The Effects of Population Density on the Incidence of Developmental Deformities in Chemosensory Organs of Tobacco Hornworm Larvae (Lepidoptera: Sphingidae).

Cultures of Manduca sexta Johanssen in our laboratory were found to have larvae with missing or deformed mouthparts or antennae. Hypothesizing that these developmental deformities were caused by crowded rearing conditions, we reared larvae in four different population densities and recorded the incidence (% of larvae affected) and types of chemoreceptor deformities. Results showed that the incidence of these deformities was directly proportional to larval population density. Deformities of the maxilla and palp were the most frequent, followed by those of the antenna, epipharynx and maxillary styloconica. Life history traits of larval mass, food consumption, and rate of development were inversely related to larval density for both normal and deformed larvae. We discuss possible causes and mechanisms of these deformities and of changes to life history traits.

Do larvae from deep-sea hydrothermal vents disperse in surface waters?

Larval dispersal significantly contributes to the geographic distribution, population dynamics, and evolutionary processes of animals endemic to deep-sea hydrothermal vents. Little is known as to the extent that their larvae migrate vertically to shallower waters and experience stronger currents and richer food supplies. Here, we first provide evidence from early life-history traits and population genetics for the surface dispersal of a vent species. Planktotrophic larvae of a red blood limpet, Shinkailepas myojinensis (Gastropoda: Neritimorpha: Phenacolepadidae), were cultured to observe their swimming behavior and to evaluate the effects of temperature on survival and growth. In addition, the population structure was analyzed based on 1.2-kbp mitochondrial DNA sequences from 77 specimens that cover the geographic and bathymetric distributions of the species (northwest Pacific, 442-1,227 m in depth). Hatched larvae constantly swam upward at 16.6-44.2 mm/min depending on temperature. Vertical migration from hydrothermal vents to the surface, calculated to take ~4-43 d, is attainable given their lengthy survival time without feeding. Fed larvae best survived and grew at 25°C (followed by 20°C), which approximates the sea surface temperature in the geographic range of the species. Little or no growth was observed at the temperature of the vent habitat where adult limpets occur (≤15°C). Population genetic analyses showed no differentiation among localities that are <1,350 km apart. The larvae of S. myojinensis most likely migrate to the surface water, where high phytoplankton biomass and strong currents enable their growth and long distance dispersal over many months. Sea surface temperature may represent a critical factor in determining the geographic distribution of many vent endemic species with a planktotrophic early development, and in turn the faunal composition of individual vent sites and regions.

Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment.

BACKGROUND: Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occurrence of this parasite in German horses was gained. For this purpose, faecal samples of 501 horses were screened for S. vulgaris with real-time PCR and an additional larval culture was performed in samples of 278 horses. A subset of 26 horses underwent multiple follow-up examinations with both methods in order to evaluate both the persistence of S. vulgaris infections and the reproducibility of each diagnostic method. RESULTS: The real-time PCR revealed S. vulgaris-DNA in ten of 501 investigated equine samples (1.9%). The larval culture demonstrated larvae of S. vulgaris in three of the 278 samples (1.1%). A direct comparison of the two methods was possible in 321 samples including 43 follow-up examinations with the result of 11 S. vulgaris-positive samples by real-time PCR and 4 S. vulgaris-positive samples by larval culture. The McNemar's test (p-value = 0.016) revealed a significant difference and the kappa values (0.525) showed a moderate agreement between real-time PCR and larval culture. CONCLUSIONS: The real-time PCR detected a significantly higher proportion of positives of S. vulgaris compared to larval culture and should thus be considered as a routine diagnostic method for the detection of S. vulgaris in equine samples.

A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms.

Resistance to fenbendazole, ivermectin, and moxidectin was explored by a fecal egg count reduction test in four meat sheep flocks in southwestern France where anthelmintic resistance was suspected. The FECR test results of the present study confirmed the presence of benzimidazole resistance in three out of the four farms and the presence of ivermectin resistance in one flock. In addition, a suspicion of moxidectin resistance was shown in this latter farm. Both conventional morphological and molecular identifications were performed on larval cultures before and after the treatment in the studied farms. A high positive correlation was found between the number of larvae counted under binocular microscope and the number of larvae estimated by the qPCR analysis (R 2 = 0.88) and a high Cohen's Kappa value (0.91) in the detection of strongylid larvae in larval cultures. According to qPCR results, Trichostrongylus species demonstrated high levels of BZ resistance and Teladorsagia circumcincta was involved in the IVM resistance in one farm. The molecular procedures used in this study have the potential to be beneficial for anthelmintic resistance surveillance in sheep industry.

Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse.

BACKGROUND: Surveillance of Strongylus vulgaris and other Strongylus spp. in equids is important for targeted intervention in parasite control, requiring reliable routine diagnostic methods. OBJECTIVES: Comparing morphological examination and PCR analyses of larval cultures to identify Strongylus spp. species based on German diagnostic samples from 2018. STUDY DESIGN: Method comparison. METHODS: During the routine diagnostic investigations, in total 712 strongyle-egg positive equine faecal samples were cultured. Third-stage larvae (L3) were morphologically differentiated. For molecular validation, samples were examined using S. vulgaris real-time PCR and Strongylus edentatus/Strongylus equinus/Strongylus asini high-resolution melting PCRs. RESULTS: Based on 28S rRNA PCR, 594 samples positive for nematode DNA were included in the study. The inter-rater reliability to compare morphological and molecular species identification was fair for Strongylus spp. without species identification and for S. edentatus, slight for S. equinus and poor for S. vulgaris. The frequency based on morphological and molecular data in this study were for S. vulgaris 0% and 0.8%, respectively, for S. edentatus 0.3% and 1.5%, respectively, and for S. equinus 2.0% and 0.2%, respectively. Based on molecular analyses, one sample obtained from a domestic horse contained S. asini DNA, which was confirmed by sequencing. MAIN LIMITATIONS: For many samples, no or only incomplete data regarding clinical history, the exact geographical location and whether samples were obtained on individual or farm level, were available. CONCLUSIONS: Results of morphological and molecular examination methods of strongyle L3 from equine samples can differ substantially. Further evaluation of these methods is required to provide reliable and cost-effective methods of screening equine parasites. Further studies using approaches suitable to detect S. asini are needed to evaluate its clinical and epidemiological relevance.

Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Practical guide for microscopic identification of infectious gastrointestinal nematode larvae in sheep from Sardinia, Italy, backed by molecular analysis.

BACKGROUND: Gastrointestinal nematodes (GIN) are ubiquitous in small ruminant farming, representing a major health and production concern. Given their differences in pathogenicity and the current problems regarding anthelmintic resistance, specific diagnosis of GIN is of significant importance. At present, the most widely applied method for this entails culture and microscopic analysis of third-stage larvae, allowing for identification at least to the genus level. Overall, a variety of keys for microscopic analysis have been published, showing substantial variation. Given this fact, this study aimed to produce a practical and updated guide for the identification of infective ovine GIN larvae. METHODS: Using existing keys and protocols, a total of 173larvae of the most common species/genera of ovine GIN from pooled faecal samples from Sardinia (Italy) were identified and extracted, and further individual molecular identification was performed. Morphometric and morphological data as well as high-quality photographs were collected and combined to produce the final guide. RESULTS: GIN microscopically and molecularly identified during this research include Trichostrongylus spp., Teladorsagia circumcincta, Haemonchus contortus, Cooperia curticei, and Chabertia ovina. Based on microscopic analysis, 73.5% of the larvae were correctly identified. Based on sheathed tail length, 91.8% were correctly classified into their respective preliminary groups. CONCLUSIONS: It is crucial for the microscopic identification of infectious GIN larvae to examine each larva in its entirety and thus to take multiple characteristics into account to obtain an accurate diagnosis. However, a preliminary classification based on sheathed tail length (resulting in three groups: A, short; B, medium; C, long) was found to be effective. Further identification within group A can be achieved based on the presence of a cranial inflexion, caudal tubercles and full body measurements (Trichostrongylus spp. < 720 µm, T. circumcincta ≥ 720 µm). Larvae within group B can be differentiated based on sheathed tail morphometry (H. contortus > 65 µm, C. curticei ≤ 65 µm), the presence of cranial refractile bodies, total body length measurements (H. contortus ≤ 790 µm, C. curticei > 790 µm) and shape of the cranial extremity. Finally, all characteristics proposed for the differentiation between Oesophagostomum spp. and C. ovina larvae (group C) were found to have considerable restrictions.

Reviving a tradition: The Development of Strongylus vulgaris in larval culture.

All horses are susceptible to the equine gastrointestinal parasite, Strongylus vulgaris, which is known to cause significant disease and death. The parasite undergoes development from the egg through the first (L1), second (L2) and third (L3) larval stages outside the horse. The L3 is the infective stage. The universally available technique for detection of S. vulgaris larvae is the larval culture method. This requires a 10-14 day culture period to induce development from egg to L3, followed by Baermannization and identification of the L3s to genus and/or species. It is unknown if the culture duration is necessary or ideal for S. vulgaris identification. The purpose of this study was to perform daily examinations of known S. vulgaris positive fecal samples in coproculture. Fresh feces were collected from a horse known to be shedding S. vulgaris eggs. A total of 140 cultures were set up using 10 g of feces. Cultures remained at room temperature and moistened every other day. Every day, 10 samples were examined, and all larvae were identified to stage, genus/species, and enumerated. Throughout the study, L1, L2, and L3 stages were observed, and S. vulgaris, Strongylus edentatus, Triodontophorus spp., and cyathostomin L3s were identified. Third stage larvae were observed on Day 5, and the mean number of L3s significantly increased on Day 10 (P < .001), and declined thereafter. Strongylus vulgaris was first observed on Day 6 with a mean count of 4.1 (95 % CI: 1.1, 7.1) S. vulgaris larvae, accounting for 4.1 % (95 % CI:1.8, 7) of the total L3s observed. The number of S. vulgaris larvae was significantly higher on Day 10 with a mean of 156.8 (95 % CI: 120.7, 192.9) S. vulgaris larvae (P < .001), and the proportion was also significantly higher with S. vulgaris comprising 50 % (95 % CI: 45.9, 54.8) (P = .006) of the total larvae. However, after 10 days, the mean number of S. vulgaris larvae declined, as did the proportion of S. vulgaris larvae compared to the total number of larvae. Using the described methods, it is possible to identify S. vulgaris as early as 6 days, and the optimal period is 10 days to detect the maximum number of S. vulgaris.

Diagnosing Strongylus vulgaris in pooled fecal samples.

Strongylus vulgaris is the most pathogenic intestinal helminth parasite infecting horses. The migrating larvae in the mesenteric blood vessels can cause non-strangulating intestinal infarctions, which have a guarded prognosis for survival. Infections are typically diagnosed by coproculture, but a PCR test is available in some countries. While it is ideal to test horses individually, many veterinarians and clients wish to pool samples to reduce workload and cost of the diagnostic method. The purpose of this study was to determine if pooling of fecal samples would negatively impact diagnostic performance of the coproculture and the PCR for determination of S. vulgaris infection. Ten horses with strongylid eggs per gram (EPG) >500 and confirmed as either S. vulgaris positive or negative were selected as fecal donors. Eight pools with feces from five horses were created with 0%, 10 %, 20 %, 30 %, 40 %, 50 %, 80 %, and 100 % S. vulgaris positive feces. From each pool, 20 subsamples of 10 g each were collected and analyzed. Half of these samples were set up for coproculture and the other half for PCR. All pools containing 50 % or greater S. vulgaris positive feces were detected positive by both PCR and coproculture. In the pools with less than 50 % S. vulgaris positive feces, the PCR detected 33 positive samples compared to 24 with the coproculture. Three samples from the 0% pool were detected as low-level PCR positives, but this could be due to contamination. These results indicate that diagnosing S. vulgaris on pooled samples is reliable, when at least 50 % of the feces in a pool are from S. vulgaris positive animals. Since S. vulgaris remains relatively rare in managed horses, however, some diagnostic sensitivity is expected to be lost with a pooled sample screening approach. Nonetheless, pooled sample screening on farms could still be considered useful under some circumstances, and the PCR generally performed better at the lower proportions of S. vulgaris positive feces.

Breeding of a Wild Population of South Pacific Bonito Sarda chiliensis chiliensis (Cuvier 1832) Broodstock under Laboratory Conditions in Pisagua, Northern Chile.

The wild population of South Pacific bonito Sarda chiliensis chiliensis, which has a wide distribution in northern Chile, is considered of importance in Chilean aquaculture. The biological feasibility of cultivation of any marine species begins with the establishment of an initial broodstock population to obtain eggs, larvae, and juveniles. In this work, 22 South Pacific bonito fishing campaigns were carried out in Pisagua, Chile, between spring in November 2011 and the summer in January 2012. At least 74 specimens were obtained of which 24 survived the capture and transport processes. Fish were stocked in a recirculating land-based aquaculture system, and at 14 months under captivity, fish began spawning. Eggs were collected, to describe some stages of development, and were placed in incubators at 20 °C and on the third-day eggs hatched. Larvae reached a total length between 1.435 and 1.7 mm, which were accurately characterized during their first morphological changes. This is the first work that describes the capture, transport, and acclimatization in captivity of a breeding population of wild Pacific bonito in Chile.

Initial ontogeny of digestive enzymes in the early life stages of captive-bred European eels during fasting: A partial characterization.

The European eel has recently been included on the Red List of the International Union for Conservation of Nature (IUCN) as a critically endangered species. The rearing of Anguilla larvae is seen as a key bottleneck to the mass production of glass eels since very little ecological information is available regarding their natural nutrition. Studies of digestive physiology and ontogenetic development in eel larvae could provide useful information for solving some of the puzzles regarding larval fish culture. The aim of this study was to characterize the ontogeny of pancreatic enzymes (trypsin, lipase and amylase) and a peptide hormone regulator of pancreatic secretion (cholecystokinin) in terms of gene expression in European eel larvae from day 0 (P0) of hatching to 5, 10, 15 and 20 days post hatching during fasting. The results in the present study showed that all the genes selected were present, with different levels of expression and increasing trends, during larval development. At P0, the increase in the gene expression of lipase and amylase was higher than that of trypsin and cholecystokinin, confirming that enzymatic activity began before mouth opening and that larvae, provided with a complete enzymatic set, might have the capacity of digesting and absorbing various nutrients.

Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle.

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance.

An automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study.

Detecting the genera and species of gastrointestinal (GI) nematode infections in faecal samples obtained from cattle requires the incubation of faeces ('larval culture') followed by identification of the third-stage larvae that are harvested after 10-14days. Substantial research in the development of PCR-based methods for the rapid and specific identification GI nematodes has been conducted for small ruminants, whilst only few such assays have been developed for cattle. In the present paper we describe the development of an automated, robotic PCR platform for the detection and genus and/or species-specific identification of GI nematodes from bovine faecal samples. This test was then validated using samples from different regions of three countries (Australia, Belgium and Scotland). The PCR platform was found to be highly sensitive and specific for the identification of the important GI nematodes in naturally infected cattle (both estimates >90%). The PCR platform can also estimate the percentage of genera or species present in a mixed-species infection, and was found superior to larval culture in terms of speed (1-2days versus 1-2 weeks for culture), sensitivity and specificity. The PCR was simple to use and the operator requires no knowledge or experience to identify the nematodes present, compared to larval culture where even experienced operators can make substantial errors due to considerable overlap in the published characteristics of key species.

The specific diagnosis of gastrointestinal nematode infections in livestock: larval culture technique, its limitations and alternative DNA-based approaches.

The specific diagnosis of gastrointestinal nematode infections in ruminants is routinely based on larval culture technique and on the morphological identification of developed third-stage larvae. However, research on the ecology and developmental requirements of different species suggests that environmental conditions (e.g., temperature and humidity) for optimal development to occur vary between the different species. Thus, employing a common culture protocol for all species will favour the development of certain species over others and can cause a biased result in particular when species proportions in a mixed infection are to be determined. Furthermore, the morphological identification of L3 larvae is complicated by a lack of distinctive, obvious features that would allow the identification of all key species. In the present paper we review in detail the potential limitations of larval culture technique and morphological identification and provide account to some modern molecular alternatives to the specific diagnosis of gastrointestinal nematode infection in ruminants.