorticoids as indicators of individual quality in a capital breeder[END_REF]. As expected, non-reproductive females, which had relatively low energetic demands during the same time-period, had substantially lower CORT levels.

In contrast with the high energetic demands of vitellogenesis, reproductive females during late-gravidity had already completed a majority of their energetic investment but were divesting considerable amounts of internal water to their developing eggs [START_REF] Stahlschmidt | The use of ultrasonography to assess reproductive investment and output in pythons[END_REF]. Regardless of water availability, reproductive females had the highest CORT levels during late-gravidity, despite lower energetic requirements at this time compared to late vitellogenesis (Fig. 1A). These findings are in contrast with the CORT-energy allocation relationship [START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF]), yet increased CORT has been associated with later stages of pregnancy in other species [START_REF] Dauphin-Villemant | In vitro steroid biosynthesis by the adrenal gland of the female Lacerta vivipara Jacquin: The metabolism of exogenous precursors[END_REF][START_REF] Lind | The relationship between plasma steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake, Crotalus oreganus[END_REF][START_REF] Lorioux | Are glucocorticoids good indicators of pregnancy constraints in a capital breeder?[END_REF][START_REF] Schuett | Sympatric rattlesnakes with contrasting mating systems show differences in seasonal patterns of plasma sex steroids[END_REF]. Increased CORT levels may also be associated with maternal physiological and behavioral shifts associated with gravidity [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): a test of the preadaptation hypothesis for maternal thermophily in snakes[END_REF]. This period of embryonic development is particularly sensitive to thermal variation and females shift their activity to increase body temperatures [START_REF] Lorioux | Maternal influences on early development: Preferred temperature prior to oviposition hastens embryogenesis and enhances offspring traits in the Children's python, Antaresia childreni[END_REF]. Additionally, CORT may serve as a hormonal stimulus for females to oviposit [START_REF] Taylor | Seasonal steroid hormone levels and their relation to reproduction in the Western Diamond-backed Rattlesnake, Crotalus atrox (Serpentes: Viperidae)[END_REF], which may explain why CORT is at its highest in late gravid females.

We found significant positive relationships between CORT and blood glucose, which is also in line with the classic role of CORT as a mediator of energy levels [START_REF] Romero | Seasonal changes in plasma glucocorticoid concentrations in free-living vertebrates[END_REF].

However, this relationship appears to be context dependent. It was only after parsing our data into treatment groups that we detected any significant relationships. We found significant relationships in reproductive females (Fig. 3A,B), but not in non-reproductive females (Fig. 3C,D), regardless of water availability. Previous research has found that female Children's pythons are able to preferentially catabolize muscle during gravidity to sustain the demands of reproduction [START_REF] Lourdais | Structural and performance costs of reproduction in a pure capital breeder, the children's python Antaresia childreni[END_REF] and that muscle catabolism increases with water deprivation [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. This, coupled with data from our study, emphasizes that these relationships (i.e., CORT and glucose levels or increased muscle catabolism) may only be detectable during periods of high physiological burden such as what occurs when an organism must face simultaneous challenges (e.g., reproduction and dehydration).

Reproductive females in our study with ad libitum access to water had significant increases in plasma osmolality (average osmolality increase: 37 mOsm kg -1 ) compared to their non-reproductive yoked pairs (6 mOsm kg -1 ) likely due to the considerable hydric demands of egg production. These increases were not significantly different from non-reproductive females without water access (44 mOsm kg -1 ) that had the hydric challenge of living without water.

Reproductive females without access to water during gravidity were faced with the combined hydric challenges of reproduction and water deprivation, and subsequently had the largest plasma osmolality increases during the experiment (66 mOsm kg -1 ; Brusch et al., 2018).

Increases in CORT throughout the experiment followed comparable trends with nonreproductive, water-provided females having the smallest change (average CORT increase: 2.5 ng mL -1 ), non-reproductive without water and reproductive with water in the middle (10.0 ng mL -1 and 13.9 ng mL -1 , respectively), and reproductive without water during gravidity having the largest change (36.0 ng mL -1 ).

We found a strong relationship between CORT and plasma osmolality, although, as with the rest of our results, it appears to be context dependent. When we examined the influence of reproductive status and water availability on osmolality, we found that CORT explained 54% of the variance in reproductive females without access to water during gravidity. The low metabolic demands of the non-reproductive females enabled us to examine the relationship between CORT and plasma osmolality (i.e., hydration) independent of the high energetic demands. Nonreproductive females showed increases in CORT when they were deprived of water, and we found a significant positive relationship between hyperosmolality (dehydration) and CORT (Fig. 2C). Juxtaposed against these results, we did not detect any similar increases or a significant relationship in non-reproductive females with water provided (Fig. 2D). While non-reproductive females in our study were exposed to a constant temperature approximately 2.5°C above their preferred body temperature [START_REF] Lourdais | Thermoregulation during gravidity in the children's python (Antaresia childreni): a test of the preadaptation hypothesis for maternal thermophily in snakes[END_REF], this was not enough to impact their CORT levels.

Our results from an ectotherm appear to be in congruence with decades of research in rodents that show CORT has important mineralocorticoid actions involved in water balance (see de [START_REF] De Kloet | Brain mineralocorticoid receptors and centrally regulated functions[END_REF]Joëls, 1997 for review). Unlike previous studies [START_REF] Dupoué | Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children's python (Antaresia childreni)[END_REF][START_REF] Moeller | Dehydration enhances multiple physiological defense mechanisms in a desert lizard, Heloderma suspectum[END_REF], we found that dehydration induced a CORT increase in non-reproductive females. Also in contrast with recent studies [START_REF] Dupoué | Water deprivation increases maternal corticosterone levels and enhances offspring growth in the snake Vipera aspis[END_REF][START_REF] Lorioux | Are glucocorticoids good indicators of pregnancy constraints in a capital breeder?[END_REF], our data suggest that CORT is linked to both energy and water balance. Our study controlled for a fundamental factor in heterothermic taxa, temperature, which may explain the discrepancy between previous results and ours. Organisms often alter their thermal preference based on their internal state. For example, dehydrated tiger snakes (Notechis scutatus) have reduced preferred temperatures, likely in an attempt to reduce water loss [START_REF] Ladyman | The influence of dehydration on the thermal preferences of the Western tiger snake, Notechis scutatus[END_REF]. Such thermal adjustments may also impact other physiological functions including hormone production and release. Given that temperature and the availability of resources are predicted to dramatically fluctuate due to climate change, further work is needed to better understand the potential inter-relationship among CORT, energy, and water balance.

Figure 1 -

 1 Figure 1 -Average plasma corticosterone (CORT, ng mL -1 ) concentrations measured in nonreproductive (NR) and reproductive (R) female Antaresia childreni (A) that were held with (W)or without (NW) access to water (B) during the duration of gravidity or its equivalent for nonreproductive yoked females. Initial blood samples were collected when reproductive females were in late-vitellogenesis, prior to water deprivation, and final blood samples were collected at late-gravidity. Error bars represent ± 1 SEM. Different letters indicate significant differences between groups and blood samples (Tukey's HSD post-hoc test).

Figure 2 -

 2 Figure 2 -Relationships between corticosterone (CORT, ng mL -1 ) and plasma osmolality (mOsm kg -1 ) measured in reproductive (R, n = 19; A,B) and non-reproductive (NR, n = 16; C,D) female Antaresia childreni. Females were held with (W) or without (NW) access to water during the duration of gravidity (i.e., prior to the second sampling point) or its equivalent for nonreproductive yoked females. Both sampling periods were combined for statistical analyses (see text for details) and a line of best fit is included for significant relationships (p < 0.05). Closed circles represent initial blood sampling, while open circles represent final blood sampling.

Figure 3 -

 3 Figure 3 -Relationships between corticosterone (CORT, ng mL -1 ) and glucose (mg dL -1 ) measured in reproductive (R, n = 19; A,B) and non-reproductive (NR, n = 16; C,D) female Antaresia childreni during the experiment. Females were held with (W) or without (NW) access to water during the duration of gravidity (i.e., prior to the second sampling point) or its equivalent for non-reproductive yoked females. Both sampling periods were combined for statistical analyses (see text for details) and a line of best fit is included for significant relationships (p < 0.05). Closed circles represent initial blood sampling, while open circles represent final blood sampling.

Table 1 -

 1 Statistical models used to examine corticosterone concentrations (ng mL -1 , CORT) in female Antaresia childreni during initial and final blood samplings (session), that were either reproductive or non-reproductive (status) and had continuous access to water or were waterrestricted (treat) during the time reproductive females were gravid.

	Factors	d.f.	F ratio	P value
	intercept	1, 31	98.29	< 0.001
	status	1, 31	26.69	< 0.001
	treat	1, 31	4.41	0.044
	session	1, 31	25.08	< 0.001
	status × treat	1, 31	1.22	0.277
	status × session	1, 31	8.29	0.007
	treat × session	1, 31	5.32	0.028
	status × treat × session	1, 31	1.19	0.283

Acknowledgments: This work would not have been possible without assistance from members of the DeNardo lab who helped with breeding colony management as well as Department of Animal Care and Technologies staff who assisted with animal care.

Funding: This material is based upon work supported by the National Science Foundation Graduate Research Fellowship (Grant # 1311230), the Peabody Family Memorial Award, and the Graduate and Professional Students Association JumpStart grant at Arizona State University for GABIV. Funding in France was provided by the CNRS and the Conseil Régional de Nouvelle Aquitaine (AQUASTRESS project).

Data availability:

The datasets supporting this article can be accessed at https://doi.org/10.6084/m9.figshare.7970819

Competing interests:

No competing interests declared.

Author contributions: GABIV, OL, and DFD designed the study and collected the samples.

GABIV conducted a majority of the assays (except for CORT), performed the statistical analyses, and led the writing of the manuscript. DD and OL contributed to revisions and gave final approval for publication.


Adriana Codrean
email: j.adriana9@gmail.com

Dan L Dumitrascu
email: dan_dumitrascu@yahoo.de

Victor Codrean
email: victorcodrean34@yahoo.com

Delia Mirela Tit

Simona Bungau
email: simonabungau@gmail.com

Selim Aleya

Marius Rus
email: rusmariusr@yahoo.com

Ovidiu Fratila
email: ovidiufr@yahoo.co.uk

Delia Carmen Nistor-Cseppento

Lotfi Aleya
email: lotfi.aleya@univ-fcomte.fr

Delia Mirela Tit
email: mirela_tit@yahoo.com

Nicoleta Negrut

Epidemiology of human giardiasis in Romania: A 14 years survey

Keywords: giardiasis, epidemiology, parasitic intestinal diseases, prevalence, digestive symptoms, zoonoses

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Giardiasis is a digestive zoonotic disease caused by the protozoan genus Giardia [G], and the most frequent species is G. duodenalis (syn. G. intestinalis, G. lamblia) [START_REF] Acha | Zoonosis y efermendades transmisibles communes al hombre y a los animals[END_REF]. It is considered as the most common intestinal protozoan and the most frequent cause of parasitic gastroenteritis around the world (Ignatiu et al., 2012), with approximately 2.8×10 8 new cases emerging every year [START_REF] Lanes | Current trends in research into the waterborne parasite Giardia[END_REF]. G. lamblia is a ubiquitous infection and is more frequent in children than adults, especially in areas with a warm climate [START_REF] Eissa | Giardia lamblia: a new target for miltefosine[END_REF]. In Romania, the infection with G. lamblia, along with other intestinal parasitic infections (ascariasis and enterobiasis), is the most frequent diagnosed intestinal parasitic disease [START_REF] Neghina | Epidemiology and history of human parasitic diseases in Romania[END_REF]. This flagellated protozoan is transmitted either through direct contact, on a fecal-oral route, or through consumption of water or foods that are contaminated with cysts of Giardia spp. [START_REF] Ehsan | Occurrence and potential health risk of Cryptosporidium and Giardia in different water catchments in Belgium[END_REF][START_REF] Hooshyar | Giardia lamblia infection: review of current diagnostic strategies[END_REF], the infectious and resistant form of the parasite in nature. Cysts are extremely resistant to vitreous environmental conditions and able to keep their ability to infect for months. Therefore, there is a real risk of contamination of and from the environment with Giardia cysts. Water samples (wastewater from any source: surface water, drinking water, swimming pools) taken anywhere in the world have confirmed this infestation, with numerous outbreaks of germination in many countries [START_REF] Sărmăşan | Identification, communication and management of risks relating to drinking water pollution in Bihor County[END_REF]. Giardia is the second most common cause of gastroenteritis caused by water infested with protozoan parasites [START_REF] Boarato-David | Giardia duodenalis[END_REF]. The presence of the Giardia spp. cysts in the water is also due to the fact that they are largely spread in the environment and are generally fairly resistant to environmental factors. In both human and animals, this protozoan has a great clinical value [START_REF] Olson | Update on Cryptosporidium and Giardia infections in cattle[END_REF]. The main zoonotic-epidemiologic issues that these parasitic zoonoses raise are those concerning the surveillance, prevention and limitation [START_REF] Suteu | Actualitati privind epidemiologia, patogenia si controlul zoonozelor protozoariene[END_REF].

It can manifest in endemic form, especially in tropical and subtropical areas, or in an epidemic form (that affects the travelers in the endemic areas, as it is believed that 2-3% of the travelers' diarrheas are caused by Giardia spp.). It can also occur in restricted communities, like families or groups from areas with precarious hygiene, or it can manifest like a hydric epidemic, after the consumption of contaminated water from uncontrolled sources. The World Health Organization (WHO) estimates that Giardia spp. is one of the parasites that is most commonly spread in humans and animals, which get infected through water with a parasitic germ that is rarely lethal [START_REF] Meyer | Giardia and giardiasis[END_REF][START_REF] Dubourg | Giardia secretome highlights secreted tenascins as a key component of pathogenesis[END_REF]. This constitutes a major concern for public health in both developed and developing countries [START_REF] Hoque | Risk of giardiasis in Aucklanders: a case control study[END_REF]. The incidence of the infection with Giardia spp. varies greatly according to geographical regions and in the different regions of the same country. The prevalence is greatly influenced by the diagnostic methods that are used and by the expertise of those who diagnose the disease.

The clinical presentation of the diseases varies greatly, primarily depending on the location of the parasite, the individual factors (the immune response) and the parasitic virulence (Judea-Pusta et al., 2019), the dose of the parasite and the duration of the infection, which can be symptomatic or asymptomatic, with chronic or acute diarrhea, along with dehydration, weight loss, abdominal pain, nausea and vomiting [START_REF] Eckmann | Mucosal defences against Giardia[END_REF]Hooshyar et al., 2009). The chronic infection with G. duodenalis can lead to chronic gastroenteritis, malnutrition, persistent dysbiosis, zinc deficiency, vitamin A deficiency, iron deficiency and growth deficiency (Thomson, 2008;[START_REF] Beatty | Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms[END_REF][START_REF] Gabor-Harosa | Proposed model for a Romanian register of chronic diseases in children[END_REF][START_REF] Choy | Prevalence and Associated Risk Factors of Giardia Infection among Indigenous Communities in Rural Malaysia[END_REF]. The extra-digestive symptoms are also an important part of the clinical panel of giardiasis, especially the allergic, neuropsychiatric and general ones [START_REF] Einarsson | An up-date on Giardia and giardiasis[END_REF][START_REF] Hooshyar | Giardia lamblia infection: review of current diagnostic strategies[END_REF].

The aim of our study was to find new epidemiological data on the infection with Giardia spp. and to look for factors influencing the incidence and clinical presentation.

Materials and Methods

Protocol

A comprehensive 14 years prospective screening from January 1, 2004 until December 31, 2017, was undertaken in a single center for infectious diseases, with large referral area. All patients admitted to this center were tested for Giardia spp. infection. All the subjects were tested, whether symptomatic or not. The records of the positive cases were analyzed. We undertook a survey of the demographic and epidemiologic data, as well as the symptoms. The diagnosis of giardiasis was the only criterion for being included in the study.

It were not necessary exclusion criteria.

The following information were processed: the incidence and prevalence of giardiasis according to age and to the month of the year, as well as prevalence on the age category, gender and background. All the patients diagnosed with Giardia spp. were included in the study. History was collected by direct interview or, for children, from their relatives. The patients included in the study were separated according to gender and age groups: 0-1 years old, [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][15][16][17][18][19][20][21][22][23][24][25][25][26][27][28][29][30][31][32][33][34][START_REF] Carvalho | A comparative study of the TF-Test®, Kato-Katz, Hoffman-Pons-Janer, Willis and Baermann-Moraes coprologic methods for the detection of human parasitosis[END_REF][START_REF] Carvalho | A comparative study of the TF-Test®, Kato-Katz, Hoffman-Pons-Janer, Willis and Baermann-Moraes coprologic methods for the detection of human parasitosis[END_REF][36][37][38][39][40][41][42][43][44][45][45][46][47][48][49][50][51][52][53][54][55][55][56][57][58][59][60][61][62][63][64][65][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84]and >85 years old. Full diagnosis at discharge was also analysed and correlated with Giardia infection.

Ethical statement

The study was approved by the Ethics Commission of the Council of the Medicine and Pharmacy Faculty, University of Oradea, and conducted in accordance with the World Medical Association Code of Ethics (Declaration of Helsinki, 1967). Each patient included in the study signed an informed-consent form.

Setting

The current study was carried out in a single tertiary center, namely the Department of Infectious Disease, Municipal Hospital Oradea "Gavril Curteanu", Bihor County, Romania.

The Bihor County is located in the north-west of Romania. It has a population of 566,435 (2017), 51% of the population living in urban areas. The Municipal Hospital from Oradea serves the entire county, both the rural and the urban areas. This hospital admits both children and adults.

Diagnosis of giardiasis

The copro-parasitological technique used to diagnose giardiasis was the Willis method. The Willis technique is considered to be a floatation method, which is based on the ability of helminth eggs to float on / to the surface of a saturated NaCl solution (ρ=1.20 g / mL) and to adhere to the glass. By this technique, a saturated solution with emulsified faeces is introduced into a round bottom flask; a meniscus forms on its surface. The flask is then covered with a glass blade/slide. After a few minutes, the glass blade/slide is removed and examined under a microscope [START_REF] Carvalho | A comparative study of the TF-Test®, Kato-Katz, Hoffman-Pons-Janer, Willis and Baermann-Moraes coprologic methods for the detection of human parasitosis[END_REF]. Faecal samples (n=3) from each subject were collected on alternate days (first, third and fifth day), in a sterile universal container, with no preservative solution. The samples were kept at a temperature of 4 °C and were processed within 24 hours of collection.

Statistical analysis

The statistical analysis and graphical representation were performed using the software package SPSS (Statistical Package for the Social Sciences), version 26 for Windows.

Today, the program is used in different area like marketing, experimental research, education, health. SPSS use different procedures like descriptive, comparative, reductive, modeling which some we use in our article: descriptive, frequency and comparative analysis. Also, Chi square test was used to calculate p-value and determine the confidence interval. Descriptive statistics (95% Confidence Interval) were calculated for each variable as appropriate. Mann-Whitney U statistics for two independent variables. A p-value of less than 0.05 was regarded as statistically significant and a strong relation between independent variables which we consider in this article.

Results

Incidence

During the 14 year interval investigated, 54,623 patients, children and adults were admitted to the tertiary center included in the study and tested for Giardia spp. infection. In this group, 2,441 patients were diagnosed with giardiasis (Figure 1), representing a prevalence of 4.47%. In the observed period of time, the annual prevalence of the infection with Giardia spp. showed variations (16.36%-0.60%). As it can be observed in Figure 1, the evolution of the infection prevalence during this interval of 14 years showed a decreasing trend. A frequent association of giardiasis with acute enterocolitis, gallstone diseases, acute gastritis and acute hepatitis was observed in patients infected with Giardia spp. (Figure 2).

The incidence of giardiasis in each calendar month can be seen in Figure 3. During the 14 years, over 10% of the cases were recorded in the moths June, July and August (10.20%, 10.65% and 10.49%). During our study, increased values were also recorded in the months January and February, namely 8.89% and 8.68%. The lowest values were recorded in October (6.96%), November (6.31%) and December (6.80%). Regarding gender, 61% of the patients infected with Giardia spp. were female, the female-male ratio being 1.5:1 (61% female, 39% male) (Figure 4).

In this study, the incidence of giardiasis according to age categories varied greatly. The incidence of giardiasis was higher in patients from urban areas, in comparison with those from rural areas. Thus, 788 of the patients from rural areas were diagnosed with giardiasis (36.30%) and 1,434 patients from urban areas (64.70%), the ratio being 1.8:1 (Figure 5).

Our research revealed that the incidence of giardiasis according to age categories varied greatly and there was a direct relation to the age of the patients. Over 60% of the patients were aged 15-54 years (66.86%). A high incidence was observed in patients aged 15-24 years: 16.14%; in patients aged 25-34 years: 17.94%; in patients aged 35-44 years: 16.80%; and in patients aged 45-54 years: 15.98%. In the other age categories, the incidence below 15%, the lowest incidence being 0.66%, in patients under one year of age and 0.16% in patients with ages over 85 (Figure 6).

Clinical presentation

As expected, the gastrointestinal and hepatobiliary symptoms were overwhelming. A small number of patients presented allergic symptoms. The most frequent complaints were abdominal pain (69.07%), especially diffuse pain, loss of appetite (71.24%) and sensitivity at palpation (54.69%), especially in the right hypochondrium, hepatomegaly (46.17%). Other symptoms were nausea (42.52%), vomiting (25.60%), diarrhea (18.48%), constipation (8.89%), intestinal distension (7.50%) and erythematous skin rash (11.35%) (Figure 7). The abdominal pain was recorded in 10.67% of the infants, 16.62% of the children were 1-4 years old, in 19.94% of the teenagers and in 13.23% of the adults aged 55-64 years. The lowest prevalence of abdominal pain was in patients with ages between 45 and 54 (4.31%) and the highest was observed in teenagers (19.94%) (p<0.001).

The loss of appetite was more frequently observed in 18.14% of the patients aged 15-24 years, the lowest prevalence being recorded in children aged 5-14 years (5.04%) (p<0.001). Nausea was reported by nearly 8.04% of the patients. Vomiting was reported by 4.84% of the patients, especially in children aged 1-4 years. Hepatomegaly was observed in nearly 9% of the patients (8.73%). We mention that hepatomegaly was more frequent in adults. Concerning the intestinal transit disorders, diarrhea was more frequent than constipation in the patients that participated in the study. Diarrhea was observed in all age groups, the highest percent being represented by infants (50%) (p<0.001). Constipation was recorded in 4% of the infants, in 5.90% of the children aged 1-4 years and 6.06% in the elderly (p<0.001). Allergic skin rash appeared in only 5% of the cases, the highest percentage being observed in children aged 1-4 years (12.06%) (p<0.001). In the other age groups, the percent varied between 0% and 3.55% (Figure 8).

All patients with positive tests for Giardia were treated according to guidelines with albendazole; 1,147 (47%) of them returned for follow-up and showed negative tests.

Discussion

Human giardiasis with universal spread is characterized by epidemics in some countries, and the spread affects between 1% and 27% of the population [START_REF] Fletcher | Epidemiology and geographical distribution of enteric protozoan infections in Sydney, Australia[END_REF]. The incidence of the infection with G. duodenalis is lower in developed countries, and recent studies show values of 1% in Germany, 3.7% in Portugal, 1.30% in England, 1.40 and 5.8% in the United States, 2.50% in North Korea, 6.75% in China [START_REF] Sagebiel | Giardiasis in kindergartens: prevalence study in Berlin, Germany[END_REF][START_REF] Almeida | Genotype analysis of Giardia isolated from asymptomatic children in northern Portugal[END_REF][START_REF] Davies | Asymptomatic carriage of protozoan parasites in children in day care centers in the United Kingdom[END_REF][START_REF] Church | Intestinal infections in humans in the Rocky Mountain region, United States[END_REF][START_REF] Painter | Giardiasis surveillance -United States, 2011-2012[END_REF][START_REF] Huh | A survey of intestinal protozoan infections among gastroenteritis patients during a 3-year period (2004-2006) in Gyeonggi-do (province), South Korea[END_REF][START_REF] Liu | Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China[END_REF]. In developing countries, including Romania, the disease has an extremely high prevalence in children enrolled in communities (pre-schools, kindergartens, schools), easily transmitting the infection to the other members of the family. The prevalence of giardiasis in the world, estimated at 7.2% in 1965, fluctuates greatly and depends on various factors such as age (the highest incidence between the ages 1-3, as high as 85%), environmental background (more frequent in communities, 5.4%-78%, as opposed to the family environment 1.5%-21%), the social economical and geographic area [START_REF] Gillon | Change in the small intestinal mucosa in giardiasis[END_REF].

In Romania, the incidence of infection in humans varies from 15% to 44%, in communities rarely reaching 37-77%. According to other authors, Giardia spp. can affect all age groups, the prevalence of the disease in the world varying between 2% and 25%, being higher in urban communities, especially in very young children [START_REF] Steriu | Parasitic infections (in Roumanian: Infectii parazitare)[END_REF].

A correct diagnosis of giardiasis is relevant for the prevention and / or treatment of diseases that are caused by this helminth. The diagnosis resulting from the laboratory analyses, in the case of Giardia spp., is based mainly on finding and identifying microscopic cysts in faecal samples. In addition, immunological-based tests along with molecular methods are used for diagnosis and / or for advanced research. These diagnostic methods exhibit different levels of sensitivity and specificity. The determination of the pathology itself depends on several factors, of which can be mentioned: the chosen test method, the ability to carry out the stages of the identification / detection operations, the skill of operations, as well as the stage in which the test was performed [START_REF] Elmi | Comparison of sensitivity of sucrose gradient, wet mount and formalin -ether in detecting protozoan giardia lamblia in stool specimens of BALB/c mice[END_REF]. There is no known traditional or ultramodern method able detect absolutely all cases of Giardia infection. In the last three decades, several immunodiagnostic tests (for the rapid diagnosis of giardiasis) based in particular on the detection of Giardia antigens in faecal samples have been developed. At the global level, due to the high sensitivity of these methods, examination of stool samples by microscopy (after using the concentration method) is the most frequent procedure performed in laboratory analysis dedicated to giardiasis. The results obtained are relevant due to their good performance in the diagnostic strategy. These methods should thus be viewed as the gold standard [START_REF] Hooshyar | Giardia lamblia infection: review of current diagnostic strategies[END_REF][START_REF] Soares | Giardiasis: an update review on sensitivity and specificity of methods for laboratorial diagnosis[END_REF]. In the present study we used the Willis technique since it is commonly used for detecting light structures (protozoan cysts, hookworm eggs) [START_REF] Cerqueira | Análise comparativa da sensibilidade da técnica de Willis no diagnóstico parasitológico da ancilostomíase[END_REF]. To increase clinic sensitivity test, 3 samples were collected on alternative days and then analysed [START_REF] Elmi | Comparison of sensitivity of sucrose gradient, wet mount and formalin -ether in detecting protozoan giardia lamblia in stool specimens of BALB/c mice[END_REF].

Our study showed a prevalence of 4.47% in a community admitted in a hospital for infectious diseases. The data are similar to those of Steriu [START_REF] Steriu | Parasitic infections (in Roumanian: Infectii parazitare)[END_REF], reported from other Romanian counties, who state that the infection with Giardia spp. has an incidence of 2-25%, respectively 4-7%, but also similar to the data collected by Basualto (Basualto et al., 2007) and Hortez and Gurvith (2011) who reported an incidence of giardiasis of 6.9%, respectively 1-11%. In a study conducted on hospitalized patients in the south-west of Romania [START_REF] Neghina | Epidemiology and history of human parasitic diseases in Romania[END_REF] it was showed an incidence of Giardia spp. of 7.80%. The studies conducted by [START_REF] Akdemir | Evaluation of parasitological laboratory results of a group of people older than 15 years in Kutahya[END_REF] on a group of 675 people over 15 years of age from Kutahya, Turkey, the study conducted by [START_REF] Degirmenci | Distribution of intestinal parasites detected in the parasitology laboratory of the Ege University Medical School Hospital, in 2005[END_REF], on a group of 3,925 patients from the University Hospital from Ege, Turkey and the studies conducted by [START_REF] Yaman | Distribution of intestinal parasites in patients presenting at the parasitology laboratory of the medical school of Erciyes University between the years of 2005 and 2008[END_REF] on a group of 28,911 patients that appeared in the Parasitology Laboratory of the University of Medicine from Erciyes, Turkey, reported much lower incidence of Giardia spp., namely 1.48%, 1.78% and 1.96%. Our study found a substantial decline of the infection during the period in which the patients with Giardia spp. were studied. The highest value was reached in the year 2004 (16.36%) and the lowest in the years 2016 (0.65%) and 2017 (0.69%). In the last two years we observed much lower values of the prevalence of giardiasis. A decline trend of the infection with Giardia spp. was also observed in the studies conducted by Painter [START_REF] Painter | Giardiasis surveillance -United States, 2011-2012[END_REF], in a retrospective study in the United States and in the studies conducted by Jejaw [START_REF] Jejaw | Status of intestinal parasitic infections among residents of Jimma Town, Ethiopia[END_REF], in a retrospective study in Jumma Town, Ethiopia, with an incidence of the Giardia spp. infection lower than 5%.

In our study, the monthly prevalence of giardiasis varies between 10.65% and 6.31%, the highest percent being recorded in the months June, July and August (10.20%, 10.65% and respectively 10.49%), and the lowest percent in the months October, November and December (6.96%, 6.31% and respectively 6.80%). [START_REF] Painter | Giardiasis surveillance -United States, 2011-2012[END_REF], in a study conducted in the United States, observed higher incidences of the infection during summer. A study conducted by Terletskaia-Ladwig [START_REF] Terletskaia-Ladwig | Epidemiological aspects of gastrointestinal infections[END_REF], in Germany, reported an incidence of 15% of infection with Giardia spp. from June until August, but disproves the incidence on the infection in October and November. [START_REF] Mbae | Intestinal parasitic infections in children presenting with diarrhoea in outpatient and inpatient settings in an informal settlement of Nairobi, Kenya[END_REF], in a two years study, conducted on children with diarrhea also observed a high incidence of the infection in the months of June and August, but also from September until November, a period in which our study showed lower incidences. The high incidence of infection with Giardia spp. in humans during the summer months could be explained by the connection between the consumption of contaminated water, the frequency of travelling and time spent outside, which is a factor for transmitting the cysts of Giardia spp. in humans.

Based on the gender, the data presented in our study revealed a higher incidence in females, namely 61% in females and 39% in males. The higher prevalence of the infection with Giardia spp. in females as opposed to that in males, as showed in our study, is in key with some previous studies (Wansaas et al., 2009;[START_REF] Persson | The relationship between irritable bowel syndrome, functional dyspepsia, chronic fatigue and overactive bladder syndrome: a controlled study 6 years after acute gastrointestinal infection[END_REF]. Other studies conducted on both adults and children show different data from ours. According to the studies conducted by Ostan [START_REF] Ostan | Health inequities: lower socio-economic conditions and higher incidences of intestinal parasites[END_REF] on a group of 294 children, by [START_REF] Kasssem | Intestinal parasitic infection among children and neonatus admitted to Ibn-Sina Hospital, Sirt, Libya[END_REF] on a group of 350 children and newborn babies and by Warrunee et al. (2007) on 1920 children show no significant differences regarding the gender of the people infected with Giardia spp. Also, a series of previous studies show a higher incidence in males than in females (Wansaas et al., 2009;[START_REF] Mellingen | Post epidemic giardiasis and gastrointestinal symptoms among preschool children in Bergen, Norway. A cross-sectional study[END_REF][START_REF] Escobedo | Hospitalization of Cuban children for giardiasis: a retrospective study in a paediatric hospital in Havana[END_REF][START_REF] Asher | Distribution of Giardia duodenalis assemblages A and B among children living in a remote indigenous community of the Northern Territory, Australia[END_REF][START_REF] Ramos | Different profile of intestinal protozoa and helminthic infections among patients with diarrhea according to age attending a rural hospital in southern Ethiopia[END_REF][START_REF] Tamer | The frequency of the presence of intestinal parasites in students of Arslanbey Primary School[END_REF][START_REF] Tamer | Genotyping and phylogenetic analysis of Giardia duodenalis isolates from turkish children[END_REF].

The data we collected in our study show a higher incidence of infection with Giardia spp. in people who come from urban areas, than in people from rural areas (63.70%, compared to 36.30%). Our data are similar to those presented by [START_REF] Neghina | Epidemiology of ascariasis, enterobiasis and giardiasis in Romanian western county (Timis), 1993-2006[END_REF], in a study conducted in Timis County, Romania, on a 14 years period of time. A study conducted by [START_REF] Korzeniewski | Prevalence of intestinal parasites in Afghan community on the example of patients treated in Ghazni Provincial Hospital[END_REF], on children and adults in a provincial hospital in Afghanistan showed an incidence of infection with Giardia spp. of 38.3%, these data being similar to the ones we have found. According to Al-Aghra and Teodorescu, in a study conducted in Palestine, the incidence of the infection with the G. intestinalis in people from urban areas is 33%, and 53% in people from urban areas (Al-Aghra and Teodorescu, 2000). Studies conducted by [START_REF] Basualdo | Intestinal parasitoses and environmental factors in a rural population of Argentina, 2002-2003[END_REF] in Argentina and by [START_REF] Quattara | Prevalence and polyparasitism of intestinal protozoa and spatial distribution of Entamoeba histolytica, E. dispar and Giardia intestinalis from pupils in the rural zone of Man in Cote d'Ivoire[END_REF] in France on individuals from rural areas, have showed much lower incidences of infection with Giardia spp. (18.1% and 17.5%). The high prevalence of the infection in urban areas can be due to the poor sanitation in some of the poor areas of the city, the access to drinking water and the poor hygiene.

In our study, the intestinal infection with Giardia spp. can be associated with age, the highest incidence of the infection being in the age groups 5-14 years old (13.40%), 15-24 years old (16.14%), 25-34 years old (17.94%), 35-44 years old (16.80%) and 45-54 years old (15.98%). They are significantly higher than in other age groups (there is an incidence of 0.66% in infants and an incidence of 3.66% in children aged 1 to 4 years old). The number of cases of infection with Giardia spp., higher in adults and teenagers, could be explained by the fact that more adults than children came to the hospital. The data from our study regarding the incidence of Giardia spp. infection in teenagers and adults is disproved by [START_REF] Degirmenci | Distribution of intestinal parasites detected in the parasitology laboratory of the Ege University Medical School Hospital, in 2005[END_REF] and Yamon et al. (2008) in studies conducted in Turkey, which reported a much lower percent (namely 1.78% and 1.96%). In the United States, [START_REF] Painter | Giardiasis surveillance -United States, 2011-2012[END_REF] observed the highest incidence in children aged 1 to 4 years old (16.4%), followed by children aged 5 to 9 years old (8.4%) and a much lower percent in adults. Yoder et al. (2010), in a study conducted over a period of 3 years, observed the highest number of cases of infection with G. duodenalis in children aged 1 to 4 years old. Asher in a study conducted on a native community in Australia, observed a much higher percent of the infection than the ones reported in our study (26% in children aged 0 to 5 years old and 30% in children aged 5 to 10 years old), [START_REF] Asher | Distribution of Giardia duodenalis assemblages A and B among children living in a remote indigenous community of the Northern Territory, Australia[END_REF]. A study conducted by Mbae et al., in Kenya on children with diarrhea, showed an incidence of 1.30% in infants under 1 year of age and a high incidence of infection in children aged 1 to 5 years old, namely 4.00%, [START_REF] Mbae | Intestinal parasitic infections in children presenting with diarrhoea in outpatient and inpatient settings in an informal settlement of Nairobi, Kenya[END_REF].

Various studies conducted on children recorded a number of more than 10% of infection with G. duodenalis. Giraldo-Gomez et al. in a study conducted on preschool children from Armenia, Columbia, observed a prevalence of infection with G. duodenalis of 13%. G. duodenalis is the most common parasite, [START_REF] Giraldo-Gomez | Prevalence of giardiasis and intestinal parasites in pre-school children from homes being attended as part of a state programme in Armenia, Columbia[END_REF]. Lindo et al.

in a study conducted on children under the age of 12 in a town from Guyana observed an incidence of infection with G. duodenalis of 10.5%, [START_REF] Lindo | Intestinal parasite among young children in the interior of Guyana[END_REF]. [START_REF] Astal | The investigation of intestinal parasites in two primary school in different social-economic districts of the city of Yozgat, Turkey[END_REF] observed a prevalence of 15.5% of infection with G. intestinalis in children from two schools in Turkey. [START_REF] Ostan | Health inequities: lower socio-economic conditions and higher incidences of intestinal parasites[END_REF] in a study conducted in Turkey, observed an incidence of infection of 13.7%, and [START_REF] Quadros | Intestinal parasites in nursery school of Lages, southern Brazil[END_REF] in a study conducted in Brazil, observed an incidence of infection with G. duodenalis of 14%. Other studies have showed an incidence of infection with G. duodenalis of less than 10% [START_REF] Astal | The investigation of intestinal parasites in two primary school in different social-economic districts of the city of Yozgat, Turkey[END_REF][START_REF] Celik | Incidence of intestinal parasites among primary school children in Malatya[END_REF][START_REF] Aksoy | Demographic status and prevalence of intestinal parasitic infections in schoolchildren in Izmir, Turkey[END_REF][START_REF] Worku | Malnutrition and intestinal parasitic infections in school children of Gondar, North West Ethiopia[END_REF][START_REF] Tamer | The frequency of the presence of intestinal parasites in students of Arslanbey Primary School[END_REF][START_REF] Hamamci | Investigation of intestinal parasites among primary school students in Kayseri-Hacilar[END_REF].

Regarding the clinical significance of the infection with Giardia spp. in the subjects that have participated in the study, we have tried to describe the association of the clinical signs with the presence of the parasitic infection, with the mention that some of the subjects, beside the parasitic infection, also suffered from other diseases that could interfere with the clinical manifestations of the Giardia infection. It is well known that the infection with G. duodenalis can progress with many acute or chronic clinical manifestations, or it can be asymptomatic (Pierce and Kirkpatric, 2009). Our priority was not to establish the exact clinical symptoms exhibited by the patients who have an infection with Giardia spp.

Regardless of the presence of another disease associated with the Giardia spp. infection, we observed a high incidence of abdominal pain (69.07%), lack of appetite (71.24%), sensitivity of the abdomen (54.69%), especially in the right hypochondrium, and hepatomegaly (47.17%), and a lower incidence of clinical symptoms such as diarrhea (18.48%), nausea (42.52%), vomiting (25.60%) and erythematous skin rash (11.35%). It has also been observed that the clinical manifestations associated with the parasitic infection have a higher frequency in adults and teenagers than in infants and small children, as [START_REF] Mellingen | Post epidemic giardiasis and gastrointestinal symptoms among preschool children in Bergen, Norway. A cross-sectional study[END_REF] also observed in a study conducted in Norway on preschool children, in which he states that the clinical manifestations of the children infected with G. duodenalis are limited. The lack of appetite has been observed in all the studied age groups, but more frequently found in adults and having a lower incidence in small children, where the basic manifestation was diarrhea.

This disproves the studies conducted by Ignatius on 583 children under the age of five in Rwanda [START_REF] Gahutu | High prevalence of Giardia duodenalis assemblage B infection and association with underweight in Rwandan children[END_REF]. In our study, the abdominal pain was frequently reported in both children and adults infected with Giardia spp., a fact observed also in the studies conducted by Younas in Pakistan, on 239 children with recurrent abdominal pain (Younas et al., 2008). The same thing has been noticed in Escobedo's retrospective study on hospitalized children in a paediatric hospital in Havana [START_REF] Escobedo | Hospitalization of Cuban children for giardiasis: a retrospective study in a paediatric hospital in Havana[END_REF]. Diarrhea or constipation have been observed in a small percentage of the subjects that have participated in the study (18.48% and 8.89%), which suggests that giardiasis can evolve, in certain cases, without changes in the intestinal transit. In the previous studies, diarrhea is the most common clinical manifestation that was observed in people infected with G. duodenalis [START_REF] Guamri | Epidemiological retrospective survey intestinal parasitism in the Provincial Hospital Center (Kenitra, Morocco): review of 10 years (1996-2005[END_REF][START_REF] Escobedo | Hospitalization of Cuban children for giardiasis: a retrospective study in a paediatric hospital in Havana[END_REF] data consistent with our data. Among the rarest clinical manifestations of the infection with G. duodenales, there are also the allergic manifestations, previous studies showing the possibility of associating the parasitic infection with the occurrence of allergic manifestations [START_REF] Escobedo | Hospitalization of Cuban children for giardiasis: a retrospective study in a paediatric hospital in Havana[END_REF][START_REF] Nenoff | Giardia lamblia -cause of urticaria and pruritus or accidental association?[END_REF]. A possible association has also been observed in our study, as 11.35% of the infected subjects presented erythematous skin rash, with the mention that we established no other cause for the skin rash.

The decrease in the prevalence of the infection with Giardia spp. observed in our study can be explained by improved hygienic dwelling conditions, both in urban and rural areas, associated with better personal hygiene. The persistence of the parasite is explained by its natural resistance to the processing of drinking water, precarious hygienic conditions in some dwellings, cohabitation with farm animals, contact with pets or street dogs.

The advanced technology and modern methods used for detecting and monitoring pathogens from different waters, obviously, ensure a better monitoring of them. The cysts from Giardia were detected from numerous environmental matrices (hydrographic basins that provide source of drinking water, the waters used for recreational activities, soil, sewage, sludge, etc.), independent of the economic income of the area in which these types of water are located. Water from the environment, regardless of its origin and use, is extremely vulnerable to contamination with Giardia, due to leakage to the ground and the uncertain wastewater disposal module, as varied as the source (industrial, agricultural, zootechnical, household, etc.). At this dispersion, major and frequent infestation, and transmission, a major role is played by faeces excreted by a large number of hosts (humans, animals), but also by the increased ability of cysts to survive in the environment, under extremely precarious conditions. The legislation and policies of many countries ensure compliance with the parameters for the quality of the source water and the protection of the environment; however, Giardia persists in being a major cause of the formation of outbreaks of pathologies transmitted through water. As an real evidence, correct knowledge and according to the reality of the way of contamination of the environment by cysts, the data on the occurrence, persistence, their survival, can optimize the understanding of the epidemiology and contribute major to the establishment of priorities, respectively of preventive measures to ensure good public health.

Conclusion

Giardia spp. is one of the most common aquatic parasites that infect humans and animals alike. Data collected between 2004 and 2017 show an increased incidence of infection with Giardia spp. in both children and adults hospitalized in the Department of Infectious Disease, in the West of Romania. Diarrhea, epigastric cramps, flatulence, nausea, vomiting and weight loss are the main symptoms of human acute giardiasis. To take action in reducing the cases of giardiasis, it is necessary to evaluate the exact factors that influence or cause the disease, followed by the identification or implementation of methods to reduce the effects of the identified factors. In this regard, intervention programs need to be developed based on systematic studies, programs and politics which must be implemented through an interdepartmental and interinstitutional synergic cooperation. MMWR Surveillance Summaries, 59, 15-25. Younas M., Shah S., Talaat A., 2008. Frequency of Giardia lamblia infection in children with recurrent abdominal pain. J Pakistan Med Assoc, 58, 171-174.

List of Figures

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Prevalence of Giardia spp. infection according to year

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 45678 Figure 4. Prevalence of Giardia spp. infection according to year and gender

  Figure 1

Figure 5 Figure 6

 56 Figure 5

Figure 7

 7 Figure 7

Figure 8

 8 Figure 8

Acknowledgements: Authors also wish to express their gratitude to Wei Huang, Associate Editor of STOTEN, and to the unknown Reviewers who helped in improving the quality of this paper.

Author contributions: All authors contributed equally to this paper.

Funding: This research received no external funding.

Conflicts of Interest:

The authors declare no conflict of interest.