Detection by microsatellite analysis of early embryonic mortality in an alligator population in Florida

In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.     

Altered histology of the thymus and spleen in contaminant-exposed juvenile American alligators

Morphological differences in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Differences in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P = 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P = 0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P = 0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals; Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P = 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P = 0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.     

Altered neonatal development and endocrine function in Alligator mississippiensis associated with a contaminated environment

Reduced reproductive success, altered reproductive tract development, and differences in circulating hormones have been documented in American alligators (Alligator mississippiensis) from Lake Apopka, FL, compared to less contaminated sites, such as the Lake Woodruff National Wildlife Refuge, FL. Comparative studies among alligators of varying size and age suggest that in ovo contaminant-induced alterations of endocrine function are further modified during postembryonic development and/or through environmental exposure. In the present study, we examined developmental and endocrine-related indices in neonatal (age, <1 mo) alligators from Lake Apopka in comparison to those of a reference population (Lake Woodruff), thereby limiting contaminant exposure to that derived via maternal contribution. We compared several reproductive and developmental parameters, including hatching success, primary sex determination, and somatic indices. Furthermore, we examined circulating testosterone concentrations and aromatase activity in an effort to establish relative gonadal endocrine function shortly after hatching. Finally, we compared phallus size among males and oviduct epithelial cell height (ECH) among females (androgen- and estrogen-dependent tissues, respectively). Significant differences between populations were noted for body size and spleen somatic index. Neonatal alligators from Lake Apopka exhibited higher plasma testosterone, but no differences were detected in gonadal aromatase activity compared to Lake Woodruff. Phallus tip length and cuff diameter were smaller in males from Lake Apopka, whereas no differences were noted in oviduct ECH. Our data establish basic indices of development and endocrine function in neonatal alligators before environmental exposure to contaminants. These results should begin to help separate developmental abnormalities resulting from in ovo exposure, presumably of maternal origin, from physiological alterations induced through environmental exposure to contaminants.     

Increased posthatching mortality and loss of sexually dimorphic gene expression in alligators (Alligator mississippiensis) from a contaminated environment

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.     

Plasma steroid concentrations in relation to size and age in juvenile alligators from two Florida lakes

Previous studies have reported a number of physiological differences among juvenile alligators from two well-studied populations (Lake Apopka and Lake Woodruff) in north central Florida. These studies obtained alligators of similar size from each lake under the assumption that the animals were of similar age. Lake Apopka is a hypertrophic lake with a 50-year history of contamination from agricultural and municipal operations, whereas Lake Woodruff is a eutrophic lake and part of a National Wildlife Refuge that receives little point source pollution. If growth rates differ among these areas, it could be argued that differences in endocrine parameters reported previously (e.g. steroid or thyroid hormone concentrations) could be the result of differences in the animals' ages. Using growth annuli in cross-sections of femurs, we estimated the ages of juvenile alligators and compared the relationship of estradiol-17beta (E(2)) and testosterone (T) to size and age within each lake and sex. No differences were detected in the relationship between size and age between the two areas indicating similar growth rates between lakes. Plasma E(2) was positively related to size in females from Lake Apopka, and age in Woodruff females. Males from Lake Apopka had elevated plasma E(2) compared with Lake Woodruff males and did not differ from Woodruff females. No significant relationships were detected for T from either lake, and no differences in plasma T were detected among lakes or sexes. Our data indicate that both size and age can have a significant relationship with steroid concentrations. However, the relationship between steroid concentrations and size or age differed between lakes. We suggest both factors should be considered when conducting physiological studies where there is evidence to suggest growth rates may differ among populations.     

Seasonal variation in plasma thyroxine concentrations in juvenile alligators (Alligators mississippiensis) from three Florida Lakes

Circulating concentrations of thyroxine (T(4)) vary seasonally in many vertebrates. This study examined the seasonal variation in plasma concentrations of T(4) in juvenile American alligators (Alligator mississippiensis) from three populations in central Florida, USA. One site, Lake Woodruff National Wildlife Refuge, is considered a reference site whereas the other two lakes, Lake Apopka and Orange Lake, are significantly impacted by human activity. Juvenile American alligators ranging from 75-150 cm in total length were hand-captured at night from November 2000-April 2002. Plasma thyroxine concentrations were analyzed using a radioimmunoassay (RIA) previously validated for alligator plasma. Juvenile American alligators display seasonal variation in circulating T(4) concentrations. Plasma T(4) concentrations decrease from August/September to November and then begin a slow rise until April, at which point they plateau. Sex of juveniles influenced plasma concentrations of T(4) in some months but did not appear to alter the pattern in seasonal variation. The pattern we observed in plasma T(4) concentrations is not directly related to an environmental factor such as ambient temperature but is similar to that seen in plasma sex steroid concentrations during the reproductive cycle of adult alligators. Although the pattern and plasma concentration of T(4) exhibits significant variation among the three lakes studied, the pattern in seasonal variation appears similar. Comparing the seasonal pattern in plasma T(4) with plasma concentrations of sex steroids (testosterone and estradiol-17beta) or corticosterone could provide important information on the peripubescent life stage of the American alligator.     

Identification of the critical timing of sex determination in <Emphasis Type="Italic">Daphnia magna</Emphasis> (Crustacea,Branchiopoda) for use in toxicological studies

Daphnia are often used as test organisms to determine toxicity of chemicals found in the environment. Fecundity and mortality are the classic endpoints in such tests; however, there is increased interest in other sub-lethal endpoints. Most Daphnia reproduce by cyclic parthenogenesis, typically reproducing asexually to produce genetically identical females. Environmental cues trigger the production of males allowing for sexual reproduction. This ability to switch reproductive strategies has been increasingly used as a bioassay endpoint in screens for sub-lethal effects of contaminants. In order to optimize such test, it is necessary to know when sex is determined during Daphnia development. A previous study using the insect juvenile hormone methyl farnesoate has shown that sex determination occurred during the egg maturation period in Daphnia. Our study exposed female Daphnia magna carrying eggs and embryos at different stages of maturation, to the insecticide toxaphene to determine if the timing of sex determination is similarly influenced by a pesticide. Our results suggest that in response to toxaphene exposure sex is determined in the immature oocyte, before the final maturation cleavage, and before the developing egg is extruded into the brood chamber. Thus, sex determination is transgenerational, being determined while the egg is still immature and in the mother’s ovary.     

Gonadal development and differentiation in Alligator mississippiensis at male and female producing incubation temperatures

The development and differentiation of the gonads of embryonic alligators incubated at 30 °C (100% female producing) and 33 °C (100% male producing) was investigated histologically. The stage of development of the gonad and differentiation into an ovary or a testis occurred at essentially the same time at both temperatures. This contrasts with the overall development of the embryos which was slower at the lower temperature. A few days prior to differentiation, gonads grew more quickly at 33 °C than they did at 30 °C. However, once differentiated into a presumptive testis, gonads reduced in volume so that at hatching presumptive testes were smaller than presumptive ovaries. It is hypothesized that synchrony/asynchrony of development of the gonad and the rest of the embryo may account for temperature-dependent sex determination.     

Egg environments have large effects on embryonic development,but have minimal consequences for hatchling phenotypes in an invasive lizard

Plastic responses of embryos to developmental environments can shape phenotypes in ways that impact fitness. The mechanisms by which developmental conditions affect offspring phenotypes vary substantially among taxa and are poorly understood in most systems. In this study, we evaluate the effects of thermal and hydric conditions on patterns of egg water uptake, embryonic development and yolk metabolism in embryos of the lizard Anolis sagrei to gain insights into how these factors shape morphological variation in hatchlings. Our 3 × 2 experimental design (3 thermal and 2 hydric conditions) revealed that developmental temperature has strong effects on rates of development and yolk metabolism, but the impacts of moisture were minimal. Increased water uptake by eggs under relatively wet conditions resulted in larger hatchlings with less internalized residual yolk than hatchlings from dry‐incubated eggs. However, the relatively small phenotypic differences among treatments may have small fitness consequences. These results demonstrate that embryos of A. sagrei can tolerate a broad range of environmental conditions without substantial impacts on critical morphological traits. Such embryonic tolerances may facilitate colonization and establishment in novel environments. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 25–41.     

The Red-Eared Slider Turtle: An Animal Model for the Study of Low Doses and Mixtures

Current risk assessment techniques for almost all chemicalsinvolve establishing a threshold dose, or the dose below whichno adverse effects are seen. But threshold doses may not applyin situations where the chemical mimics the actions of an endogenouscompound—such as a steroid hormone—that is importantto development. In such cases, any exposure may exceed the threshold.Recent studies with the red-eared slider turtle have shown thatexogenous estradiol—even when applied to eggshell in dosesas low 0.4 ng—will affect sex development during embryogenesis.Considering that only 0.2% of the estradiol applied to the eggshellends up in the embryo, it becomes apparent that even very lowdosages of steroid hormones or their mimics can have profoundbiological effects. We tested this idea using eight compoundsidentified in the yolk of alligator eggs from Lake Apopka, Fla.Five of the compounds—the PCB mixture Aroclor 1242,trans-Nonachlor,cis-Nonachlor,p,p'-DDE, and chlordane—altered sex ratio outcomes whenapplied to eggshells during development. Aroclor 1242 producedthe most powerful effects, shifting the ratio of females almosttwofold, while chlordane had the greatest effect when combinedwith estradiol. Administration of all eight compounds togetheralso increased the ratio of females to males. However, comparingthe single-compound exposures at the same dosages indicate thatthese compounds behave differently in combination than theydo singly, emphasizing the need for further studies using chemicalmixtures reflecting proportions found in nature. The effectof chlordane and Aroclor 1242 on aromatase activity levels duringembryogenesis in the brain and adrenal-kidney-gonad (AKG) complexwas also examined. Chlordane, a suspected anti-androgen in thisspecies, did not affect aromatase activity in either the brainor the AKG. However, Aroclor 1242 significantly altered aromataseactivity levels in the red-eared slider turtle brain—butnot in the AKG—during a crucial developmental period.After this crucial period, Aroclor 1242 caused an increase inaromatase activity in the AKG of embryos just prior to hatch.Additionally, hatchling males treated during embryogenesis withAroclor 1242 and chlordane exhibited significantly lower testosteronelevels than controls in response to follicle-stimulating hormoneadministration, while chlordane-treated females had significantlylower progesterone, testosterone, and 5-dihydrotestosteronelevels relative to controls. These results are similar to thosefound in juvenile alligators from Lake Apopka. Males treatedwith Aroclor 1242 and trans-Nonachlor displayed an elevatedestradiol response to FSH administration vs. control males.Taken together, these results suggest that EDCs exert effectsduring embryonic development that extend beyond birth. Theyalso suggest that the alterations in sex steroid hormone levelsobserved in animals from contaminated areas may result fromEDC-induced alterations in the neuroendocrine axis controllinggonadal sex steroid hormone production.     

‘Endocrine Disruption’ and the ‘Wildlife Connection’

A ‘wildlife connection’ has been proposed to link several human sexual-endocrine abnormalities with synthetic chemicals in the environment. A review of several frequently cited examples of ‘endocrine disruption’ in wildlife indicates, however, that any connection is remote at best. The reported loss of sexuality by bald eagles is attributed to misinterpretations of the wording in a popular article; no supporting data can be found. The existence of feminized male western gulls remains hypothetical; an alternative and plausible explanation for a skewed sex ratio and female-female pairing in this species is supported by field data. Dioxin-related effects in wildlife complement an extensive human data base but provide no obvious insight to the causes of the sexual-endocrine dysfunctions. Sexual abnormalities of the Lake Apopka alligators are attributed to leakage of the nematicide DBCP from a manufacturing facility; DBCP is no longer used because of its capacity to reduce human sperm counts to zero. While concern about effects of low-level exposure of people to synthetic chemicals that exert biological activity continues to be justified, human health risk assessment protocols might incorporate both an extra amount of skepticism and an enhanced respect for scientific rigor.     

Effects of Egg and Circulating Testosterone on Ring‐Necked Pheasant (Phasianus colchicus) Male Traits and Combat Outcome

Studies of avian species have shown that maternal effects mediated by the transfer of egg hormones can profoundly affect offspring phenotype and fitness. We previously demonstrated that the injection of a physiological amount of testosterone (T) in the eggs of ring‐necked pheasants (Phasianus colchicus) disrupted the covariation among male morphological traits at sexual maturity and positively affected male mating success. Here, we investigate whether egg T exposure affected adult male circulating T levels at the onset of the breeding season (reflecting gonadal maturation), and the relationship between circulating T and male traits. Egg T exposure did not affect pre‐mating plasma T. T levels were not associated with the expression of secondary sexual and non‐sexual traits or socio‐sexual behaviour (social rank, overall fighting ability and mating success). However, wattle brightness decreased with increasing circulating T in males hatched from T‐eggs (T‐males) but not among control males. In dyadic encounters during the peak mating period, control males with higher pre‐mating T levels had higher chances of being dominant over other control males. However, higher pre‐mating T levels did not predict success in male‐male competition in encounters involving T‐males. We suggest that the long‐term effects of egg T on male phenotype do not originate from differential gonadal maturation according to egg T treatment. Rather, prenatal androgens may have priming effects on functioning of target tissues, translating into differential phenotypic effects according to androgen exposure during embryonic development.     

Clinical and necropsy findings associated with increased mortality among American alligators of Lake Griffin,Florida

From December, 1997, through November, 2000, 306 deaths were documented among adult and subadult American alligators (Alligator mississippiensis) of Lake Griffin, Florida (USA). Some live alligators were lethargic and unresponsive to approach. To determine the cause, we examined ten alligators captured from Lake Griffin between December 1997 and June 1999. Initially, four alligators, three of which were clinically unresponsive, were sacrificed for routine diagnostic necropsy. The other six Lake Griffin alligators, and five control alligators captured from Lake Woodruff National Wildlife Refuge, Florida, where mortality was negligible, were studied extensively by clinical neurologic examination, electromyography, hematology, serum chemical analyses, and blood culture, then sacrificed and necropsied. Samples of brain, spinal cord, peripheral nerves, skeletal muscle, and major internal organs were examined by light microscopy for abnormalities. Samples of nervous tissue also were examined by electron microscopy, and samples of various tissues were collected for toxicologic analyses. Clinical signs included swimming in circles, inability to submerge, lethargy, weakness, unresponsiveness, slow reflexes, dragging the dorsal surfaces of the hind feet, head tilt, and anisocoria. Lake Griffin alligators had significantly lower distal sciatic nerve conduction velocities than Lake Woodruff alligators, and the most severely affected alligators had the lowest velocities; but morphologic abnormalities in peripheral nerves were not evident in most cases. Three severely affected alligators had acute focal necrosis of the torus semicircularis in the midbrain, two had skeletal myofiber atrophy, another had diffuse nonsuppurative encephalomyelitis, and one mildly affected alligator had skeletal myodegeneration. The cause or causes have not yet been identified.     

Effects of electromagnetic fields on the reproductive success of American kestrels

Reduced reproductive success of birds nesting near power lines has been documented but never directly attributed to electromagnetic fields (EMFs). Laboratory studies have identified EMF effects on embryonic development, but reproductive success of wild birds is dependent on additional factors, including fertility, egg size, hatching, and fledging success. We tested whether EMFs affect reproductive success of birds. Captive American kestrels (Falco sparverius) were bred for one season per year for 2 yr under either controlled or EMF conditions. EMF exposure was equivalent to that experienced by wild reproducing kestrels and was weakly associated with reduced egg laying in 1 yr only. In both years fertility was higher, but hatching success was lower in EMF pairs than control pairs. Fledging success was higher in EMF pairs than control pairs in 1995 only. Egg composition and embryonic development were examined in 1 yr only, but hatchlings were measured in both years. EMF eggs were larger, with more yolk, albumen, and water, but had thinner egg shells than control eggs. Late-term EMF embryos were larger and longer than control embryos, although hatchlings were similar in body mass and size. EMF exposure affected reproductive success of kestrels, increasing fertility, egg size, embryonic development, and fledging success but reducing hatching success.     

Organochlorine pesticides and thiamine in eggs of largemouth bass and American alligators and their relationship with early life-stage mortality

Thiamine deficiency has been linked to early mortality syndrome in salmonids in the Great Lakes. This study was conducted to compare thiamine concentrations in American alligators (Alligator mississippiensis) and Florida largemouth bass (Micropterus salmoides floridanus) eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Lakes Apopka and Griffin, and Emeralda Marsh, Florida, USA) to those from sites that have historically exhibited low embryo mortality and low OCPs (Lakes Woodruff and Orange, Florida). During June-July 2000, 20 alligator clutches were collected from these sites, artificially incubated, and monitored for embryo mortality. Thiamine and OCPs were measured in one egg/clutch. During February 2002, 10 adult female bass were collected from Emeralda Marsh and Lake Woodruff and mature ovaries analyzed for thiamine and OCP concentrations. Although ovaries from the Emeralda Marsh bass contained almost 1,000-fold more OCPs compared with the reference site, Lake Woodruff, there were no differences in thiamine concentrations between sites (11,710 vs. 11,857 pmol/g). In contrast, alligator eggs from the reference site had five times the amount of thiamine compared with the contaminated sites (3,123 vs. 617 pmol/g). Similarly, clutches with >55% hatch rates had significantly higher concentrations of thiamine compared with clutches with <54% hatch rates (1,119 vs. 201 pmol/g). These results suggest that thiamine deficiency might be playing an important role in alligator embryo survival but not in reproductive failure and recruitment of largemouth bass. The cause(s) of this thiamine deficiency are unknown but might be related to differences in the nutritional value of prey items across the sites studied and/or to the presence of high concentration of contaminants in eggs.     

Pathology, physiologic parameters, tissue contaminants, and tissue thiamine in morbid and healthy central Florida adult American alligators (Alligator mississippiensis)

An investigation of adult alligator (Alligator mississippiensis) mortalities in Lake Griffin, central Florida, was conducted from 1998-2004. Alligator mortality was highest in the months of April and May and annual death count peaked in 2000. Bacterial pathogens, heavy metals, and pesticides were not linked with the mortalities. Blood chemistry did not point to any clinical diagnosis, although differences between impaired and normal animals were noted. Captured alligators with signs of neurologic impairment displayed unresponsive and uncoordinated behavior. Three of 21 impaired Lake Griffin alligators were found to have neural lesions characteristic of thiamine deficiency in the telencephalon, particularly the dorsal ventricular ridge. In some cases, lesions were found in the thalamus, and parts of the midbrain. Liver and muscle tissue concentrations of thiamine (vitamin B(1)) were lowest in impaired Lake Griffin alligators when compared to unimpaired alligators or to alligators from Lake Woodruff. The consumption of thiaminase-positive gizzard shad (Dorosoma cepedianum) is thought to have been the cause of the low tissue thiamine and resulting mortalities.     

The Maestro (Mro) Gene Is Dispensable for Normal Sexual Development and Fertility in Mice

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.     

Bicephality,a seldom occurring developmental deformity in Tegenaria atrica caused by alternating temperatures

The experiment was aimed at demonstrating the relationship between deformities of the front part of the prosoma accompanied by changes in the brain structure in bicephalous Tegenaria atrica and exposure of their embryos to temperature fluctuations. By exposing spider embryos to alternating temperatures of 14 and 32 °C for the first 10 days of embryonic development, we obtained eight two-headed individuals, subsequently divided into three groups according to morphological differences. We described in detail morphological abnormalities of the prosoma identified in members of each group. Histological examination confirmed a close relationship between morphological deformities and the brain structure of teratogenically changed spiders. The fusion of appendages (pedipalps and chalicerae) was accompanied by the fusion of corresponding ganglia. The absence of appendages (pedipalps) was accompanied by the absence of corresponding ganglia. This correlation may have resulted from previously impaired neuromere development which led to changes in the morphological structure of the prosoma. Since no deformities were identified in control animals, it can be concluded that bicephaly was caused by exposing embryos to alternating temperatures.     

Effects of elevated yolk androgens on perinatal begging behavior in yellow-legged gull (Larus michahellis) chicks

Maternal hormones may represent an important pathway by which mothers can adaptively adjust offspring traits and performance to suit the prevailing environmental conditions. Earlier studies of birds have shown that egg androgens of maternal origin may enhance post-natal offspring 'begging' displays, functioning to solicit parental care. Here we investigate the effects of elevated egg androgen levels on the prenatal begging behavior of yellow-legged gull (Larus michahellis) chicks. At laying, we experimentally increased the concentration of yolk testosterone (T) within the natural range of variation, and, shortly before hatching, we compared the structural properties, rate, and loudness of vocalizations of embryos developing in T- and oil-injected (control) eggs. In addition, we compared the early post-hatch begging rate (measured as the pecking rate towards a dummy gull head) in chicks of the two experimental groups. We found that T embryos produced louder embryonic vocalizations than controls, whereas structural properties and the calling rate did not differ between T and control embryos. The post-hatch begging rate was unaffected by T treatment, but strongly decreased with increasing chick body mass, suggesting that intensity of the begging display was sensitive to chick state and may therefore reliably indicate the need of food in this species. Therefore, the results of this study show for the first time that prenatal T exposure modulates the quality of embryonic vocalizations, but are not in accordance with previous findings reporting increased post-hatching begging intensity following increased prenatal exposure to androgens.