Larvae Research Articles

Ramming the parasites: Evaluation of quarantine procedures against Haemonchus contortus at sheep markets in Sweden

In Sweden, it is recommended to treat rams at sheep markets with ivermectin and then keep them in quarantine to minimise the risk of transmission of Haemonchus contortus between farms. The aim of this study was to evaluate the effectiveness of this practise. Accordingly the gastrointestinal parasite status of 50 rams purchased at two sheep markets in central Sweden in autumn 2019 was investigated using faecal samples collected on the day of sale (test 1), 12 ± 3 days after ivermectin injection (test 2, in quarantine) and a few months later before inclusion in the new flocks (test 3). We used both traditional diagnostic methods (i.e. identification of nematode eggs in faeces or larvae in cultures when H. contortus eggs could not be identified) and a molecular test based on the digital droplet PCR platform to further identify positive samples. In test 1, conducted in autumn, 40 % of rams were FEC-positive, but only 12 % (six rams) were infected with H. contortus according to conventional routine diagnostics. In test 2, 8 % (four rams) were FEC-positive, including one with 1050 eggs, 90 % of which were identified as H. contortus. This ram was therefore returned to the supplier. However, ivermectin was found to be effective when this animal and a group of lambs from the same farm were treated and tested again. Test 3 was performed on 44 of the same rams (in addition to the returned ram, two rams died in quarantine and the samples from three rams were never provided by the owners). The proportion of FEC-positive animals was 42 %, with an even higher proportion (27 %) of animals being H. contortus-positive than in test 1. The corresponding results for tests 1, 2 and 3 with the ddPCR assay were 18 %, 4 % and 76 %, respectively. This study demonstrates the superiority of DNA detection over microscopy, which is the mainstay in most diagnostic laboratories. Although the combined results confirm that H. contortus survived quarantine in two rams, in the other cases it is not clear whether the spring infection rates are due to re-emergence of persistent larvae from quarantine or reinfection on the new farm. These results suggest not only that we should recommend that sheep farmers use a more sensitive molecular test when purchasing and introducing new animals to their flock, but also that the reliability of injectable ivermectin as a quarantine treatment for removal of adult and larval stages needs further investigation.

Monitoring of mosquito breeding sites in the municipality of Cascais, Portugal

Abstract Culex pipiens is the primary West Nile Virus vector in Portugal. Additionally, in 2017, Aedes albopictus, vector of various arboviruses, was detected in Northern Portugal. To mitigate viral transmission risks, effective surveillance is crucial, including monitoring egg, larva, and pupa stages. Located on the west of Lisbon, Cascais implemented a surveillance based on monitoring invasive species at Points of Entry and, complementarily, on detecting and intervening in breeding sites. Active searches for new breeding sites and monitoring those that were already identified are conducted weekly during the peak mosquito season (May to October) and biweekly during the colder season (November to April), following the methodology outlined below: • Upon detection, samples of immature stages are collected for submission to the National Institute of Health (INSA); • Corrective measures are requested to the managing entities of the locations where the sites are found; • Date of action, location, collection point, date of report, and status (pending or intervened) are recorded on a database; • Locations with pending status undergo continuous monitoring. In 2023, 16 artificial breeding sites were identified. These findings resulted from 23 actions conducted across 8 locations. 35 samples of immatures were collected, followed by their submission and notification to the respective managing entities. Recurrent detections were noted in 9 breeding sites. The managing entities implemented measures at 13 of the identified sites, leaving 3 with pending intervention. The identification of breeding sites demands continuous monitoring efforts and requires an effective coordination between the Public Health services and the management entities. Based on the achieved outcomes, it is evident that the implementation of corrective measures has been successful, making a significant contribution to reducing mosquito density. Key messages • Effective surveillance and intervention strategies are crucial in mitigating West Nile Virus and arbovirus transmission risks in Portugal. • Continuous monitoring and coordination between public health services and management entities are key for maintaining effective vector control measures.

Processing of Larvae of Alphitobius diaperinus and Tenebrio molitor in Cooked Sausages: Effects on Physicochemical, Microbiological, and Sensory Parameters

Proteins from insect production represent an interesting (environmentally friendly) option or supplement to commercial livestock farming. At present, however, the larval stages of T. molitor (mealworm) and A. diaperinus (buffalo worm) have been authorized as food for human consumption EU-wide, as have the nymph and adult stages of Locusta (L.) migratoria (Locusta migratoria, Linnaeus, 1758) and Acheta (A.) domesticus (house cricket, Acheta domesticus, Linnaeus, 1758). However, there is the problem that insects that are recognizable as a whole tend to be avoided by consumers, especially in the European region, as they are reminiscent of living things and can cause aversion and disgust in consumers. Against this background, in the present study, five batches of two types of cooked sausages were produced: on the one hand, with turkey, and on the other hand, with pork lean meat as a base. In different formulations, 10% and 20% of the meat contents (turkey or pork) in these meat products were replaced by deep-frozen, pulverized T. molitor and A. diaperinus larvae. The effects of the addition of these insects in the products on the microbiological and physicochemical parameters of these cooked sausages, compared to a product without insect content, directly after heating, were investigated. After production, a storage trial was also carried out to determine whether possible insect ingredients could influence the growth of inoculated bacterial species (Bacillus (B.) cereus, Escherichia (E.) coli, Listeria (L.) monocytogenes, and Campylobacter (C.) jejuni) and how the addition of insect larvae affectsthe sensory and physicochemical properties during storage. The study showed that the products with insects had reduced lightness (turkey p C = 0.025), increased yellowness (pork p S = 0.0009, p C < 0.0001 and turkey p C = 0.0027) and a reduced red color (pork p S < 0.0001, p C = 0.0001) after heating when compared to the cooked sausages without insects. However, no significant differences between the various cooked sausages with or without insects in terms of cooking loss, firmness, and protein, ash, and fat or water contents were found. The microbiological tests showed, on the one hand, that the prior microbial reduction (e.g., in the form of blanching) of the insect larvae was essential in order to guarantee the flawless microbiological quality of the cooked sausages and, on the other hand, that the addition of insects to the cooked sausages did not significantly affect the growth of the inoculated bacterial species and that no sensory differences could be detected during storage. Despite the significant color effects on the product, A. diaperinus and T. molitor larvae would be suitable as protein or meat alternatives in cooked sausages, but they would have to undergo pre-treatment, primarily with regard to microbiological safety. The extent to which a complete replacement of meat is possible has to be investigated in further studies.

Exposure to copper metal enhances the tolerance of An. gambiae s.s. over multiple generations while reducing both fertility and fecundity in this primary malaria vector.

Background Anopheles s.l. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness. This study investigated the larval metal exposure on larval development and reproduction of An. gambiae s.s., a laboratory susceptible strain, kisumu. Methods Stage 2 larvae of Anopheles gambiae, Kisumu were exposed to C1 = 484 μg L-1, C2 = 300 μg L-1 and 0 μg L-1 (control) of copper chloride. Larval mortality, pupation time, pupation rate, gonotrophic cycle length, fecundity and fertility of larvae/adults were assessed over six generations. Results Results revealed that larval mortality rate was significantly higher in the C1 groups of each group (p = 0.000), but this mortality rate decreased over generations. Pupation time was extended to 13 and 14 days respectively for C2 and C1 groups (p = 0.000) compared to the control group. Similar results were observed for the gonotrophic cycle, which increased from 4 days at G0 to more than 6 days at generation 5 in adults of C1. The pupation rate in generation 4 (C1) and generation 5 of the same group (p = 0.000) as well as the emergence rate in generation 4 (C2, p = 0.000) and generation 5 (C1 and C2, p = 0.000) decreased significantly compared to the control group. The average number of eggs laid was lower in the test groups from generation 4 to generation 5 (C1 and C2, p = 0.00) and egg fertility was also negatively affected by exposure of the larval stage of An. gambiae s.s. to copper. Conclusion Although studies have already shown that copper induces resistance in An. gambiae s.l. to insecticides, this study has shown that this adaptation requires a non-negligible biological cost in the life of the insect.

First Modern Morphological and Molecular Description of Saccocoelium Cephali Larvae Stages (Digenea: Haploporidae) from the Black Sea.

Knowledge of haploporid trematode larvae is very poor. Until recently, only scattered reports from the Black and Medditeranean Seas were known. The present research is the first report of haploporid cercariae S. cephali from the gastropod Hydrobia acuta inhabiting the Black Sea. Thus, the present study aimed to investigate the larvae of one species from the Haploporidae family. Hydrobia acuta molluscs were collected in the estuary of the Chernaya River (the Black Sea) during 2011-2013 and examined for trematode larvae. Found parthenitae were drawn and preserved for DNA extractions. Morphological features were compared to nominal species, and molecular comparisons were made of the subunit ribosomal DNA with other Haploporidae species. The haploporid parthenitae were found in the gonads of Hydrobia acuta molluscs. Most of the morphological characteristics of the specimens investigated agree with those of Saccococoelium sp. and are closest to those of S. cephali by shape and length of the body, ratio of suckers, ratio of tail length to body, shape, and size of the pharynx. Phylogenetic analysis from our study generated with Bayesian algorithm showed that studied cercariae specimens from the Black Sea were within the hapolporine clade and closely related to S. cephali from Spain. Morphological characteristics of cercariae emerging from Hydrobia acuta from the Black Sea and the analysis of partial 28S rDNA sequences support the conspecificity of the parasite with S. cephali mature worms from the Mediterranean Sea.

Hyperosmolality tolerance of freshwater largemouth bass Micropterus salmoides to brackish in early ontogenetic stages of eggs, embryos, and yolk-sac larvae

Hyperosmolality tolerance of freshwater largemouth bass Micropterus salmoides to brackish in early ontogenetic stages of eggs, embryos, and yolk-sac larvae

Potential of zodia (Evodia suaveolens) as natural repellents for Culex sp

Background: Adult Culex sp mosquitoes as disease vector also cause discomfort around humans. Various efforts have been made to prevent the transmission of diseases carried by mosquitoes, including the use of mosquito nets and the use of chemical insecticides in the larval and adult stages. Objectives: This Reserach aim to investigates the potential of Zodia (Evodia suaveolens) as natural repellents. Materials and Methods: An experimental study was conducted to test the effectiveness of Zodia extract at concentrations of 1%, 2%, and 3% against Culex sp. mosquitoes. The tests were conducted under controlled conditions in the Laboratory of Parasitology, University of Binawan. The effectiveness was measured by the percentage of mosquitoes repelled after exposure. Results: The effectiveness of Zodia repellent increased with concentration, showing 76.4% effectiveness at 1%, 81.3% at 2%, and 91.05% at 3%. These results indicate a concentration-dependent increase in repellency, with the highest concentration approaching typical efficacy rates of synthetic repellents. Conclusions: The Zodia repellent (Evodia suaveolens) with a concentration of 1%, 2% and 3% has been demonstrated to be effective as a natural repellent against Culex sp. mosquitoes.

Bacillus thuringiensis Cry14A family proteins as novel anthelmintics against gastrointestinal nematode parasites.

Bacillus thuringiensis crystal (Cry) proteins have been expressed in commercial transgenic crops for nearly 30 years, providing safe and effective control of insect pests and significantly reducing the application of hazardous chemical pesticides. B. thuringiensis crystal proteins have also been shown to target parasitic nematodes, including plant parasitic nematodes. Recently, transgenic soybean crops expressing Cry14Ab have been shown to provide control against the soybean cyst nematode Heterodera glycines, marking the first time a crystal protein is being commercialized in transgenic crops for control of a nematode pest. However, apart from H. glycines and the free-living nematode, Caenornabditis elegans, the breadth of nematode activity of Cry14Ab, e.g., against gastrointestinal parasitic nematodes (GINs), has not been reported. Here we study the efficacy of Cry14Ab against a wide range of gastrointestinal nematode parasites (GINs) in vitro and in vivo. We find that Cry14Ab is effective in vitro against the barber's pole worm Haemonchus contortus larvae, small strongyles cyathostomin larvae, the hookworm Ancylostoma ceylanicum adults, the roundworm Ascaris suum L4 larvae, and the whipworm Trichuris muris adults. In rodents infected with GIN parasites, Cry14Ab is effective as an in vivo anthelmintic against the hookworms A. ceylanicum and N. americanus, against the mouse parasite Heligmosomoides polygyrus bakeri, and against the roundworm A. suum. Cry14Ab also variably reduces the reproduction of the whipworm T. muris in vivo. Using optimized profile Markov Models, we looked for other putative anthelmintic Cry proteins and, within this list, identified a Bt crystal protein, GenBank accession no. MF893203, that we produced and demonstrated intoxicated GINs. This protein, with 90% amino acid identity to Cry14Ab, is active against C. elegans, A. ceylanicum adults, and A. suum L4 larvae in vitro. MF893203 was given the official designation of Cry14Ac. Cry14Ac is also an effective in vivo anthelmintic against A. ceylanicum hookworms in hamsters and intestinal A. suum in mice. Taken together, our results demonstrate that Cry14Ab and Cry14Ac have wide therapeutic utility against GINs.

Volatile Organic Compounds as Early Detection Indicators of Wheat Infected by Sitophilus oryzae

The rice weevil, Sitophilus oryzae (L.), is a major pest that poses a considerable threat to grain safety storage. Early detection is of great significance in reducing grain losses. Studies have demonstrated that pest infestation causes alterations in grain volatiles, potentially indicating the presence of pests. In this study, we detected volatile organic compounds (VOCs) in non-infected and pest-infected wheat on the 3rd, 9th, 17th, 22nd, and 40th days, corresponding to the developmental stages of the rice weevil at the egg, young larval, old larval, pupal, and adult stages, respectively. A total of 126 VOCs were identified, including 96 hydrocarbons, 7 alcohols, 5 aldehydes, 9 ketones, 9 esters, and 18 other compounds, 62 of which are newly produced compared to non-infected wheat. Six characteristic volatiles, namely dodecane, pentadecane, hexadecane, heptadecane, 2, 6, 10-trimethylpentadecane, and squalene, may be related to the degradation of lipids and the expression of wheat stress tolerance and underwent significant changes as infestation progressed, according to the VIP value. This study assists in interpreting the effects of rice weevil infestation on wheat at the metabolic level and establishes a foundation for storage inspection based on VOC analysis.

Graphene oxide in low concentrations can change mitochondrial potential, autophagy, and apoptosis paths in two strains of invertebrates with different life strategies

Nanoparticles, like graphene oxide (GO), are particles with unique physiochemical properties that enable their wide application in various areas of life. The effects of GO on individual cell organelles like mitochondria and the effects of interactions are worth investigating, as they can activate multiple cellular processes, such as autophagy or apoptosis. Mitochondrial injury plays an essential role in the majority of cell death routines. In the project, we investigated cell health status measured as mitochondrial inner membrane depolarization, autophagy, and apoptosis induction during long-term GO administration in food (0.02 μg g−1 and 0.2 μg g−1 of food). Two unique Acheta domesticus strains that differ in life strategy were used: wild-type and long-lived at three different life stages (larva, young adult, mature adult). The changes in mitochondrial trans-membrane potential were marked in the wild-type strain. The autophagy was lower in all GO-treated groups in both strains, and the apoptosis was lower in both strains in the mature adult crickets. Low GO concentrations treatment for the whole life, despite mitochondrial dysfunction, may lead to inhibition of autophagy and apoptosis by arresting the cell cycle for the duration of repair, and other repair tools are involved in the process of restoring homeostasis.

Calcification in sea urchin larvae is associated with low metabolic costs.

The energetic costs to generate calcium carbonate skeletons and shells in marine organisms remain largely speculative due to the scarcity of empirical data. However, this information is critical to estimate energetic limitations of marine calcifiers that can explain their sensitivities to changes in sea water carbonate chemistry in past, present and future marine systems. The cost of calcification was evaluated using larval stages of the purple sea urchin, Strongylocentrotus purpuratus. We developed a skeleton re-mineralization assay, in which the skeleton was dissolved in live larvae followed by a re-mineralization over a few days. During skeleton re-mineralization, energetic costs were estimated through the measurement of key metabolic parameters including whole animal metabolic rates, citrate synthase (CS) enzyme activities and mRNA expression as well as mitochondrial densities in the calcifying primary mesenchyme cells (PMCs). Minor increases in a CS activity and a 10-15% increase in mitochondrial densities in PMCs were observed in re-mineralizing larvae as compared to control larvae. Re-mineralization under three different pH conditions (pH 8.1, pH 7.6 and pH 7.1) decreased with decreasing pH accompanied by pronounced increases in CS expression levels and increased mitochondrial densities in PMCs at pH 7.6. Despite a prominent increase in mitochondrial density of primary mesenchyme cells, particularly in the calcifying cohort of this cell type, this work demonstrated a low overall metabolic response to increased mineralization rates on the whole animal level under both, high and low pH conditions. We conclude that calcification in sea urchin larvae is compromised under low pH conditions, associated with low energetic efforts to fuel compensatory processes.

First contribution to the genera Branchiobaetis and Megabranchiella (Ephemeroptera, Baetidae) in China, with descriptions of two new species

Branchiobaetis Kaltenbach, Kluge & Gattolliat, 2022 and Megabranchiella Phlai-ngam & Tungpairojwong, 2022 (Ephemeroptera: Baetidae) are newly recorded in China. Two new species, Branchiobaetis borealissp. nov. based on larval stage and Branchiobaetis megasinussp. nov. based on larval and imaginal stages associated by laboratory rearing, are described. Megabranchiella longusa Phlai-ngam & Tungpairojwong, 2022, previously only distributed from Thailand, is recorded from China for the first time.

A pictorial key to the adult and larval nasal mites (Halarachnidae) of marine mammals

Mites in the family Halarachnidae are common endoparasites infesting the nasal tissues of a variety of marine mammals. These mites are easily transmissible and compromise the health of their hosts, especially in captive environments. While these mites are noted by marine mammal caretakers, they may easily be misidentified due to repeated revisions to halarachnid mite taxonomy and reclassification of misidentified specimens. Species identification currently requires multiple taxonomic keys, knowledge of revisions to species classifications through time, and training in acarology, which is impractical for marine mammal clinicians. Therefore, to summarize the known taxonomy and aid in future identification of halarachnid mites, we present a pictorial key composed of illustrations based on existing literature and images obtained by scanning electron microscopy (SEM) and high-resolution light microscopy (LM). Illustrations are organized into flow charts for the identification of both adult and larval stages. Dorsal shield silhouettes are also provided to facilitate the identification of adults. We hope that this key be used to simplify future taxonomic research, provide a standard for species identification, and aid in the diagnosis of halarachnid infestations in captive and rehabilitated marine mammal populations.

A novel broad-spectrum antibacterial and anti-malarial Anopheles gambiae Cecropin promotes microbial clearance during pupation.

Anophelinae mosquitoes are exposed to a variety of microbes including Plasmodium parasites that cause malaria. When infected, mosquitoes mount versatile immune responses, including the production of antimicrobial peptides. Cecropins are one of the most widely distributed families of antimicrobial peptides in insects and all previously studied Anopheles members are playing roles in adult mosquito immunity. We have identified and characterized a novel member of the Anopheles gambiae cecropin family, cecropin D (CecD), that is uniquely expressed and immune-responsive at late larval stages to promote microbial clearance through its broad-spectrum antibacterial activity during larval-pupal developmental transition. Interestingly, Cecropin D also exhibited highly potent activity against Plasmodium falciparum sporozoites, the malaria parasite stage that is transmitted from mosquitoes and infects humans and thereby holds promise as a malaria transmission-blocking agent. Finally, we have defined unequivocal cecropin-specific molecular signatures to systematically organize the diversity of the cecropin family in malaria vectors.

Micromanagement: conditions influencing antipathogen function of the skin microbiome in spotted salamanders, Ambystoma maculatum

The emerging fungal pathogen Batrachochytrium salamandrivorans (Bsal) threatens the diversity of amphibians, particularly in North America where it is projected to invade. Amphibian skin defenses include a mucosal layer containing microorganisms that can potentially modulate host response to pathogens such as Bsal. In this study, we focused on the composition of the skin microbiome across life stages of spotted salamanders (Ambystoma maculatum). We also evaluated the stress hormone corticosterone and skin microbiome response to inoculations with Bsal and probiotics at both the larval and juvenile developmental stages, and the response to different environmental conditions. Results indicated that both bacterial and fungal communities found on the skin significantly differed in structure and diversity between life stages of A. maculatum. Exposure to three different probiotics (Bacillus thuringiensis, Chryseobacterium rhizoplanae, and Penicillium sp.) and Bsal evoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer of A. maculatum. Despite changes in the microbiome, all tested probiotics and Bsal were unable to persist on the skin. Larval bacterial microbiomes shifted in response to Bsal and B. thuringiensis with no significant impacts on antifungal function or bacteria richness, however fungi strongly responded to Bsal and B. thuringiensis application. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such as B. thuringiensis, a widely used mosquito larvicide. Overall, experimental results indicate that life stage, growth and development, and environmental conditions appeared to be the main factors driving changes in the amphibian skin microbiome and potential anti-Batrachochytrium function.

Hibernation changes tissue specific lipophilic profile of Chilo partellus (Swinhoe)

Chilo partellus undergo hibernation to survive under harsh winter conditions. We investigated changes in lipophilic profiles of larval and pupal whole body and tissues (haemolymph, fat bodies and integument) of hibernation and non-hibernation C. partellus strains. Significantly higher amount of lipids was found in whole body of pre-hibernation and hibernation larvae than non-hibernation larvae. At tissue level, total lipids were significantly higher in haemolymph and fat bodies of pre-hibernation, and integument of non-hibernation larvae. In whole body of larvae and pupae of hibernation and non-hibernation strains, most abundant fatty acids were oleic acid (16.4%–23%), palmitic acid (14.6%–19.3%), linoleic acid (9.3%–11.6%), palmitoleic acid (3.7%–7.3%) and stearic acid (1.7%–2.8%). Further, at tissue level, myristic, palmitoleic, palmitic, linoleic and oleic acids in fat bodies; and stearic acid in the integument of hibernation larvae were higher as compared to other body parts and larval stages of C. partellus. In pupal tissues, myristic, palmitic and stearic acids were higher in the haemolymph and fat bodies of non-hibernation pupae than post-hibernation pupae. Among the unsaturated fatty acids (UFAs), the palmitoleic acid significantly increased in all the larval tissues, and linoleic acid in fat bodies and integument, while oleic acid significantly decreased in integument of hibernation as compared to non-hibernation larvae of C. partellus. These information on adaptive remodelling of lipids at tissue level might help in future physiological, molecular, ecological and pest management studies on C. partellus.

Regulation Mechanisms of the Glutamate Transporter in the Response of Pacific Oyster upon High-Temperature Stress

Glutamate transporters (GLTs) are integral to the glutamatergic system, modulating glutamate homeostasis to enhance resilience and resistance against environmental stress. There are six GLTs identified in the Pacific oyster (Crassostrea gigas), which were categorized into two subfamilies: excitatory amino acid transporters (CgEAATs) and vesicular glutamate transporters (CgVGLUTs). The CgEAATs harbor a GltP domain, while CgVGLUTs feature an MFS domain, both with conserved sequence and structural characteristics. The expression of CgGLTs is elevated during the planktonic larval stage compared to the fertilized egg stage and is constitutively expressed in various tissues of adult oysters, suggesting its critical role in both larval development and the physiological processes of adult oysters. Transcriptomic analysis revealed diverse expression patterns of GLTs in oyster gills after 7 days of high-temperature stress, with CgEAAT3 showing a significant upregulation. A KEGG pathway enrichment analysis identified glutathione metabolism and ferroptosis as prominently enriched pathways. At 48 h after high-temperature stress, the expression levels of Glutathione Peroxidase 4 (CgGPX4) and CgEAAT3, along with elevated Fe content in the gills, significantly increased. Moreover, the RNAi-mediated the inhibition of CgEAAT3 expression under high-temperature stress, resulting in a significant reduction in CgGPX4 expression and a further increase in Fe accumulation in oyster gills. These results indicate that CgEAAT3 contributes to the regulation of ferroptosis and redox homeostasis by modulating CgGPX4 expression. This study provides new insights into the adaptive mechanisms of bivalves to environmental stress.

Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1.

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that direct mRNA repression. miRNAs are also the subject of regulation. For example, some miRNAs are destabilized through a pathway in which pairing to specialized transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. Here, we found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). The impact of EBAX-1 depended on the developmental stage, with the greatest effect on the miRNA pool (14.5%) observed in L1 larvae and the greatest number of different miRNAs affected (17) observed in germline-depleted adults. The affected miRNAs included the miR-35-42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major miRNA destabilization pathway in the worm. The excess miR-35-42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to EBAX-1 loss had no consistent pattern of either trimming or tailing. Replacement of the 3' region of miR-43 substantially reduced EBAX-1 sensitivity, a result that differed from that observed previously for miR-35. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that a role for miRNA 3' sequences is variable in the worm.

Case Report: An exceptional localisation in the thigh of a giant primary subcutaneous hydatid cyst.

Hydatid disease is a zoonotic disease caused by the larval stage of Echinococcus granulosus and is found globally. Although the liver and lungs are the most prevalent sites of involvement, sub-cutaneous hydatid cysts are rare especially in the primary occurrence. Sub-cutaneous hydatid cysts usually are noticed as slow growing, painless, and mobile masses beneath the overlying skin which is usually normal and are generally found on the trunk and proximal extremities. To the best of our knowledge, very few cases of primary subcutaneous hydatid cysts of the thigh have been documented. We present a case of a 30-year-old woman from a rural setting, who has a large, painless swelling on her thigh. Imaging studies confirmed the presence of a primary subcutaneous hydatid cyst, which is an atypical presentation of the disease. The entire lesion was excised surgically without any evidence of recurrence for a long period postoperatively. This case illustrates the difficulty in establishing a diagnosis and the importance of early surgical management in these cases.

Insect responses to seasonal time constraints under global change are facilitated by warming and counteracted by invasive alien predators.

In seasonal environments, organisms with complex life cycles not only contend with seasonal time constraints (TC) but also increasingly face global change stressors that may interfere with responses to TC. Here, we tested how warming and predator stress imposed during the egg and larval stages shaped life history and behavioural responses to TC in the temperate damselfly Ischnura elegans. Eggs from early and late clutches in the season were subjected to ambient and 4°C warming temperature and the presence or absence of predator cues from perch and signal crayfish. After hatching, larvae were retained at the same thermal regime, and the predator treatment was continued or not up to emergence. The late eggs decreased their development time, especially under warming and when not exposed to predator cues. However, the late eggs increased their development time when exposed to predator cues, especially to crayfish cues. The TC decreased survival of late larvae that were as eggs exposed to crayfish cues, indicating a carry-over effect. The TC and warming additively reduced late larvae development time to emergence. Independent of the TC, predator cue effects on development time were stronger during the egg than during the larval stage. The late individuals expressed lower mass at emergence, which mirrored the size difference between field-collected mothers. Warming caused a higher mass at emergence. The late individuals increased their boldness and showed a higher number of moves, whereas warming caused a decreased boldness. There was no predator cue effect on larval behaviour. The results indicate that late individuals compensate for late season egg laying, which is facilitated under warming but counteracted under predation risk, especially when imposed by the crayfish.