Unhatched Eggs Research Articles

Expression interference of a number of Heterodera avenae conserved genes perturbs nematode parasitic success in Triticum aestivum

The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24–72.4 % reduction in H. avenae multiplication in T1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant.

Organochlorine pesticides and polychlorinated biphenyls in common kestrel eggs from the Canary Islands: Spatiotemporal variations and effects on eggshell and reproduction

Organochlorine (OC) pesticides were widely used on the Canary Islands (Spain) for intensive crop production and against plagues of African locust. A previous study performed in 1988–1994 showed a high concentration of p,p’-DDE in the eggs of common kestrels (Falco tinnunculus) from the island of Tenerife. The present study shows OC pesticide and polychlorinated biphenyl (PCBs) levels in 40 unhatched common kestrel eggs collected from southern Tenerife between 2009 and 2016. The protoporphyrin IX in eggshells has also been analysed in order to explore the use of this pigment as a biomarker. Egg biometry, status of embryo development, eggshell thickness and mass of extractable lipids of each egg were recorded. Surrounding land use and reproductive parameters (hatching and fledging rates) were obtained for each nest. The most abundant compound was p,p’-DDE (15.0 μg/g d.w), followed by PCBs (0.46 μg/g d.w.). The decline in p,p’-DDE levels in southern Tenerife (with 23.6 μg/g d.w. in 1988–1994) was 36.4%. p,p’-DDE levels were positively associated with the surface of active and abandoned cropland in a 200 m-radius around the nest and with proximity to urban areas. PCB levels were associated with proximity to roads. Shell thickness was negatively affected by the p,p’-DDE concentration. The concentration of protoporphyrin IX in the eggshell was negatively associated with the concentration of hexachlorobenzene in the egg content. Despite the total ban on the use of p,p’-DDT in Spain since 1986, p,p’-DDE levels remain elevated in those areas in which that use was formerly intensive.

Variation in quality of newly hatched chicks from Japanese breeder quail fed guava extract

The aim of this study was to evaluate the effects of guava extract as a dietary supplement for Japanese quail hens on incubation parameters, intestinal development, and the quality of newly hatched chicks. Six hundred and forty-eight eggs were collected from Japanese quail hens fed diets with 0.0, 3.0, 6.0, and 9.0 g/kg of guava extract. The study consisted of four treatments, six replicates, and 27 eggs per replicate. The guava extract contained 2% ellagic acid. The eggs were incubated at 37.5 °C and 65% relative air humidity. The hatch window, hatchability, embryo development in unhatched eggs, quality of newly hatched quails, and intestinal histomorphometry of a duodenum segment were evaluated. Differences among the treatments in hatchability and the hatch window were not significant. The guava extract did not affect mortality rate, live pecking, dead pecking, and contaminated and inverted eggs. The number of infertile eggs was lower in the groups that received 3.0 and 9.0 g/kg of extract in their diets. Use of up to 3.0 g/kg of guava extract in the diet increased the weight of both eggs and newly hatched chicks. The quality score of newly hatched quail was similar across treatments. The inclusion of 3.0 g/kg of guava extract in the diet of Japanese quail breeder hens increased the hatchling weight of the chicks and improved the villus to crypt ratio of their duodenum.
 Keywords: embryology, embryo metabolism, hatchery, incubation, breeder nutrition

A Mathematical Modeling Method Elucidating the Integrated Gripping Performance of Ant Mandibles and Bio-inspired Grippers

The ability to grip unhatched eggs is a skill exploited by the ants Harpegnathos venator, as they care their brood in tunneled nests, which is of extreme difficulty to keep the eggs intact while gripping. In this paper we propose a mathematical modeling method to elucidate the mechanism of such a gripping behavior in the ant mandibles. The new method can be subdivided into following steps. As a preliminary, the concavity geometry and mandible kinematics are examined experimentally. Second, coordinate transformation is used to predict the real-time spatial topology of the concavity. Third, we come up with a new method to quantify the workspace required to grip and the contact area between the concavity and ant egg. Our model indicates that the biaxial rotation fashion with specialized concavities can reduce workspace by 40% and increase contact area by 53% on average compared with the uniaxial rotation pattern, which augments success rate of gentle gripping. This methodology may have applications in evaluating mechanical performance in both natural and artificial grippers.

Ecological characteristics of Thaumetopoea pityocampa Denis & Schiffermüller, 1775 (Lepidoptera: Notodontidae) in egg stage in Thasos Island, Greece

In September 2017, 96 egg batches of Thaumetopoea pityocampa were collected from Aleppo pine (Pinus halepensis) trees at four sites of Thasos Island in Greece. In the different localities, the average number of eggs in batches varied from 196.5 to 223.9 (212.4 for the Thasos Island). The length of P. halepensis needles with egg batches differed in size - between 84 to 210 mm. Approximately 75% of batches were laid close to the middle of needles, between 121 and 180 mm. The egg masses were formed mainly on two needles (84.4%) and the rest - on one, three or four needles (13.6%) or on fine shoots (2%). In most cases (88.3%), the female moths started to lay eggs from the tip of the needle. The distance from the base of the needle to the beginning of the egg batches was established between 0 and 180 mm, as most of them (62.7%) were clustered around the average value (87.3 mm) in the diapason of 60-120 mm. The average rate of T. pityocampa survival in egg stage was 48%. The parasitized eggs were 43.8%, and the rest included unhatched eggs, predominately undeveloped eggs with dried-up yolk. No correlation was found between the percentage of parasitized eggs and the distance of the egg batches from the base of needles.

Spatio-temporal variation in seed traits affects the occurrence and body-size pattern of a seed-feeding beetle (Chrysomelidae: Bruchinae) in Brazilian Cerrado

Seed quality is an important bottom-up factor driving life-history traits of seed beetles (Chrysomelidae: Bruchinae). However, information on how seed traits change spatiotemporally and affect herbivores' survival is incipient in Neotropics. We evaluated how the spatio-temporal variation in seed weight, hardness, size, and water content of Mimosa setosa var. paludosa (Fabaceae: Mimosoideae) affects the occurrence and body-size pattern of the seed beetle, Acanthoscelides quadridentatus (Schaeffer, 1907), in Brazilian Cerrado. We collected 400 fruits from four populations of M. setosa var. paludosa in Cerrado roadsides from Goiás and Minas Gerais states and then we assessed in laboratory seed traits along with the weight and body size of A. quadridentatus. Additionally, we collected a total of 76 fruits (immature plus mature) in beginning and end of the fruiting phase and counted the frequencies of unhatched and hatched eggs of the seed beetle to evaluate the effect of seed hardening on beetle posture. We found greater seed damage in the population with softer, smaller and lighter seeds. Larger and heavier seeds were harder than smaller and lighter seeds; and male and female reared from larger and heavier seeds were respectively 15 and 25% larger than that reared from smaller and lighter seeds. We also found that weight and body size of A. quadridentatus were positively correlated with seed length, weight and water content. Unhatched eggs were found only in beginning of fruiting phase when seeds were immature and softer, suggesting the occurrence of a window of time in which females lay their eggs before seed hardening to facilitate the entry of first instar larvae. These results highlight that larger and heavier seeds are most protected against herbivory, but individuals of A. quadridentatus that overcome plant hardness can grow larger in size. We suggest that seed traits change across a spatio-temporal scale and affect life-history traits of A. quadridentatus.

Assessing the evidence of ‘infertile’ sea turtle eggs

There is increasing concern about feminization of sea turtle populations resulting from female-biased production of hatchlings due to climate change and selective loss of males from other anthropogenic drivers. Extreme female-biased breeding populations would reduce the likelihood of successful mating and potentially result in high rates of infertile eggs. Infertile eggs are those in which none of the events between sperm penetration of the ovum and syngamy have occurred. Distinguishing between fertile and infertile eggs is challenging, especially in field conditions, and researchers often have relied on physical evidence gathered from unhatched eggs at the end of the incubation period, which likely have experienced tissue decomposition. We argue that infertility in sea turtle eggs can be demonstrated only by the absence of holes caused by sperm penetration of the inner perivitelline membrane; sperm bound between the inner and outer perivitelline membranes; nuclei in the blastodisc; embryonic tissue or membranes in egg contents; and/or the characteristic white spot on the egg exterior. Unhatched eggs can be examined at the end of the incubation period, but we recommend that studies specifically investigating infertility examine at least 20 oviposited eggs each from clutches laid by at least 20 different turtles at the peak of the nesting season.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Isolation and molecular-based identification of bacteria from unhatched leftover eggs of ducks in selected mini-hatcheries of Kishoreganj, Bangladesh.

Objectives:The study was designed for isolation and identification of the bacteria present in unhatched leftover eggs of duck in selected mini-hatcheries of Kishoreganj, Bangladesh.Materials and Methods:A total of 54 unhatched discarded eggs were collected as samples from different mini-hatcheries of Tarail and Itna Upazilas of Kishoreganj and aseptically carried to the laboratory in the icebox. Surface washings (n = 54) and inner contents (n = 54) were collected and enriched in Luria–Bertani broth followed by the isolation of pure colonies of different bacteria onto eosin methylene blue agar, mannitol salt agar, Salmonella–Shigella agar, and blood agar plates. Identification of the bacterial isolates was done by cultural properties, staining, and biochemical tests followed by molecular detection by Polymerase chain reaction.Results:Of 108 samples, 62 were found positive for Salmonella spp. (76%), 59 for E. coli (54%), 52 for Staphylococcus spp. (48%), and 5 for Clostridium spp. (9%). From the egg surface samples, Staphylococcus spp. were recovered in the highest (67%) followed by Salmonella spp. (59%), E. coli (56%), and Clostridium spp. (9%). From the inner contents of eggs, Salmonella spp. were recovered in the highest (56%), followed by E. coli (53%) and Staphylococcus spp. (30%).Conclusion:The isolated bacteria might be associated with the decreased hatchability and embryo mortality in the mini-hatcheries of duck.

Toxicity of the organophosphate insecticide sumithion to embryo and larvae of zebrafish

Sumithion, a synthetic organophosphate, is widely used as an agricultural insecticide and for control of tiger bug (Cicindela spp.) in larval rearing for aquaculture. An experiment was conducted to examine the effects of sumithion on embryological and larval development of zebrafish Danio rerio. Fertilized egg (n = 100) and larvae (n = 100) were exposed to six concentrations of sumithion (0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg L−1) in three replicates. LC50 values for embryos and larvae were calculated by probit analysis. The 24 h LC50 value of sumithion for embryo was 0.235 (0.079-0.428) mgL−1. Increasing sumithion concentrations decreased hatching success and increased embryonic mortality. In embryos, sumithion induced several malformations including immature yolk sac, dark yolk sac, yolk sac bud, broken eggshell and notochord, unhatched eggs. Larval LC50 values at 24, 48 and 72 h of various doses of sumithion exposure were 0.620 (0.436-0.963), 0.475 (0.302-0.801) and 0.341 (0.177-0.617) mgL−1, respectively. Various physical deformities, including edema, notochord deformity, yolk sac damage, body arcuation, lordosis and black pigmentation on the yolk sac were evident in response to different concentrations of sumithion. The results of the current study indicate that sumithion exerts developmental toxicity to zebrafish embryos and larvae. It is expected that current findings will increase sensitivity about the toxic effect of sumithion in early development, as well the possibility of similar actions induced by other insecticides and pesticides.

DETECTION OF SOME VIRULENCE GENES IN SALMONELLA SPECIES ISOLATED FROM DUCKS AND DUCK EGGS

Four hundred and fifty (450) samples were collected, 200 from internal organs (liver, caecum, gall bladder and spleen) of 50 ducks, 200 from internal organs of 50 ducklings and 50 unhatched duck eggs (dead in shell embryo) from different farms and hatcheries in Dakahlia Province. All samples were cultured on specific media for isolation and serological identification of Salmonellae spp. A total of 71 samples were positive to Salmonella with total percentage (15.8%). 29, 37 and 5 with (14.5%, 18.5% and 10%) positive samples were isolated from ducks, ducklings and duck eggs, respectively. Serotyping of the isolated Salmonellae from ducklings were five S.infantis, four S.kentucky, ten S.typhimurium, six S.newport, six S.enteritidis and six untypeable Salmonella with a percentage of (13.5%), (10.8%), (27 %), (16.2%), (16.2%) and (16.2%), respectively. Three S. derby, six S.typhimurium, five S.kentucky, three S. shubra, four S. enteritidis, three S.newport and five untypeable Salmonella were isolated from ducks with a percentage of (10.3%), (20.7%), (17.2%), (10.3%), (13.8%), (10.3%) and (17.2%), respectively. On the other hand only one of each S.typhimurium, S.enteritidis, S. newport, S. infantisand untypeable Salmonella were isolated from duck eggs with a percentage of (20%) for each one of these isolates. S.typhimuriumwas the most prevalence isolates and showed high resistance to streptomycin (100%), lincomycin (88%), erythromycin (82%), oxytetracycline (76%) and trimethoprim-sulfamethoxazole (70%). Moderate resistance to neomycin (59%), gentamycin (59%), amoxicillin (53%), ampicillin (53%) and chloramphenicol (47%) we reported. Meanwhile, the lowest resistances were against doxycycline (41%), flumequine (35%), norfloxacin (18%), ciprofloxxacin (6%) and colistin (6%). Conventional PCR assay detected virulence genes (invA, mgtC, sop B and bcfC) using specific primer for each gene in all examined S.typhimurium samples. This study highlights that, there was significant association between invA, mgtC, sop B and bcfC virulence genes and pathogenicity of S.typhimurium which showed high multiple antibiotic resistance which require strict regulations of the use of antibiotics in duck farms to minimize the resistant bacterial strains.

Unhatched eggs represent the invisible fraction in two wild bird populations.

Prenatal mortality is typically overlooked in population studies, which biases evolutionary inference by confounding selection and inheritance. Birds represent an opportunity to include this ‘invisible fraction’ if each egg contains a zygote, but whether hatching failure is caused by fertilization failure versus prenatal mortality is largely unknown. We quantified fertilization failure rates in two bird species that are popular systems for studying evolutionary dynamics and found that overwhelming majorities (99.9%) of laid eggs were fertilized. These systems thus present opportunities to eliminate the invisible fraction from life-history data.

Control strategies for a population dynamics model of Aedes aegypti with seasonal variability and their effects on dengue incidence

Aedes aegypti female mosquitoes are the principal transmitters of dengue and other vector-borne infections. This species is closely associated with human habitation, due to its blood-feeding habits and the presence of breeding sites widely available around households. In this paper, we introduce a mathematical model for the life cycle of Aedes aegypti mosquitoes comprising two stages, aerial and aquatic, that reflects seasonal changes in the mosquito abundance. This model is further amended by three season-dependent control actions. Two coercive actions are introduced during the hot seasons characterized by higher abundance and enhanced growth rates of mosquitoes. They consist in the application of two chemical substances, insecticide and larvicide, acting upon the aerial and aquatic mosquito stages, respectively. During the cool seasons, characterized by the slower growth rates of mosquitoes and abundance of quiescent unhatched eggs, we introduce a preventive vector control measure consisting in mechanical elimination of mosquito breeding sites. Using the framework of optimal control in combination with the cost-benefit approach and epidemiological assessment, we identify the most efficient strategy capable of essentially reducing the population of adult and immature mosquitoes during both seasons and provide a sketch for its modus operandi.

PSVII-21 Determination of apparent and standardized ileal digestibility of amino acids in various hatchery by-products for broiler chickens

Abstract The Korean poultry industry produces more than 4,000 tons of hatchery by-products (HBPs) every year. There is an increasing interest in the use of HBPs as a feed ingredient in The Korean feed industry. However, limited information regarding nutritional values for HBPs has been available. Thus, we conducted an experiment to measure apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids (AAs) in 4 different HBPs for broiler chickens. Four HBPs included infertile eggs (IFE), unhatched eggs (UHE), low grade and dead chicks (LDC), and mixture (MIX) containing 55% IFE, 10% UHE, 10% LDC, and 25% hatched eggshells. A total of three-hundred-thirty-six 21-d-old Ross 308 broiler chickens were randomly allotted to 1 of 4 dietary treatments with 7 replicates per treatment. Each diet contained individual HBP as a sole source of AAs. Additional 125 birds were used to estimate ileal endogenous losses of AAs. Results indicated that the AID and SID of 5 essential AAs (Arg, Met, Trp, His, and Val) for LDC were greatest (P < 0.05), but those (Arg, Met, and Trp) for MIX were the least (P < 0.05) among 4 HBPs. The AID and SID of most AAs in IFE were comparable to those in UHE. However, the AID and SID of 3 essential AAs (Lys, Leu, and Phe) were not different among 4 HBPs. The average AID and SID of both essential and nonessential AAs (18 AAs) for LDC were greatest (P < 0.05), but those for MIX were the least (P < 0.05) among 4 HBPs. In conclusion, HBPs used in this experiment contain high amounts of digestible AAs, which are comparable to other animal by-products. Generally, AAs in LDC are more digestible than AAs in other 3 HBPs (UHE, IFE, and MIX).

PSVII-28 Additivity of energy concentrations and phosphorus and calcium digestibility in hatchery byproducts fed to nursery pigs

Abstract The objective of this study was to determine the additivity of energy concentrations and phosphorus (P) and calcium (Ca) digestibility in hatchery byproducts. In our previous studies, digestible energy (DE), metabolizable energy (ME), and apparent total tract digestibility (ATTD) and standardized total tract digestibility (STTD) of P and Ca were determined using 10-kg barrows. Test ingredients were infertile eggs, unhatched eggs, culled chicks, and a mixture (20% infertile eggs, 20% unhatched eggs, and 60% culled chicks). It was hypothesized that determined nutritional values of the mixture is not different from predicted values calculated from nutritional values in each ingredient. A t-test was used to determine the additivity on nutritional values in the mixture. The predicted DE in the mixture was not different from the determined value (4,203 and 4,224 kcal/kg as-is; P = 0.890), and the predicted ME was not different from the determined value (3,990 and 3,998 kcal/kg as-is; P = 0.959). Predicted ATTD and STTD of P in the mixture (46.4 and 58.4%, respectively) were greater than determined values (35.5 and 47.4%, respectively; P < 0.05). However, predicted ATTD and STTD of Ca in the mixture (45.5 and 51.7%, respectively) were less than determined values (58.0 and 64.5%, respectively; P < 0.05). In conclusion, energy concentrations in a hatchery byproduct mixture could be predicted as a weighted mean of energy values in infertile eggs, unhatched eggs, and culled chicks, whereas phosphorus and calcium digestibility values in the mixture were not additive.

PSIV-14 Calcium digestibility in hatchery byproducts fed to nursery pigs

Abstract The objective was to determine standardized total tract digestibility (STTD) of calcium (Ca) in hatchery byproducts fed to nursery pigs. Ten barrows with mean body weight of 10.2 ± 0.7 kg were individually housed in metabolism crates. Four corn-based diets were formulated to contain 10% of infertile eggs (40% CP, 11.6% Ca, and 4,630 kcal/kg GE as-is), 10% of unhatched eggs (40% CP, 12.7% Ca, and 4,296 kcal/kg GE as-is), 25% of culled chicks (66% CP, 1.8% Ca, and 5,897 kcal/kg GE as-is), and 15% of a mixture of 3 ingredients aforementioned (56% CP, 6.2% Ca, and 5,342 kcal/kg GE as-is), respectively. A calcium-free diet was also prepared to measure basal endogenous losses of Ca. Ten animals were assigned to a replicated 5 × 4 Latin square design with 5 diets, and 4 periods. Each period consisted of a 5-d adaptation period and 5-d collection period. The marker to marker method was employed for total feces collection. The STTD of Ca in culled chicks (75.6%) was greater than that in the mixture (64.5%; P < 0.05). The ATTD and STTD of Ca in unhatched eggs (38.3 and 43.4%, respectively) were less than those in infertile eggs (48.5 and 53.3%, respectively; P < 0.05). Basal endogenous losses of Ca were determined at 659 mg/kg of dry matter intake. In conclusion, culled chicks had the greatest standardized total tract digestibility of calcium among test ingredients and standardized total tract digestible calcium in infertile eggs, unhatched eggs, culled chicks, and the mixture were 6.2, 5.5, 1.4, and 4.0%, respectively.

PSII-37 Determining the possible anthelmintic properties of allicin and pumpkin seed oil for mitigation of anthelmintic resistance found in gastrointestinal nematodes infecting ruminants

Abstract Anthelmintics are a widely utilized treatment option for gastrointestinal nematodes infecting ruminants. Recently, these parasites have exhibited increasing resistance towards these drugs. Haemonchus controtus, a gastrointestinal nematode commonly found in small ruminants, has developed resistance towards multiple classes of anthelmintics, leading to huge economic losses worldwide. There are few novel drugs currently being developed, but certain plant-derived products have demonstrated anthelmintic properties when administered in various forms to ruminants. By utilizing these plant products, producers have the opportunity to decrease the rate of development of anthelmintic resistance, and prevent eggs from hatching when excreted by infected livestock. Therefore, it is hypothesized that specific plant-derived products will act as ovicidal agents in H. contortus eggs. The objective of this study was to determine the ovicidal activity of allicin (from Allium sativum) and pumpkin seed oil (PSO). H. contortus eggs were extracted from a pooled fecal sample of Boer and Spanish goats naturally infected with H. contortus. The eggs were then exposed to allicin or PSO at different concentrations. Experiments were replicated four times per concentration. To determine ovicidal activity, samples were observed for unhatched eggs and hatched worms for 24 hours. The 1:100 allicin dilution treatment tended (P = 0.053) to inhibit hatching more than the control. Furthermore, any worms that hatched in allicin treatments exhibited limited signs of motility. Pumpkin seed oil does not appear to possess the same ovicidal potency as allicin, but may still have potential as a larvicidal agent. Based on the current results, allicin is a potential ovicidal agent that may be used independently or incorporated with current treatment methods for H. contortus control.

Effectiveness of Hot-Water Immersion Against Brevipalpus yothersi (Acari: Tenuipalpidae) as a Postharvest Treatment for Lemons.

Citrus leprosis is a destructive disease of citrus caused by several viruses (CiLVs) that are quarantine pests in the United States. Brevipalpus yothersi Baker (Acari: Tenuipalpidae) vectors the most virulent strain of CiLV. This mite is present in the United States and could facilitate the spread of the disease if CiLV reaches the country. Postharvest treatments could mitigate B. yothersi on imported commodities from areas where CiLV exists. The current study explores the effectiveness of hot-water immersion as a postharvest treatment against B. yothersi. Lemons were immersed in water at 21, 48, 53, or 63°C for 5, 10, and 15 min. Immersions at 53 and 63°C for all time schedules dislodged over 99% of adult mites. Lemon fruit quality and B. yothersi egg viability after hot-water immersion were also evaluated. Fruit quality significantly decreased in lemons treated at 63°C resulting in decay (grade 3, rejection), while at 53°C there was a quality reduction (grade 2, minimum acceptable market level) compared to lemons immersed at 21°C or nontreated controls (grade 1). None of the eggs hatched when the lemons were immersed in water at 63°C and an average of 1.5% hatched at 53°C for all time schedules. Immersion in water at 53°C for 5 min dislodged 99.71% and 57.14% of adult and immature mites, respectively, and resulted in 98.11% unhatched eggs without significant fruit quality reduction. Hot-water immersion could be a key component in a systems approach to control B. yothersi on imported citrus fruits from countries where citrus leprosis is present.

Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes

Summary Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.

Embryonic mortality in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle nests increases with cumulative exposure to elevated temperatures

As climate change continues, sea turtle nests will be increasingly exposed to elevated incubation temperatures. Higher incubation temperatures influence many aspects of sea turtle development including sex determination and incubation length, but also survival. If temperatures in the nest increase above a thermal tolerance limit, then embryonic mortality may increase. The purpose of this research was to determine if there are differences in vulnerability to elevated temperatures across different stages of embryonic development and between loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles. Temperature dataloggers recorded nest temperature in the approximate center of loggerhead and green nests laid on the Boca Raton, Florida beach during the 2016 and 2017 nesting seasons. All unhatched eggs were collected from these nests following hatchling emergence. The eggs were dissected and the developmental stage at embryonic death was determined. The point of embryonic death was compared to the nest temperatures during that stage of the incubation period to determine if death corresponded to specific periods of elevated temperatures. Elevated nest temperatures increased embryonic mortality, but no developmental stage had higher mortality rates when exposed to any specific elevated temperatures compared to embryos that had not been exposed to that temperature. The most significant relationship was between mortality and the percent of time embryos were exposed to temperatures above 34 °C. This resulted in greater mortality of more developed embryos, as those embryos had a longer cumulative exposure to elevated temperatures. Loggerhead turtles exhibited higher rates of mortality compared to green turtles for almost all temperature exposure periods above 34 °C. Although few green nests reached 34 °C, green sea turtle embryos in south Florida may also have a higher thermal tolerance than loggerheads. Due to the increased embryonic mortality, and therefore, decreased hatching success, future management strategies may need to protect sea turtle nests from extended periods at elevated temperatures.