Fourth-stage Larvae Research Articles

Prodigiosin, a promising biocontrol agent against Thaumetopoea wilkinsoni (Tams, 1926) (Lepidoptera: Notodontidae)

Serratia marcescens Bizio (Enterobacteriaceae: Serratia) is an entomopathogenic bacterium that produces hydrolytic enzymes and toxins. It also produces a pigment with various biological properties called prodigiosin. The study was conducted at Bilecik Seyh Edebali University in 2023. In this study, the effects of medium, incubation temperature and time on the process of prodigiosin production by S. marcescens strain Se9 and the extraction efficiency of different solvents were optimized for the first time using the orthogonal Taguchi array design. The optimal yield of pigment was achieved by methanol extraction from bacteria grown in tyriptic soy broth medium at 30ºC for 96 hours. The yield of prodigiosin pigment was 83.4±1.7 mg/L in the validation experiment conducted under the optimum conditions determined. The insecticidal potential of pigment against the larvae of Thaumetopoea wilkinsoni (Tams, 1926) (Lepidoptera, Notodontidae) was demonstrated for the first time. While the mortality rate in larvae exposed to 1000 ppm of the pigment was only 40%, it was observed that doubling the applied concentration led to a significant increase in larval mortality, reaching 91%. The LC50 value of the pigment for the fourth larval stage of T. wilkinsoni was determined to be 1192 ppm. The study showed that the pigment prodigiosin may be a promising biocontrol agent for the control of T. wilkinsoni.

Biological activities of novel 2-pyrazolin-5-one derivatives and their toxicity on certain pests infesting field crops in laboratory conditions

A variety of highly potent derivatives of 2-pyrazoline-5-one, including chromene-3-carbohydrazide 2, 2-thioxothiazole-5-carbohydrazide 3, 2-oxopyridine-3-carbonitriles (4–6), 3-aryl-2-cyanoacrylohydrazide (7, 8), and 3-amino-5-arylpyrazole-4-carbohydrazide (9, 10), were synthesized as pesticide agents using acetohydrazide 1, a versatile and easily obtainable compound. The compounds structures were entirely ascertained utilizing different spectroscopic methods, including Fourier-transform infrared (FTIR), nuclear magnetic resonance spectroscopy, and analysis of elements. In the laboratory, we assessed the impact of nine 2-pyrazoline-5-one derivatives on the fourth larval stage of the cotton leaf worm (Spodoptera littorals), Monacha obstructa, and Tetranychus urticae adults. We used abamectin as the reference compound. The LC50 values of compound 3 for S. littorals, M. obstructa, and T. urticae are 0.90, 0.70, and 0.52 mg a.i./L, respectively. The LC50 values for the same compound for S. littorals, M. obstructa, and T. urticae are 0.80, 0.60, and 0.45 mg a.i./L, respectively. Therefore, it is a remarkably potent compound. Compound 6 exhibited lower levels of danger, as indicated by LC50 values of 1.94, 1.90, and 1.83 mg a.i./L against the three tested pests. Ten days after treatment, all three pests were moderately toxic to the remaining 2-pyrazolin-5-one derivatives, 1, 2, 4, 5, and 7–10. Conversely, the mortality rate of the tested compounds increased when the treated individuals were exposed to high concentrations. These 2-pyrazolin-5-one derivatives are suggested as suitable alternatives and foundational structures for developing novel insecticides.

Myocyte enhancer factor 2 exerts a pivotal role in larval development in Henosepilachna vigintioctopunctata

Myocyte enhancer factor-2 (MEF2) plays essential roles in a variety of developmental processes. Prominent amongst multiple MEF2 functions is the regulation of muscle differentiation in Drosophila melanogaster. Nevertheless, the physiological importance of MEF2 has not been explored in non-Drosophilid insect species. Here we found HvMef2 was highly expressed at the first-instar larvae, 0-day-old second- and fourth-instar larvae, pupae and adults, especially in adult muscle in a Coleopteran potato defoliator Henosepilachna vigintioctopunctata. Injection of 200, 500 or 1000 ng of dsMef2 into the third instar larvae significantly reduced the expression of HvMef2 and impaired larval-pupal-adult transition, in a dose-dependent manner. About 68 %, 77 % and 87 % of the resulting larvae arrested as stunted prepupae, which were gradually blackened and eventually died. Around 32 %, 23 % and 13 % of the HvMef2 depleted beetles formed pupae. Among these HvMef2 depleted pupae, 22 %, 16 % and 7 % became shriveled pupae until death; roughly 10 %, 7 % and 6 % emerged as malformed adults. The growth of the adult thorax muscles was repressed. The length and width of dorsal–ventral muscles were significantly reduced. Knockdown of HvMef2 at the fourth larval instar stage caused similar defects. Likewise, introduction of dsMef2 at a dose of 200, 500 or 1000 ng into pupae led to 27 %, 20 % and 11 % emergence percentages respectively, with deformed elytra and hindwings. Therefore, MEF2 plays vital roles during metamorphosis, especially in myogenic regulation network in H. vigintioctopunctata.

A review of moxidectin vs. other macrocyclic lactones for prevention of heartworm disease in dogs with an appraisal of two commercial formulations.

Macrocyclic lactones (MLs) are the only drug class currently licensed for heartworm disease prophylaxis. Macrocyclic lactones kill third- and fourth-stage larvae of Dirofilaria immitis, thus preventing the development of adult worms in dogs, which are responsible for heartworm disease, a potentially life-threatening condition. Despite considerable overlap in terms of endectocide spectrum, several important differences distinguish moxidectin from other MLs. Moxidectin has beneficial pharmacokinetic characteristics, such as a longer half-life and greater tissue distribution compared to ivermectin. Additionally, moxidectin has a greater margin of safety compared to ivermectin in dogs with ABCB1 (previously MDR1) gene-defect, which is commonly recognized in collies and other breeds. Multiple laboratory studies have shown that moxidectin is more effective than other commonly used heartworm preventives against resistant strains of D. immitis. This improved efficacy benefits individual dogs and helps reduce the risk of spreading resistant strains within the community. Despite the presence of proven resistant strains in the United States, non-compliance with preventive measures remains a major factor contributing to the diagnosis of heartworm disease in dogs. In retrospective analyses, the oral moxidectin combination product Simparica Trio® (sarolaner, moxidectin, and pyrantel) was associated with increased compliance, resulting in more time of protection compared to dogs receiving flea/tick and heartworm preventive products separately. Compliance with the extended-release moxidectin injectables ProHeart® 6 and ProHeart® 12 was higher than with monthly heartworm preventives, as they provide 6 months or a full year of protection with one single injection, respectively, and revenues remain in the veterinary clinics as injectable moxidectin cannot be sourced through online retailers.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Distribution and Identification of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) in the Kurdistan Region of Iraq

The specimens utilized for this investigation comprise adult individuals of Culex pipiens and their immature forms. The Culex pipiens mosquitoes are captured using a net trap, while larvae are collected using conventional dipping techniques. The samples are selected at random from fourteen locations in the provinces of the Kurdistan area of Iraq, specifically Erbil, Duhok, Sulaimaniyah and Halabja. From September 15, 2020 to November 20, 2021, a total of 291 female Culex pipiens mosquitoes were collected from the four provinces stated above. The breakdown of the samples is as follows: 135 from one province, 82 from another province, 23 from a third province, and 51 from the fourth province. The diagnosis key is provided for adult individuals and fourth-stage larvae, which originate from a species of mosquito. To illustrate this study, a concise morphological description was utilized, and photos were included to provide a vivid depiction.The findings of the present investigation indicate that adult females had a greater wing length compared to males. The wing length of females ranged from 3.3 to 4 mm, with an average of 3.55 mm. When adult Culex pipiens were looked at, the costa and sub-costal intersections were found at or beyond the point where R2+3 splits in most of the samples. The siphons of fourth-instar larvae are elongated and taper gradually towards the end. On average, their length is 1.4 mm, with a range of 1.3 to 1.5 mm.. KEYWORDS :Culicidae, Culex pipines, Diptera, Kurdistan Region, Iraq, Survey.

Assessment of development and consumption rate of Macrolophus caliginosus Wagner predator on different prey stages

BackgroundMacrolophus caliginosus Wagner is one of the most important polyphagous predatory mirid bug that attack many agriculture insect pests.MethodsThe present study was carried out to show the effect of different prey stages as Tuta absoluta (eggs and fourth larval stage), Bemisia tabaci (eggs and three nymph stages) and Aphids gossypii (1st nymph stage) on the life cycle and consumption rate of male and females of M. caliginosus as predator under laboratory condition. The experiments were started by collecting the first stage of M. caliginosus nymph and placed separately on acalypha plants situated on saturated cotton pads inside Petri dishes with an excess of each prey.ResultsThe results showed that the developmental period of the total immature stages of M. caliginosus female are affected by type and stage of tested prey. So, the duration of larval stages of female was significantly recorded the longest in case of feeding on eggs of T. absoluta compare to 1st nymph of A. gossypii. In addition to extension the adult longevity of female M. caliginosus, which fed on the 1st nymphal instar of B. tabaci more than the other pests. The consumption rate of total immature stages and adult M. caliginosus female were fed on eggs of B. tabaci was more than the other test pests. While the consumption rate of total immature stages and adult of male M. caliginosus were fed on eggs of B. tabaci was higher than that of the other pests.ConclusionsThe search was given the highlight focus on ability of mirid bugs to consume different insect species “stages” that might be facilitate rear predatory bugs under laboratory condition.

First ontogenesis and bioecological study of Sciobia bouvieri (Orthoptera, Gryllidae), a North African endemic species

This study aimed to investigate the bioecology and life cycle of the Algerian endemic Sciobia bouvieri, which remained unknown until recently. A combined approach of field sampling through direct searching and captive rearing of S. bouvieri nymphs under natural conditions was employed to determine the population ontogeny. The life cycle was found to comprise three stages: egg, nymph, and adult. The embryonic development period ranged from 22 to 30 days. Females laid an average of 9.4 ± 1.3 eggs per clutch. The nymphal stage consisted of six instars, lasting an average of 306.5 days. Adult longevity varied, with males living 32.3 ± 16.2 days and females living 23.9 ± 22.4 days. The duration of the ontogenesis cycle of Sciobia bouvieri in nature showed that it has a univoltine life cycle with summer and winter diapauses occurring during the fourth and fifth larval stages, respectively. These diapauses are likely controlled by photoperiod and temperature. This is the first study of the bioecology and life cycle of this North African endemic species.

Ascaris suum infection in juvenile pigs elicits a local Th2 response in a setting of ongoing Th1 expansion.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficientlylarge L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Evaluating Mosquito Biocontrol Effectiveness by Isolating and Characterizing Some Fungi Against Culex Pipiens and Anopheles Stephensi in Sothern Part of Iraq

Objectives: Isolation Aspergillus tamarii, Cladosporium herbarum, and Verticillium lecanii fungi from naturally infected Culex pipiens and Anopheles stephensi insects were morphologically and molecularly identified. Methods: In this study, populations of mosquitoes were cultured and examined to determine fungal infection and evaluated as potential agents against C. pipiens and A. stephensi. Results: A variety of fungal isolates demonstrated differing degrees of pathogenicity 24 hours after treatment against C. pipiens and A. stephensi eggs, four-instar mosquito larvae, and adults. and as a biological control, it was found that the fungal suspension of each of the used fungi affected the life roles of the two mosquitoes. as it was more than the fungus suspension A. tamarii, Cl. herbarum on V. lecanii. The highest percentage of eggs mortality were (36.72, 48.97) %, (29.14, 42.25) %, and (24.45, 35.70) % of C. pipiens and A. stephensi when using the highest concentration of 1*105 spore/ml, 3*105 spore/ml and 2*105 spore/ml of fungicide A. tamarii, C. herbarum, V.lecanii and respectively after 24 hours. The highest mortality rate was for the fourth larval stages, and the mosquito A. stephensi is more sensitive to type C. pipiens infection., as, in order (75.6, 67.19, 56.8) %, (74.18, 59.81, 50) %, and (65, 52,42.71) %. Conclusions: Results highlight the significance of a mosquito's natural fungal opponent. All isolates had an impact on adults and larvae, although they were less successful against eggs. Both have the potential to develop, particularly against the larvae of the primary arbovirus, malaria, and lymphatic filariasis vectors, A. stephensi, and C. pipiens.

Riparian invader: A secondary metabolite of Impatiens glandulifera impairs the development of the freshwater invertebrate key species Chironomus riparius

Invasive species represent a significant threat to native biodiversity. The Himalayan Balsam Impatiens glandulifera is an annual plant, which is invasive in Europe and often inhabits the riparian zone. It produces several secondary metabolites causing, for example, growth inhibition of terrestrial plants and invertebrates. One of these metabolites is the quinone 2-methoxy-1,4-naphthoquinone (2-MNQ). The compound gets washed out from the above-ground parts of the plant during precipitation and may then leach into nearby waterbodies. Despite some evidence for the allelopathic effect of plant secondary metabolites on terrestrial invertebrates, little is known about how 2-MNQ affects the survival or development of aquatic dipteran larvae, despite the importance of this functional group in European freshwaters. Here, we investigated the effects of 2-MNQ on larvae of the river keystone species Chironomus riparius in acute and chronic scenarios. The toxicity of 2-MNQ towards the first and the fourth larval stage was determined in a 48-hour acute exposure assay. We show that 2-MNQ has a negative impact on the development, growth and survival of C. riparius. The LC50 of 2-MNQ was 3.19 mg/l for the first instar and 2.09 mg/l for the fourth instar. A ten-day chronic exposure experiment, where the water was spiked with 2-MNQ, revealed that 2-MNQ had a significantly negative impact on larval body size, head capsule size, body weight, development and survival. These results demonstrate the negative impact of the secondary metabolite 2-MNQ from the terrestrial plant I. glandulifera on a crucial macroinvertebrate inhabiting the adjacent stream ecosystem in riverine ecosystems. This may lead to a decline in population size, resulting in cascading effects on the food web.

Molecular identification of suppressor of cytokine signaling 2 (SOCS2) and its response to BmNPV in Bombyx mori (Lepidoptera: Bombycidae)

The suppressor of cytokine signaling 2 (SOCS2), an essential member of the SOCS family, is a classic negative regulator of the JAK-STAT-IFN signaling pathway. Current research focuses on mammalian systems, particularly in humans, with comparatively less emphasis on invertebrate models. We discovered two unique isoforms of SOCS2 in Lepidoptera, such as Bombyx mori, named SOCS2S and SOCS2L, respectively. The protein sequences of the two isoforms differ only in the N-terminal region. SOCS2 expression profiles in Bombyx mori are highest during the embryo or the first larval instar stage, decreasing from the second to the fourth larval instar stage and reaching the lowest point during the fifth larval instar stage. Its expression is up-regulated during metamorphosis development stages, indicating that they mainly play roles in embryonic development and metamorphosis. An exception is observed in the expression level of SOCS2L in silkworm strain 306, which does not show a downward trend during the larval stage. The response modes of two SOCS2 proteins to Bombyx mori nucleopolyhedrovirus (BmNPV) are different, with SOCS2S showing a more intense response. Subcellular localization shows that they exist in the cytoplasm. The study provides a foundation for further study on the function of SOCS2 in silkworms.

GC/GC-MS analysis and biological activities of Lantana Camara Linn.

Medicinal plants have been a part of human history for thousands of years and are still used as healthcare throughout the world. The current research aims to explore the chemical constituents of the methanol soluble extract (LC-Me) and petroleum ether soluble fraction (LCM-PES) from the leaves of Lantana camara Linn by GC/ GC-MS. This chemical analysis revealed the existence of 16 and 23 phytoconstituents in LC-Me and LCM-PES respectively. The major constituents in LC-Me were found to beethyl 9,12,15-octadecatrienoate (31.9%), hexadecanoic acid, ethyl ester (12.6%), n-hexadecanoic acid (11.1%), linoleic acid ethyl ester (9.1%), squalene (8.7%), di-n-octyl phthalate (6.2%), 9,12-octadecadienoic acid (Z,Z)- (4.2%), (E)-9-octadecenoic acid ethyl ester (2.7%) andcyclopropanebutanoicacid,2-[[2-[[2-[(2-pentylcyclopropyl)methyl]cyclopropyl]methyl]cyclopropyl]methyl]-, methyl ester (2.3%). The chief bioactive compounds in petroleum ether soluble fraction were found to beandrost-8-en-3-ol, 4,4,14α-trimethyl-17-(2-bromo-1-methylethyl (57.9%), 14,17-nor-3,21-dioxo-β-amyrin, 17,18-didehydro-3-dehydroxy- (13.0%), barringtogenol B (2.5%), olean-12-ene-3,16,21,22,28-pentol, 21-(2-methyl-2-butenoate), [3β,16α,21β(Z),22α]-(1.7%),perhydrocyclopropa[e]azulene-4,5,6-triol, 1,1,4,6-tetramethyl (1.7%), ethyl iso-allocholate (1.6%) and 1,2-benzenedicarboxylic acid, diisooctyl ester (1.6%). Both the extract and its fraction have exhibited very significant antibacterial, antifungal,mosquito repellent and larvicidal propertiesoriginated by numerous bioactive metabolites. Twenty eight (20 Gram-positive and 8 Gram-negative) bacteria were tested against LC.Me and LCM-PESwith noteworthy zone of inhibition.The significant in vitro antifungal activity was observed against fifteen fungi in LC-Me and LCM-PES. Very robust initial repellency was observed for LC-Me and LCM-PES (94% and 80% respectively) against the dengue-carrying mosquito (Aedes aegypti) at 2% concentration. The extract and its fraction were also found to be an efficient larvicidal agent against fourth-stage larvae of Aedes aegypti. The effective larvicidal property was noted in methanol soluble extract as compared topetroleum ether soluble fraction and standard with LC50value of 20 and 400 ppm respectively.

Functional Characterization of CYP6QE1 and CYP6FV21 in Resistance to λ-Cyhalothrin and Imidacloprid in Bradysia odoriphaga.

Cytochrome P450 monooxygenases (P450s) belong to a family of metabolic enzymes that are involved in the detoxification of insecticides. In this study, our bioassay results showed that a field-collected population of Bradysia odoriphaga displayed a moderate resistance to λ-cyhalothrin and imidacloprid. Compared to susceptible population, CYP6QE1 and CYP6FV21 were significantly overexpressed in the field population. The expression of CYP6QE1 and CYP6FV21 was more abundant in the third and fourth larval stages, and CYP6QE1 and CYP6FV21 were most highly expressed in the midgut and Malpighian tubules. Exposure to λ-cyhalothrin and imidacloprid significantly increased the expression levels of CYP6QE1 and CYP6FV21. Furthermore, the silencing of CYP6QE1 and CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to λ-cyhalothrin and imidacloprid. The overexpression of CYP6QE1 and CYP6FV21 significantly enhanced the tolerance of transgenic Drosophila melanogaster lines to λ-cyhalothrin and imidacloprid. In addition, molecular docking revealed that these two P450 proteins have strong binding affinity toward λ-cyhalothrin and imidacloprid insecticides. Taken together, these results indicate that the overexpression of CYP6QE1 and CYP6FV21 is responsible for resistance to λ-cyhalothrin and imidacloprid in B. odoriphaga.

Analysis of P-gp genes relative expression associated to ivermectin resistance in Haemonchus contortus larval stages from in vitro cultures (L3 and xL3) and from gerbils (Meriones unguiculatus) (L4) as models of study.

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes (Hco-pgp) between fourth (L4), infective (L3), and transitory infective (xL3) larval stages as laboratory models to study ivermectin (IVM) resistance. The H. contortus resistant to IVM (IVMr) and susceptible to IVM (IVMs) strains were used to develop xL3in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L4 stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL3 and L4 was determined between comparison of IVMr stages and from IVMr vs IVMs stages. Seven Hco-pgp genes (1, 2, 3, 4, 9, 10, and 16) were analysed by RT-qPCR using L3 stage as control group, per strain, and GAPDH and β-tubulin as constitutive genes. Morphological changes were confirmed between xL3 and L4 developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences (p < 0.05). The Hco-pgp1, 2, 3, and 4 genes (p < 0.05) were upregulated from 7.1- to 463.82-fold changes between IVMr stages, and Hco-pgp9 (13.12-fold) and Hco-pgp10 (13.56-fold) genes showed differences between L4 and xL3, respectively. The comparative study between IVMr vs IVMs strains associated to xL3 and L4 displayed significant upregulation for most of the Hco-pgp genes among 4.89-188.71 fold-change. In conclusion, these results suggest the use of H. contortus xL3 and L4 as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

Occurrence of Hysterothylacium Species (Nematoda: Raphidascarididae) from Some Marine Fishes from Iraqi Waters

Parasitological investigation of 22 species of marine fishes in territorial Iraqi waters, during the period from October 2019 till September 2020, revealed recording of six different species of Hysterothylacium Ward &amp; Magath, 1917 in different developmental stages. These nematodes including two species in adult stage of Hysterothylacium viz, H. reliquens (Norris &amp; Overstreet, 1975) and Hysterothylacium sp. (females only), two species of fourth larval stages (Hysterothylacium sp. type BH and Hysterothylacium sp. type BI) and two species of third larval stages (Hysterothylacium sp. type BA and Hysterothylacium sp. type BC). Identification of Hysterothylacium sp. was based on female only isolated from Blackfin stonefish Pseudosynanceia melanostigma Day, 1875; Hysterothylacium sp. type BH 4th larval stage isolated from Arabian backspot threadfin Polydactylus mullani (Hora, 1926) and Hysterothylacium sp. type BI 4th larval stage from purple-spotted bigeye Priacanthus tayenus Richardson, 1846; all these parasitic nematodes recorded for the first time in the Arabian Gulf. In addition, 14 fish species are considered as new hosts for Hysterothylacium sp. type BC 3rd larval stage. Description of female Hysterothylacium sp., Hysterothylacium sp. type BH and BI both 4th larval stages are given. The interest record of inverse intestinal caecum in some specimens of 3rd larval stage of Hysterothylacium type BC in some marine fishes of the current study is given.

Bioefficacy of the ethanolic crude extract of the wild leek, Allium ampeloprasum L. (Amaryllidaceae), against the third and fourth larval stages of Aedes aegypti L. (Culicidae)

Insecticide resistance is a threat to vector control worldwide. In the Philippines, dengue burden remains significant, thus prioritizing the need to develop eco-friendly control strategies and tools against mosquito vectors. Various Allium species have been found to possess larvicidal activity against dengue-carrying mosquitoes. In this study, the larvicidal activity of the crude extract of Allium ampeloprasum L. (Asparagales: Amaryllidaceae) was studied in concentrations ranging from 1 to 10,000 mg/L against the third (L3) and fourth (L4) larval instars of Aedes aegypti L. Larval mortality at 48 h were subjected to probit analysis and Kruskal-Wallis H test to estimate lethal concentrations and to determine significant means among the groups, respectively. Results show that the crude extract of A. ampeloprasum L. demonstrated larvicidal activity against the L3 and L4 Ae. aegypti L. Concentrations corresponding to 50% mortality (Lethal Concentration 50 [LC50]) among L3 and L4 larvae were estimated at 2,829.16 and 13,014.06 mg/L, respectively. Moreover, 90% mortality (LC90) in the L3 and L4 larvae were estimated at 9,749.75 and 57,836.58 mg/L, respectively. Only 1,000 and 10,000 mg/L for L3, and the 10,000 mg/L for L4, had comparable larvicidal action to the commercial larvicide used as a positive control. The results support the presence of bioactive compounds with larvicidal properties, thus suggesting A. ampeloprasum L. as a potential source of active ingredients for the development of a plant-based larvicide.

Hypobiosis and Development of Haemonchus contortus and Trichostrongylus colubriformis Infection in Lambs under Different Levels of Nutrition

The influence of four levels of dietary supplementation on the development of the immune response and on the biology of Haemonchus contortus and Trichostrongylus colubriformis was evaluated in Dorper lambs under serial artificial infections with 1000 infective larvae (L3) of each species; the supplement was given every 3 days for 12 weeks. For each of the four diets formulated, one infected group (n = 7) and one control group (n = 4) were set up. Diets 1, 2, 3, and 4 contained 0%, 25%, 50%, and 75% of concentrate, respectively. There was an evident immune response to infection in all groups, manifested through eosinophilia and the production of anti-L3 immunoglobulins. There was a significant inverse relationship between the levels of nutrients in the diets and the numbers of late fourth-stage larvae, immature adult stages, and adults of H. contortus. Following 12 weeks of serial infections, most of the H. contortus population was found at the early fourth stage, indicating the occurrence of hypobiosis. The early fourth stage represented 59.5%, 75.8%, 83.3%, and 86.3% of the total Haemonchus worm burden, respectively, in Diets 1, 2, 3, and 4. In the case of T. colubriformis, hypobiosis was not observed, and diet had no influence on the establishment of parasites.

Anisakid Presence in the European Conger, Conger conger, from Spanish Mediterranean Waters.

The European conger, Conger conger, is a benthic marine fish species with a geographical distribution extending through the northeastern Atlantic and the Mediterranean. Despite being extensively distributed and widely appreciated by Spanish consumers, studies regarding parasite presence in this fish are scarce. In the present work, a hundred and eight specimens from the Mediterranean coast of northeastern Spain (Catalan waters) were surveyed for the presence of nematode parasites. Several species were morphologically identified: third-stage larvae of Anisakis type I (sensu Berland, 1961) (n = 131), third-stage larvae of Anisakis type II (sensu Berland, 1961) (n = 18), third- and fourth-stage larvae and adults of Hysterothylacium spp. (n = 48), adults of Cucullanus sp. (n = 391), and adults of Cristitectus congeri (n = 69). Moreover, some fish and decapode species were also observed as part of the host's diet, with the most detected preys being Micromesistius poutassou, Sardina pilchardus, Macropipus sp., and Goneplax rhomboides. This represents the first survey of nematode parasites infecting C. conger from the northeastern Spanish Mediterranean waters. Among the parasite species detected, the presence of Anisakis species should be highlighted as the ingestion of C. conger parasitized with these larvae could potentially lead to anisakiasis in consumers.

Physiological Effects of Oxidative Stress Caused by Saxitoxin in the Nematode Caenorhabditis elegans.

Saxitoxin (STX) causes high toxicity by blocking voltage-gated sodium channels, and it poses a major threat to marine ecosystems and human health worldwide. Our work evaluated the neurotoxicity and chronic toxicology of STX to Caenorhabditis elegans by an analysis of lifespan, brood size, growth ability, reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, and the overexpression of green fluorescent protein (GFP). After exposure to a series of concentrations of STX for 24 h, worms showed paralysis symptoms and fully recovered within 6 h; less than 5% of worms died at the highest concentration of 1000 ng/mL for first larval stage (L1) worms and 10,000 ng/mL for fourth larval stage (L4) worms. Declines in lifespan, productivity, and body size of C. elegans were observed under the stress of 1, 10, and 100 ng/mL STX, and the lifespan was shorter than that in controls. With STX exposure, the productivity declined by 32-49%; the body size, including body length and body area, declined by 13-18% and 25-27%, respectively. The levels of ROS exhibited a gradual increase over time, accompanied by a positive concentration effect of STX resulting in 1.14-1.86 times higher levels compared to the control group in L4 worms. Conversely, no statistically significant differences were observed between L1 worms. Finally, after exposure to STX for 48 h, ATP levels and GFP expression in C. elegans showed a significant dose-dependent increase. Our study reports the first evidence that STX is not lethal but imposes substantial oxidative stress on C. elegans, with a dose-responsive relationship. Our results indicated that C. elegans is an ideal model to further study the mechanisms underlying the fitness of organisms under the stress caused by paralytic shellfish toxins including STX.