A Literature Review of Biological and Bio-Rational Control Strategies for Slugs: Current Research and Future Prospects.

Terrestrial gastropod molluscs (slugs and snails) (Mollusca: Gastropoda) cause significant crop damage around the world. There is no formal approach for differentiating between slugs and snails; however, an organism is usually considered a slug when there is no external shell, or when the shell is small in comparison to the body, and a snail when there is a large external shell. Although snails are an important pest of many crops, this review focuses on slug pests and their nonchemical control measures. A recent study by the UK Agriculture and Horticulture Development Board concluded that the failure to control slugs could cost the UK agriculture industry over GBP 100 million annually, with similar figures reported around the world. Whilst slugs are mostly controlled using chemical molluscicide products, some actives have come under scrutiny due to their detrimental environmental effects and impact on nontarget organisms. This has resulted in the ban of actives such as methiocarb in the UK and EU, and, more recently, the ban of metaldehyde in the UK. Therefore, there is an urgent need to find alternative and effective nontoxic solutions in the interest of global food security. In this paper, we have integrated extant literature on the three main biological control agents of slugs, namely nematodes, carabid beetles and sciomyzid flies, and various promising bio-rational slug control strategies. The review also highlights current research gaps and indicates some relevant potential future directions towards developing environmentally benign slug control solutions.

Phasmarhabditis safricana n. sp. (Nematoda: Rhabditidae), a parasite of the slug Deroceras reticulatum from South Africa.

During a survey for mollusc-associated nematodes in South Africa, a new Phasmarhabditis species was isolated from the invasive slug, Deroceras reticulatum, collected from a nursery near George in the Western Cape province. The nematode was identified using a combination of morphological, morphometric, molecular, and phylogenetic techniques. The new species, P. safricana n. sp., is characterised by the cupola-shaped tail of the female with a spike, small, non-protruding phasmids, a fingerprint-like pattern of the cuticle covering the female tail, toothlike cephalic structures of the infective juveniles, and the distinct molecular characteristics of the species. The molecular phylogeny of the new species, as inferred from its SSU and LSU rRNA gene, places P. safricana n. sp. in close proximity to P. papillosa. Virulence tests were conducted, which demonstrated that P. safricana n. sp. caused significant mortality to the European invasive slug, D. reticulatum. The new species brings the total complement of the genus to eleven species.

Pregnancy and oestrogen regulate sinoatrial node calcium homeostasis and accelerate pacemaking.

Aims: During pregnancy, there is a significant increase in heart rate (HR) potentially associated with an increased risk of arrhythmias or exacerbation of pre-existing cardiac conditions endangering both mother and foetus. Calcium homeostasis plays an important role in regulating automaticity of the sinoatrial node (SAN); however, its contribution to the accelerated HR during pregnancy remains unknown. Methods and results: Using murine SAN cells, we showed that pregnancy increased L-type Ca2+ current (ICaL) and CaV1.3 mRNA expression, whereas T-type Ca2+ current (ICaT) and its underlying channel were unchanged. Analysis of SAN intra-cellular Ca2+ oscillations showed that the rate of spontaneous Ca2+ transients was significantly higher in pregnant mice along with a higher mRNA expression of ryanodine receptor. Assessment of supra-ventricular arrhythmias using programmed electrical stimulation protocols on anaesthetized mice revealed higher susceptibility in pregnancy. Of note, the modifications associated with pregnancy were reversible following delivery. Furthermore, chronic administration of 17ß-estradiol (E2) to nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), control mice, oestrogen-receptor-ß knockout (ERKOß) but not ERKOα mice, accelerated cardiac automaticity, recapitulating the pregnancy phenotype in both mouse and human SAN cell models. Conclusion: Together, these results indicate that pregnancy considerably alters intra-cellular Ca2+ homeostasis sustaining faster HR during pregnancy. Importantly, these changes were dependent on an oestrogen receptor α (ERα) mechanism that resulted in increased ICaL and spontaneous Ca2+ release from the sarcoplasmic reticulum, highlighting a novel role for oestrogen in regulating HR.

Angiostoma norvegicum n. sp. (Nematoda: Angiostomatidae) a parasite of arionid slugs in Norway.

Angiostoma norvegicum n. sp. (Angiostomatidae) is described from the oesophagus, crop and the buccal mass of five species of slugs of the family Arionidae, Arion vulgaris (Moquin-Tandon), Arion ater (L.), Arion fasciatus (Nilsson), Arion fuscus (Müller) and Arion rufus/Arion ater hybrid), collected throughout Norway. Angiostoma norvegicum n. sp. was found parasitising arionids at seven of the 30 sample sites examined (23.3%), and 9.9% of all Arion spp. were infected with this nematode. The new species is characterised by its large size (4.0-8.6 mm long) and in having: lateral alae; 6 + 6 papillae at the cephalic end; a large circular mouth aperture; a spacious stoma; a pharyngeal basal bulb without valvular apparatus; an excretory pore near the base of bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1+2/3+3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses demonstrated that although Angiostoma spp. cluster together, A. norvegicum n. sp. forms a tight monophyletic clade with the milacid nematode parasites Angiostoma margaretae Ross, Malan & Ivanova, 2011 and Angiostoma milacis Ivanova & Wilson, 2009.

LKB1 deletion causes early changes in atrial channel expression and electrophysiology prior to atrial fibrillation.

AIMS: Liver kinase B1 (LKB1) is a protein kinase that activates the metabolic regulator AMP-activated protein kinase (AMPK) and other related kinases. Deletion of LKB1 in mice leads to cardiomyopathy and atrial fibrillation (AF). However, the specific role of the LKB1 pathway in early atrial biology remains unknown. Thus, we investigated whether LKB1 deletion altered atrial channel expression and electrophysiological function in a cardiomyocyte-specific knockout mouse model. METHODS AND RESULTS: We performed a systematic comparison of αMHC-Cre LKB1(fl/fl) and littermate LKB1(fl/fl) male mice. This included analysis of gene expression, histology, and echocardiography, as well as cellular and tissue-level electrophysiology using patch-clamp recordings in vitro, optical mapping ex vivo, and ECG recordings in vivo. At postnatal day 1, atrial depolarization was prolonged, and Nav1.5 and Cx40 expression were markedly down-regulated in MHC-Cre LKB1(fl/fl) mice. Inward sodium current density was significantly decreased in MHC-Cre LKB1(fl/fl) neonatal atrial myocytes. Subsequently, additional alterations in atrial channel expression, atrial fibrosis, and spontaneous onset of AF developed by 2 weeks of age. In adult mice, abnormalities of interatrial conduction and bi-atrial electrical coupling were observed, likely promoting the perpetuation of AF. Mice with AMPK-inactivated hearts demonstrated modest overlap in channel expression with MHC-Cre LKB1(fl/fl) hearts, but retained normal structure, electrophysiological function and contractility. CONCLUSIONS: Deletion of LKB1 causes early defects in atrial channel expression, action potential generation and conduction, which precede widespread atrial remodelling, fibrosis and AF. LKB1 is critical for normal atrial growth and electrophysiological function.

Age and ovariectomy abolish beneficial effects of female sex on rat ventricular myocytes exposed to simulated ischemia and reperfusion.

Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca(2+) in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5-6-month-old rats that had an ovariectomy at 3-4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca(2+) and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca(2+) transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.

Angiostoma margaretae n. sp (Nematoda: Angiostomatidae), a parasite of the milacid slug Milax gagates Draparnaud collected near Caledon, South Africa.

Angiostoma margaretae n. sp. (Angiostomatidae) is described from the oesophagus of the slug Milax gagates Draparnaud collected near Caledon in the Western Cape Province of South Africa. The new species closely resembles another parasite of a milacid slug, A. milacis Ivanova & Wilson, 2009, with a similar head, stoma and spicule shape, the presence of distally outstretched ovaries, coiled oviducts, the same number of caudal papillae and enlarged rectal glands. However, A. margaretae differs from the latter by having: a shorter, wider tail with a rounded vs pointed tip; the distal parts of both ovaries with a particular hook-like shape due to an expansion closely following the short initial zone; ovoviparous females; and a different arrangement of male papillae. A. margaretae is comparable with A. limacis Dujardin, 1845, A. asamati (Spiridonov, 1985), A. coloaense (Pham Van Luc, Spiridonov & Wilson, 2005) and A. stammeri (Mengert, 1953), which have a similar stoma shape and size, but can be readily differentiated by the presence of distally outstretched vs reflexed ovaries and the presence vs lack of enlarged rectal glands. The new species has a similar arrangement of the ovaries to A. kimmeriense Korol & Spiridonov, 1991 and A. zonitidis Ivanova & Wilson, 2009, but is clearly differentiated by the lack of an off-set lip region and presence of a large bowl-shaped vs tubular stoma and less numerous male caudal papillae (seven pairs vs nine in A. kimmeriense and 10 in A. zonitidis).

Sex differences in mechanisms of cardiac excitation-contraction coupling in rat ventricular myocytes.

Components of excitation-contraction (E-C) coupling were compared in ventricular myocytes isolated from 3-mo-old male and female rats. Ca(2+) concentrations (fura-2) and cell shortening (edge detector) were measured simultaneously (37 degrees C). Membrane potential and ionic currents were measured with microelectrodes. Action potentials were similar in male and female myocytes, but contractions were smaller and slower in females. In voltage-clamped cells, peak contractions were smaller in females than in males (5.1 +/- 0.7% vs. 7.7 +/- 0.8% diastolic length, P < 0.05). Similarly, Ca(2+) transients were smaller in females than in males and the rate of rise of the Ca(2+) transient was slower in females. Despite smaller contractions and Ca(2+) transients in females, Ca(2+) current density was similar in both groups. Sarcoplasmic reticulum Ca(2+) content, assessed with caffeine, did not differ between the sexes. However, E-C coupling gain (rate of Ca(2+) release/Ca(2+) current) was smaller in females than in males (157.0 +/- 15.6 vs. 338.4 +/- 54.3 (nM/s)/(pA/pF), P < 0.05). To determine whether the reduced gain in female cells was due to changes in unitary Ca(2+) release, spontaneous Ca(2+) sparks were evaluated (fluo-4, 37 degrees C). Spark frequencies and widths were similar in both groups, but spark amplitudes were smaller in females than in males (0.56 +/- 0.01 vs. 0.64 +/- 0.01 DeltaF/F(0), P < 0.05). Spark durations also were shorter in females than in males (full duration at half-maximum = 14.86 +/- 0.17 vs. 16.25 +/- 0.27 ms, P < 0.05). These observations suggest that decreases in the size and duration of Ca(2+) sparks contributes to the decrease in E-C coupling gain in female myocytes. Thus, differences in cardiac contractile function arise, in part, from differences in unitary Ca(2+) release between the sexes.

Molecular phylogeny of slug-parasitic nematodes inferred from 18S rRNA gene sequences.

Terrestrial molluscs are diverse and are infected by many nematodes. We propose a phylogeny of slug-parasitic nematodes using 18S rRNA gene sequences from nematodes isolated from slugs collected from six countries. Eight species, representing six families of nematodes were identified and trees inferred placed them within four (I, III, IV and V) out of the five clades of Nematoda, indicating multiple origins of slug parasitism. Five species representing three families formed a monophyletic group in clade V. Although these species are closely related, their morphology has changed greatly, suggesting adaptive radiation to fill different niches within the host.

Beta-adrenoceptor stimulation exacerbates detrimental effects of ischemia and reperfusion in isolated guinea pig ventricular myocytes.

We investigated whether beta-adrenoceptor stimulation exacerbates detrimental effects of ischemia and reperfusion on electrical and contractile function and on intracellular Ca(2+) homeostasis in isolated guinea pig ventricular myocytes. Myocytes were exposed to 20 min of simulated ischemia (37 degrees C) in the absence or presence of isoproterenol (10 nM, applied prior to and during ischemia) and reperfused with Tyrode's solution for 30 min. Unloaded cell shortening, Ca(2+) transients (fura-2), and cell viability were recorded at 5 min intervals in field-stimulated cells (2 Hz). In experiments using microelectrodes, membrane potentials, contractions, and transmembrane currents also were recorded at 5 min intervals. In the absence of ischemia, 10 nM isoproterenol had little effect on either contractile function or Ca(2+) homeostasis. In contrast, when cells were exposed to ischemia, isoproterenol increased the size of contractions and Ca(2+) transients and augmented the increase in diastolic Ca(2+) concentration during ischemia in field-stimulated myocytes. Exposure to isoproterenol also promoted contractile depression in reperfusion. In voltage clamp experiments, isoproterenol abolished the decrease in the magnitude of L-type Ca(2+) current caused by ischemia. Isoproterenol also increased the incidence of abnormal contractile activity and induced delayed afterdepolarizations and the arrhythmogenic transient inward current in ischemia. Additionally, the decline in cell viability in ischemia and reperfusion was exacerbated by isoproterenol. This study shows that beta-adrenoceptor stimulation strongly potentiates adverse effects of ischemia and reperfusion on electrical and contractile function. These adverse effects of isoproterenol are likely caused by an increase in intracellular Ca(2+) accumulation during ischemia.