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An approximate 1:1 sex ratio of American lobsters can be skewed due to environmental factors or fisheries management. Substantial skewness can impact mating behaviour and lower reproduction which could have far-reaching ecological and economic consequences. The aim was to investigate the sex ratio patterns of lobsters in two lobster fishing areas (LFAs) in southwestern Nova Scotia, Canada and identify factors associated with skewed sex ratios. This study analyzed biological data from more than 270,000 lobsters sampled over ten years (2010-2019) by the Fishermen and Scientists Research Society. A mixed effect logistic regression model evaluated the effect of spatial, temporal and environmental factors as well as size on the sex ratio of lobsters. There were significant temporal patterns in sex ratios that differed by LFA. After the effects of sampling month, year and LFA were accounted for, lower bottom temperature and deeper water depth were associated with a higher prevalence of females, especially in larger lobsters. We present the first long term analyses of sex ratio patterns in H. americanus in Atlantic Canada's most commercially important region for this species and provide evidence that these patterns are influenced by environmental factors and fisheries. In view of future climate change scenarios, monitoring the population dynamics of this iconic fishery species is crucial to ensure sustainable fisheries and healthy lobster stocks.

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Next-generation sequencing has been a huge benefit to investigators studying non-model species. High-throughput gene expression studies, which were once restricted to animals with extensive genomic resources, can now be applied to any species. Transcriptomic studies using RNA-Seq can discover hundreds of thousands of transcripts from any species of interest. The power and limitation of these techniques is the sheer size of the dataset that is acquired. Parsing these large datasets is becoming easier as more bioinformatic tools are available for biologists without extensive computer programming expertise. Gene annotation and physiological pathway tools such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology enable the application of the vast amount of information acquired from model organisms to non-model species. While noble in nature, utilization of these tools can inadvertently misrepresent transcriptomic data from non-model species via annotation omission. Annotation followed by molecular pathway analysis highlights pathways that are disproportionately affected by disease, stress, or the physiological condition being examined. Problems occur when gene annotation procedures only recognizes a subset, often 50% or less, of the genes differently expressed from a non-model organisms. Annotated transcripts normally belong to highly conserved metabolic or regulatory genes that likely have a secondary or tertiary role, if any at all, in immunity. They appear to be disproportionately affected simply because conserved genes are most easily annotated. Evolutionarily induced specialization of physiological pathways is a driving force of adaptive evolution, but it results in genes that have diverged sufficiently to prevent their identification and annotation through conventional gene or protein databases. The purpose of this manuscript is to highlight some of the challenges faced when annotating crustacean immune genes by using an American lobster (Homarus americanus) transcriptome as an example. Immune genes have evolved rapidly over time, facilitating speciation and adaption to highly divergent ecological niches. Complete and proper annotation of immune genes from invertebrates has been challenging. Modulation of the crustacean immune system occurs in a variety of physiological responses including biotic and abiotic stressors, molting and reproduction. A simple method for the identification of a greater number of potential immune genes is proposed, along with a short introductory primer on crustacean immune response. The intended audience is not the advanced bioinformatic user, but those investigating physiological responses who require rudimentary understanding of crustacean immunological principles, but where immune gene regulation is not their primary interest.

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Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.

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Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

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The Gram-positive bacterium Aerococcus viridans var. homari is a well-documented causative agent of the lethal systemic disease gaffkemia in both the American lobster, Homarus americanus, and the European lobster, Homarus gammarus. Previous phenotypic characterization has been unsuccessful at differentiating avirulent from virulent strains without performing lethal animal infection trials. Recent genetic characterization of A. viridans strains through 16S rRNA sequencing and random amplification of polymorphic DNA fingerprinting has revealed the presence of two subtypes. However, subtype 1 contains both virulent and avirulent strains which are genetically identical. The purpose of this study was to determine the proteomic mediators of virulence in A. viridans. Quantitative proteomic mapping of these two strains has revealed 29 differentially expressed protein spots, seven of which are only expressed in the virulent strain and could act as virulence factors. One protein, chaperonin 60 (Cpn60), is uniquely expressed in the virulent strain and has been shown to act as a virulence factor in many other bacteria. The proteomic mapping strategy employed in this study is the first to show phenotypic differences between virulent and avirulent strains. Cpn60 expression represents a potentially useful tool for identifying the virulent strains of A. viridans in epidemiological studies.

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Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

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We tested the hypothesis that vocal fold paresis leads to a substantial reduction in activity from the internal branch of the superior laryngeal nerve (iSLN) during respiration and evoked vocalization. The iSLN afferent activity was measured before and after recurrent laryngeal nerve paresis by cold block in decerebrate cats during spontaneous respiration and electrically evoked vocalization. Response rate patterns of 33 iSLN single units from 11 cats were categorized into 5 groups based on responses to vocalization. Only 24% of single units during spontaneous respiration and 18% during evoked phonation displayed activity pattern changes as a result of paresis. Those fibers affected were heterogeneous in discharge pattern, but none of the units that followed voice frequency lost this characteristic when the motor nerve was cooled. The relative insensitivity of iSLN activity to motor paralysis suggests that the receptors studied are coupled to tissue such that passive interaction rather than active muscular contraction is the major stimulus.

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The purpose of this project was to identify and categorize patterns of activity of the internal branch of the superior laryngeal nerve during vocalization evoked by midbrain stimulation in cats anesthetized with alpha-chloralose. Unit activity was isolated from the cut distal end of the internal branch of the superior laryngeal nerve by means of floating bipolar electrodes that retained their contact with nerve fibers despite movement due to vocalization. The phases of respiration and vocalization were determined with a diaphragm electromyogram, a photoglottogram, and a microphone recording. Five groups of discrete afferent activities were defined according to relationships between the spike activity and the phases of vocalization. Group 1 cell activity peaked just before phonation, during expiratory airflow (n = 26). Group 2 cells (n = 19) followed a vocal fold vibratory pattern during phonation. Group 3 cell activity (n = 6) peaked during phonation, but did not follow vocal fold vibration. Group 4 cells (n = 3) were active during inspiration between phonations. Group 5 cells (n = 4) showed both inspiratory and expiratory modulation. These results indicate that laryngeal afferent activity responds to phonation-specific events during vocalization. This stereotyped afferent information might be used by the central nervous system to modulate vocalization.

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The goal of this study was to determine whether sensory fibers in an intact recurrent laryngeal nerve (RLN) are influenced by respiration or vocalization. Patterns of RLN afferent activity were examined during respiration and evoked vocalization by means of midbrain electrical stimulation in cats anesthetized with alpha-chloralose. Nerve bundles were dissected from an intact RLN, with motor function preserved. The bundles were cut and the laryngeal end was placed on floating bipolar electrodes. Fifteen right RLNs were examined. A total of 9 single and multiunit afferent fibers from 4 cats were isolated and examined during respiration. Four units, analyzed from 3 fibers, showed respiratory phase modulation. Eight units, analyzed from 4 fibers in 1 cat, were observed during vocalization and showed no vocalization phase modulation. The RLN afferents could contribute to reflex modulation of the respiratory cycle, but more extensive sampling would be necessary to preclude effects from vocalization.

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OBJECTIVE: To determine if exposure to electromagnetic fields influences regeneration of the transected facial nerve in the rat. DESIGN AND METHODS: The left facial nerve was transected in the tympanic section of the fallopian canal in 24 rats randomly assigned to 2 groups. The cut ends of the facial nerve were reapproximated without sutures within the fallopian canal to maximize the potential for regeneration. Rats in the experimental group (n= 12) were then exposed to pulsed electromagnetic stimulation (0.4 millitesla at 120 Hz) for 4 hours per day, 5 days per week, for 8 weeks. Rats in the control group (n=12) were handled in an identical manner without pulsed electromagnetic stimulation. Four other rats were given sham operations in which all surgical procedures were carried out except for the actual nerve transection. Two of these rats were placed in each group. Nerve regeneration was evaluated using electroneurography (compound action potentials), force of whisker and eyelid movements, and voluntary facial movements before and at 2-week intervals after transection. Histological evaluation was performed at 10 weeks after transection. Each dependent variable was analyzed using a 2-way analysis of variance with 1 between variable (groups) and 1 within repeated measures variable (days after transection). RESULTS: Statistical analysis indicated that N1 (the negative deflection of depolarization phase of the muscle and/or nerve fibers) area, N1 amplitude, and N1 duration, as well as absolute amplitude of the compound action potentials, were all significantly greater 2 weeks after transection in the experimental than in the control group of rats. The force of eye and whisker movements after electrical stimulation was statistically greater in the experimental group of rats 4 weeks after transection. Voluntary eye movements in the experimental group were significantly better at 5 and 10 weeks, while whisker movements were better at 3 and 10 weeks. There was no statistical difference between the 2 groups for any histological variable. CONCLUSION: Results of this study indicate that pulsed electromagnetic stimulation enhances early regeneration of the transected facial nerve in rats.

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Controversy exists regarding voice recovery after the use of laser vs. microforceps techniques in the removal of benign vocal fold lesions. The purpose of this study is to compare recovery of voice and healing between groups of cats undergoing vocal fold epithelium removal by CO2 laser and those having vocal fold stripping. Fourteen adult female cats underwent standardized unilateral vocal fold injuries by CO2 laser ablation or stripping. After a 6-week recovery period, phonations were evoked by electrical stimulation of the midbrain periaqueductal gray area. Phonations were recorded for acoustic analysis. The larynges were harvested, fixed, and sectioned for histologic correlation. Acoustic analysis showed the mean signal-to-noise ratios in the laser group (19.72) to be significantly higher than those in the stripped group (13.51) (p = 0.04). The stripped group showed significantly greater amplitude perturbation (8.68% vs. 2.43%, p = 0.02). No between-group difference was found for period perturbation. Histologically, the laser group showed minimal Reinke's space scarring and near-normal epithelial regeneration, and the stripped group showed marked subepithelial scarring, often involving the vocalis muscle. These results demonstrate superior recovery of voice and healing in animals undergoing vocal fold epithelium removal with the CO2 laser. Inferior outcomes seen in the stripped group may be related to difficulty in preserving Reinke's space during epithelium removal.

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Cysteamine bitartrate capsules (Cystagon) have been approved by the US Food and Drug Administration for use in patients with nephropathic cystinosis. Plasma cysteamine concentrations were virtually identical at various times following ingestion of either cysteamine hydrochloride or Cystagon capsules in 24 normal control subjects. A transfer study was done with eight cystinosis patients who had been receiving either cysteamine hydrochloride or phosphocysteamine for many years. The plasma cysteamine concentration was significantly higher 2h after Cystagon and the leukocyte cystine content was significantly lower at all times after Cystagon compared to older forms of the drug. These differences are probably the result of greater patient compliance in taking the capsules compared to the older, liquid forms of the drug. A new method for following the course of renal glomerular deterioration in diseases such as cystinosis has been published recently. This method was used to re-analyse data on the efficacy of cysteamine treatment and to re-analyse new data on treating cystinosis patients with either of two doses of cysteamine (1.30 g/m2 per day and 1.95 g/m2 per day). This new method agrees well with other methods and shows that both doses of drug are equally effective in maintaining glomerular function.

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Hemangiopericytomas are unusual vascular tumours that rarely occur in the paranasal sinuses and nasal cavity. They are thought to arise from pericytes that surround capillaries, however, there is no proven etiology of these tumours. The diagnosis cannot be made based on gross morphologic characteristics nor on frozen section biopsy. Instead, the diagnosis is dependent on careful histologic examination and reticulum staining. The diagnosis can be confirmed by electron microscopy. The histologic picture is greatly varied from tumour to tumour and within a given tumour itself. The clinical course of hemangiopericytomas ranges from benign to malignant with possible distant metastases and cannot be reliably predicted by histologic criteria. Hemangiopericytomas of the nose and paranasal sinuses are thought to behave less aggressively than those occurring in other parts of the body. Lymph node metastasis is rare and elective neck dissection is not indicated. The accepted treatment of these tumours is wide surgical excision, however adequate surgical margins are usually difficult in the sinonasal region. As a result, hemangiopericytomas can exhibit a high recurrence rate. Therefore, it is mandatory that these patients be followed carefully for the remainder of their lives.

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Recent changes in reimbursement for tonsillectomy and adenoidectomy have resulted in a large number of these procedures being done on an outpatient basis. There is still considerable controversy, however, as to the safety of this concept. We reviewed the charts of 1000 consecutive patients who underwent these procedures. Three hundred ninety-six procedures were performed as inpatient surgery and 604 were performed as outpatient surgery. Patients were considered as candidates for outpatient surgery if they met specific criteria, such as good overall medical health, no central apnea, normal bleeding history and profile, and had adequate social considerations. The inpatient group included those patients who did not meet the criteria to be outpatients. We compared the complication rates of these two groups with regard to age, type of procedure performed (tonsillectomy, adenoidectomy, adenotonsillectomy), and indication for surgery in order to determine if there was any increased risk of outpatient surgery, despite strict selection criteria. The two groups were similar in their distribution with regards to age, sex, type of procedure, and indication for surgery. The overall complication rate for the entire group was 7.9%, with an 11.8% complication rate for inpatients and 4.1% complications for outpatients. The higher complication rate among the inpatient group is probably a direct result of the selection process because this group included the higher-risk patients. On the basis of these findings, we believe that surgery of the tonsils and adenoids can be performed safely as an outpatient procedure, regardless of age, indication, or procedure, if the candidates for ambulatory surgery are carefully selected by the surgeon.

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Proper radiographic evaluation is essential to confirm the presence and location of a mandibular fracture. The panoramic view of the mandible is considered to be a very accurate technique and has been heavily relied on by many clinicians as the initial or sole means of diagnosing mandibular fractures. Three cases of mandibular fractures are presented in which a panoramic view failed to demonstrate fractures of the mandible that were obvious on plain film radiographs. Diagnosis and treatment planning should not be based on the information obtained from a single roentgenogram such as a panoramic view. The combination of the mandibular series with the panoramic view provides increased diagnostic information that should enable accurate diagnosis of fractures in all areas of the mandible.

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Cysteamine (beta-mercaptoethylamine, MEA) is currently used to treat children with nephropathic cystinosis. In this study MEA was compared to phosphocysteamine (MEAP), a phosphorothioester that tastes and smells better than MEA, with respect to its ability to elevate plasma MEA and deplete leukocytes of cystine. Studies were performed in six children with nephropathic cystinosis ranging in age from 2 to 10 yr. After equimolar oral doses of either MEA or MEAP plasma cysteamine was determined at various times for 6 h. MEA was determined by sodium borohydride reduction followed by high-performance liquid chromatography separation and electrochemical detection. Leukocyte cystine was measured before and 1 and 6 h after drug administration. Peak plasma MEA was obtained 30 min to 1 h after a dose and was not significantly different when MEA (48.6 +/- 10.7, mean +/- SD) or MEAP (54.1 +/- 20.2) was given. Significant plasma MEA concentrations were seen as early as 15 min after an oral dose, indicating rapid absorption. Analysis of vomitus indicated that hydrolysis of the phosphate group of MEAP occurs in the stomach. The percent decrease in leukocyte cystine content obtained with MEA administration (61.9%) was not significantly different from the decrease observed when MEAP was administered (65.3%). MEA and MEAP appear to be equally effective in their cystine-depleting properties.

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Cystine exodus from partially purified granular fractions of normal leucocytes is stimulated by MgATP. N-Ethylmaleimide, an inhibitor of the lysosomal H+-translocating ATPase, eliminated the stimulated exodus, but had no effect on basal exodus. As the initial content of cystine was increased, the initial velocity of both the basal and ATP-stimulated egress increased. However, as saturation with substrate was approached, the ATP stimulation disappeared leaving only the N-ethylmaleimide-insensitive basal exodus. The increased initial velocity in the presence of ATP may represent improved binding of cystine to the partially saturated inner transporter as a result of conformational or charge optimization brought about by the action of the H+-translocating ATPase.

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Heterozygotes for the autosomal recessive disease cystinosis are currently detected by measuring the cystine content of mixed-leukocyte preparations. The present study was designed to reassess the accuracy of this method and to determine whether measuring the cystine content of purified polymorphonuclear leukocytes would improve heterozygote detection. Blood samples were obtained from 29 obligate heterozygotes for nephropathic cystinosis, one obligate heterozygote for benign cystinosis, and 18 individuals presumed to be normal. When the cystine content of mixed-leukocyte preparations was measured, three heterozygote values overlapped the normal range. When polymorphonuclear-leukocyte cystine content was measured, no heterozygote values were within the normal range. Measurement of the cystine content of purified preparations of polymorphonuclear leukocytes affords a simple method that improves the sensitivity of heterozygote detection for cystinosis.

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