Biological characteristics of a new long-chain fatty acid transport protein 1 from Trichinella spiralis and its participation in lipid metabolism, larval moulting, and development.

Long-chain fatty acid transport protein 1 (FATP1) is a member of the fatty acid transporter family. It facilitates transmembrane transport of fatty acids and participates in lipid metabolism. Lipids are essential components of the cell and organelle membranes of Trichinella spiralis. The nematode has lost the capacity to synthesise the necessary lipids de novo and has instead evolved to obtain fatty acids and their derivatives from its host. This study aims to ascertain the primary biological characteristics and roles of T. spiralis FATP1 (TsFATP1) in lipid metabolism, larval moulting, and the development of this nematode. The results show that TsFATP1 is highly expressed at enteral T. spiralis stages, mainly localised at the cuticle, the stichosome and the intrauterine embryos of the parasite. The silencing of the TsFATP1 gene by TsFATP1-specific dsRNA significantly decreases the expression levels of TsFATP1 in the worm. It reduces the contents of ATP, triglycerides, total cholesterol, and phospholipids both in vitro and in vivo. RNAi inhibits lipid metabolism, moulting, and the growth of this nematode. The results demonstrate that TsFATP1 plays an essential role in lipid metabolism, moulting, and the development of T. spiralis. It could also be a target candidate for the anti-Trichinella vaccine and drugs.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Comparative evaluation of different moulting methods on productive performance, egg quality, and antibody response of leghorn hens.

The aim of the study was to compare three moulting procedures and their effect on productive performance, egg quality, and antibody response of leghorn hens. For this, a total of 324 laying hens were distributed into three treatment groups having 12 replicates of 9 birds each according to completely randomized design. Treatments consisted of three moulting procedures based on feed and light restriction. The targeted weight at the end of moulting was 1450-1470 g. As soon as the moulting procedure is complete, the comparative analysis of post-moult productive performance (feed intake, egg production, egg weight, egg mass, feed per dozen eggs, feed per kg egg mass, livability), egg quality characteristics (egg weight, egg length and width, shape index, surface area, volume, albumen height, weight, Haugh unit score, yolk width, height, index, egg shell pore number, shell weight, thickness, breaking strength), and antibody response against Newcastle disease and avian influenza (H-9) were evaluated. Birds subjected to moulting procedure 3 (8 days fasting and gradual decrease in light) showed improvement in productive performance, egg geometry and quality traits, and antibody response against Newcastle disease virus. Birds experienced moulting procedure 1 (11 days fasting) had improved feed intake, egg production, and livability. However, birds moulted with procedure 2 (6 days fasting) revealed intermediate result in all the studied parameters. In conclusion, moulting through feed and light restriction with 8 days fasting and gradual reduction in light has potential to improve performance of leghorn hens.

The miR-282-5p regulates larval moulting process by targeting chitinase 5 in Bombyx mori.

Moulting is critical for growth, development and survival in insects. As the main components of cuticle, dynamic change of chitin is consistent with the moulting process. Chitinase is the main enzyme to mediate chitin metabolism in the old cuticle. To avoid over-degrading chitin from the new cuticle, the expression of chitinase must be precisely regulated. In this study, we performed microRNA-sequencing to investigate expression change of microRNAs in silkworm epidermis during the moulting process. A comparative microRNA transcriptomic analysis from different moulting stages and 20-hydroxyecdysone (20E) treatment identified bmo-miR-282-5p as a candidate. By the bioinformatic analysis, chitinase 5 (BmCht5) was predicted to be a target of bmo-miR-282-5p. Meanwhile, a temporal expression analysis revealed that BmCht5 only expressed at moulting D3 stage, whereas bmo-miR-282-5p showed a converse pattern, in which its transcript signal disappeared at this time point. Furthermore, a luciferase assay and agomir treatment demonstrated that bmo-miR-282-5p suppressed transcript of BmCht5 in vivo. As a result, injection of 282-5p agomir triggered 40% death due to moulting failure. In addition, RNA interference (RNAi)-mediated silencing of BmCht5 caused 30% developmental defect. Taken together, our data demonstrate the coordinated regulation of chitinase 5 by conserved miR-282-5p, and the 20E signalling pathway is essential for the normal moulting process in the domesticated silkworm.

Neuronal calcium sensor 2 is key to moulting and oocyte development in the brown planthopper, Nilaparvata lugens.

Intracellular calcium (Ca2+ ) is vital for signal transduction in many cellular events. Several Ca2+ -binding proteins mediate the transduction of intracellular calcium signals. The EF-hand motifs containing neuronal calcium sensor (NCS) proteins are mainly expressed in the nervous system, where they have important roles in the regulation of a variety of neuronal functions. NCS1 has four EF-hand motifs and well-defined neuronal development functions in a variety of eukaryotes. However, NCS2 has only been identified in invertebrates such as insects and nematodes thus far. The functions of NCS2 remain largely unknown. Here, we identified an orthologous NCS2 in the hemipteran Nilaparvata lugens. Based on qRT-PCR, this gene was found to be primarily expressed in the brain. Knockdown of NCS2 in each nymphal instar by RNA interference led to lethality and caused aggradation and disordered arrangement of lipid droplets in the ovaries and testes of adults, which were associated with the absence of mature oocytes in female ovaries and reduction of spermiation in male adults. Our findings revealed a novel function for NCS2 as a regulator in development and reproduction and suggested that this protein had an important role in modulating lipid droplet remodelling in ovary and testis of N. lugens adults.

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.

Practices and issues of moulting programs for laying hens: a review.

1. Moulting is a natural physiological process in birds when they shed their old feathers and replace them with new ones, and it is followed by reproductive quiescence resulting in reduced egg production. Different birds undergo moulting at different points in their life. Some birds have seasonal moulting while some moult at the end of their breeding cycle. This review will mainly focus on moulting practices associated with commercial layer birds because, in all other bird types, this is not managed.2. Commercial farms commonly analyse the cost-benefit ratio to decide the time and method to adopt for moulting. Commercial layer farms adopt different practices to force birds out of moult and restart the production cycle. Animal welfare groups consider this as stressful and against animal welfare, raising questions about the ethics of this practice.3. Many studies have been conducted using complete or partial feed withdrawal and non-feed withdrawal programs to measure their effectiveness in maintaining animal welfare, economy, and post-moult performance in mind.4. Animal welfare should not be compromised during moulting. The United States Egg Producers and other such groups from the United Kingdom and Europe have decided to sell eggs produced only through a non-feed withdrawal moulting programs.

Composition of feathers and pulp of two broiler genotypes.

1. Cobb and Ross broilers (200 of each sex and breed) were fed four phases of diets ad libitum formulated with balanced protein to match their amino acid requirements throughout growth. Ten birds per genotype were sampled and euthanised at two-weekly intervals from 14 to 112 d of age. All feathers were dry-plucked from each of the seven tracts (specific skin areas) and pulp (the centre of the feather filament) was removed from primary and secondary remiges.2. Daily losses of feathers were collected from an additional 20 individually-caged broilers of each breed. These feathers were separated into natal down, contour feathers, remiges and rectrices and then pooled by type, sex and genotype to quantify water and protein contents. Only those feathers collected from male Cobb 500 MX broilers were analysed for amino acid content.3. Amino acid contents of feathers from the seven tracts were measured only in Cobb males on days 1, 28 and 70; for pulp on days 28 and 70; and for the four types of moulted feathers.4. Protein content on a dry matter basis remained relatively constant over all ages and tracts during growth. Water content decreased with age in both sexes and genotype. Lysine and methionine content in feathers decreased with age while cystine, valine, leucine and serine increased. Lysine, methionine and histidine levels were higher in pulp than in mature feathers whereas cystine and valine were higher in mature feathers than in pulp.5. These results, together with information about moulting patterns in broilers, enabled the effects of age of the bird and of the type of feather, to be taken into account when determining the rate of deposition of amino acids in feathers.

Sex-specific growth is mirrored in feeding rate but not moulting frequency in a sexually dimorphic snake.

Sexual size dimorphism (SSD), commonly observed in snakes, may arise from a different growth rate between the sexes. This indicates a sex-specific resource intake that is in fact observable in free-living snakes. It is not so well known whether the sexes can express differential feeding rates under conditions unconstrained by spatial accessibility, competition, etc. Here, I studied sex-specific variation in growth, its correlate-moulting frequency, and feeding rate in a captive group of sexually dimorphic banded water snakes (Nerodia fasciata) with access to food unconstrained by predation, competition or space. I showed that the sexes did indeed differ in relative mass growth in that females grew faster than males (p = 0.02), but such differences were not apparent in the moulting rate (p = 0.19). Such differential growth was mirrored in the sex-specific feeding rate, with females ingesting a larger number of meals than males (p = 0.004). Such variation in feeding rate may be governed by an individual's energy expenditure and can be interpreted as a behavioural tendency that contributes to SSD development, independently of other behavioural characteristics. Sex-specific resource demands may drive the differential effects of increasing resource scarcity on both sexes.

Transcriptome of pleuropodia from locust embryos supports that these organs produce enzymes enabling the larva to hatch.

BACKGROUND: Pleuropodia are limb-derived glandular organs that transiently appear on the first abdominal segment in embryos of insects from majority of "orders". They are missing in the genetic model Drosophila and little is known about them. Experiments carried out on orthopteran insects 80 years ago indicated that the pleuropodia secrete a "hatching enzyme" that digests the serosal cuticle to enable the larva to hatch, but evidence by state-of-the-art molecular methods is missing. RESULTS: We used high-throughput RNA-sequencing to identify the genes expressed in the pleuropodia of the locust Schistocerca gregaria (Orthoptera). First, using transmission electron microscopy we studied the development of the pleuropodia during 11 stages of the locust embryogenesis. We show that the glandular cells differentiate and start secreting just before the definitive dorsal closure of the embryo and the secretion granules outside the cells become more abundant prior to hatching. Next, we generated a comprehensive embryonic reference transcriptome for the locust and used it to study genome wide gene expression across ten morphologicaly defined stages of the pleuropodia. We show that when the pleuropodia have morphological markers of functional organs and produce secretion, they are primarily enriched in transcripts associated with transport functions. They express genes encoding enzymes capable of digesting cuticular protein and chitin. These include the potent cuticulo-lytic Chitinase 5, whose transcript rises just before hatching. Unexpected finding was the enrichment in transcripts for immunity-related enzymes. This indicates that the pleuropodia are equipped with epithelial immunity similarly as barrier epithelia in postembryonic stages. CONCLUSIONS: These data provide transcriptomic support for the historic hypothesis that pleuropodia produce cuticle-degrading enzymes and function in hatching. They may also have other functions, such as facilitation of embryonic immune defense. By the genes that they express the pleuropodia are specialized embryonic organs and apparently an important though neglected part of insect physiology.

Evolution of the Torso activation cassette, a pathway required for terminal patterning and moulting.

Embryonic terminal patterning and moulting are critical developmental processes in insects. In Drosophila and Tribolium both of these processes are regulated by the Torso-activation cassette (TAC). The TAC consists of a common receptor, Torso, ligands Trunk and prothoracicotropic hormone (PTTH), and the spatially restricted protein Torso-like, with combinations of these elements acting mechanistically to activate the receptor in different developmental contexts. In order to trace the evolutionary history of the TAC we determined the presence or absence of TAC components in the genomes of arthropods. Our analyses reveal that Torso, Trunk and PTTH are evolutionarily labile components of the TAC with multiple individual or combined losses occurring in the arthropod lineages leading to and within the insects. These losses are often correlated, with both ligands and receptor missing from the genome of the same species. We determine that the PTTH gene evolved in the common ancestor of Hemiptera and Holometabola, and is missing from the genomes of a number of species with experimentally demonstrated PTTH activity, implying another molecule may be involved in ecdysis in these species. In contrast, the torso-like gene is a common component of pancrustacean genomes.

20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, Bombyx mori.

Insect chitinases are hydrolytic enzymes required for the degradation of chitin. They are essential for insect moulting and metamorphosis. In this study, the regulation mechanism of a chitinase gene, Bombyx mori chitinase 5 (BmCHT5), was studied. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that BmCHT5 was up-regulated during the larval-larval and larval-pupa transitions and notably induced by 20-hydroxyecdysone (20E). Analysis of the BmCHT5 promoter revealed the presence of one Bombyx mori Broad-Complex Zinc-Finger Isoform 4 (BR-C Z4), two BR-C Z2 and two ecdysone-induced protein 74A (E74A) cis-regulatory elements (CREs) that are related to 20E. qRT-PCR showed that the expression of both BmBR-C Z4 and BmBR-C Z2 during metamorphosis, and when induced by 20E, was anastomotic with the variations in BmCHT5 mRNA level. In contrast, BmE74A did not follow this trend. An electrophoretic mobility shift assay did not retrieve a binding partner for the two BR-C Z2 CREs in the BmN cell line nuclear extract, whereas BR-C Z4 CRE specifically bound to BmBR-C Z4. Besides, luciferase activity analysis confirmed that BmBR-C Z4 could enhance the activity of the BmCHT5 promoter with BR-C Z4 CRE and could not enhance the promoter activity by mutating BR-C Z4 CRE. Taken together, these data suggest that the transcription factor BmBR-C Z4 enhances the expression of BmCHT5 during metamorphosis.

A clip domain serine protease involved in moulting in the silkworm, Bombyx mori: cloning, characterization, expression patterns and functional analysis.

Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signalling cascades in various biological processes, especially in innate immunity and the embryonic development of insects. Additionally, CLIPs may have additional non-immune functions in insect development. In the present study, the clip domain serine protease gene Bombyx mori serine protease 95 (BmSP95), which encodes a 527-residue protein, was cloned from the integument of B. mori. Bioinformatics analysis indicated that BmSP95 is a typical CLIP of the subfamily D and possesses a clip domain at the N terminus, a trypsin-like serine protease (tryp_spc) domain at the C terminus and a conserved proline-rich motif between these two domains. At the transcriptional level, BmSP95 is expressed in the integument during moulting and metamorphosis, and the expression pattern is consistent with the fluctuating 20-hydroxyecdysone (20E) titre in B. mori. At the translational level, BmSP95 protein is synthesized in the epidermal cells, secreted as a zymogen and activated in the moulting fluid. Immunofluorescence revealed that BmSP95 is distributed into the old endocuticle in the moulting stage. The expression of BmSP95 was upregulated by 20E. Moreover, expression of BmSP95 was downregulated by pathogen infection. RNA interference-mediated silencing of BmSP95 led to delayed moulting from pupa to moth. These results suggest that BmSP95 is involved in integument remodelling during moulting and metamorphosis.

Living with a leaky skin: upregulation of ion transport proteins during sloughing.

Amphibian skin is a multifunctional organ providing protection from the external environment and facilitating the physiological exchange of gases, water and salts with the environment. In order to maintain these functions, the outer layer of skin is regularly replaced in a process called sloughing. During sloughing, the outermost layer of the skin is removed in its entirety, which has the potential to interfere with skin permeability and ion transport, disrupting homeostasis. In this study, we measured, in vivo, the effects of sloughing on the cutaneous efflux of ions in toads Rhinella marina kept in freshwater conditions. We also measured transepithelial potential, cutaneous resistance, active ion transport and the distribution, abundance and gene expression of the key ion transport proteins sodium-potassium ATPase (NKA) and epithelial sodium channel (ENaC) during sloughing. We hypothesised that the increase in transepithelial efflux of ions during sloughing is a consequence of increased permeability and/or a reduction in the abundance or expression of cutaneous ion transport proteins, resulting in disruption of internal ion homeostasis. There was a significant increase in sodium and chloride efflux during sloughing in R. marina However, although in vitro skin resistance decreased after sloughing, active sodium transport increased commensurate with an increase in NKA and ENaC protein abundance in the skin. These changes in skin function associated with sloughing did not affect the maintenance of internal electrolyte homeostasis. These results suggest that during sloughing, amphibians actively maintain internal homeostasis by increasing cutaneous rates of ion uptake.

Aquaporin in different moult stages of a freshwater decapod crustacean: Expression and participation in muscle hydration control.

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs.

A critical review of the use and performance of different function types for modeling temperature-dependent development of arthropod larvae.

Temperature-dependent development influences production rates of arthropods, including crustaceans important to fisheries and agricultural pests. Numerous candidate equation types (development functions) exist to describe the effect of temperature on development time, yet most studies use only a single type of equation and there is no consensus as to which, if any model predicts development rates better than the others, nor what the consequences of selecting a potentially incorrect model equation are on predicted development times. In this study, a literature search was performed of studies fitting development functions to development data of arthropod larvae (99 species). The published data of most (79) of these species were then fit with 33 commonly-used development functions. Overall performance of each function type and consequences of using a function other than the best one to model data were assessed. Performance was also related to taxonomy and the range of temperatures examined. The majority (91.1%) of studies were found to not use the best function out of those tested. Using the incorrect model lead to significantly less accurate (e.g., mean difference±SE 85.9±27.4%, range: -1.7 to 1725.5%) predictions of development times than the best function. Overall, more complex functions performed poorly relative to simpler ones. However, performance of some complex functions improved when wide temperature ranges were tested, which tended to be confined to studies of insects or arachnids compared with those of crustaceans. Results indicate the biological significance of choosing the best-fitting model to describe temperature-dependent development time data.

Development and deposition of resilin in energy stores for locust jumping.

Locusts jump by using a catapult mechanism in which energy produced by slow contractions of the extensor tibiae muscles of the hind legs is stored in distortions of the exoskeleton, most notably (1) the two semi-lunar processes at each knee joint and (2) the tendons of the extensor muscles themselves. The energy is then suddenly released from these stores to power the rapid, propulsive movements of the hind legs. The reliance on the mechanical storage of energy is likely to impact on jumping because growth occurs by a series of five moults, at each of which the exoskeleton is replaced by a new one. All developmental stages (instars) nevertheless jump as a means of forward locomotion, or as an escape movement. Here, I show that in each instar, resilin is added to the semi-lunar processes and to the core of the extensor tendons so that their thickness increases. As the next moult approaches, a new exoskeleton forms within the old one, with resilin already present in the new semi-lunar processes. The old exoskeleton, the tendons and their resilin are discarded at moulting. The resilin of the semi-lunar processes and tendons of the new instar is initially thin, but a similar pattern of deposition results in an increase of their thickness. In adults, resilin continues to be deposited so that at 4 weeks old the thickness in the semi-lunar processes has increased fourfold. These changes in the energy stores accompany changes in jumping ability and performance during each moulting cycle.

Experimental evidence that sperm maturation drives protandry in an ectotherm.

Protandry, i.e., the earlier arrival to breeding areas of males than females, has attracted a lot of scientific attention. However, evidence for the evolutionary hypotheses of protandry is surprisingly scarce. Here, we experimentally manipulate the time of emergence from hibernation of males, relative to females, in the common lizard, Zootoca vivipara. We test whether the timing of emergence affects sperm maturation and mating success, to disentangle among proposed selective advantages of protandry. Our results experimentally demonstrate that the timing of emergence affects the date of sperm presence. Moreover, the degree of protandry affected whether males had sperm upon their first encounter with females, but it did not affect the probability of copulating. Mating occurred independent of male fertility and mating during infertility was least common in early emerging males. Early emergence from hibernation by males, relative to females, thus increases the male's chance of fertilising eggs and later emergence from hibernation by females reduces the female's probability of mating with infertile males. These results point to direct reproductive benefits of protandry in males and females, where earlier emergence is predicted to increase the male's opportunities to inseminate mates, and later emergence reduces the female's probability of copulating with infertile males. This suggests that protandry evolved due to the time required for sperm maturation after emergence from hibernation.

Precopulatory mate guarding influences the development of quiescent deutonymph females in the two-spotted spider mite (Acari: Tetranychidae).

Individuals of some organisms have a specific stage sensitive to environmental cues that initiate developmental plasticity which subsequently influences their entire development. Females may use male behaviour such as precopulatory mate guarding as an environmental cue to change their developmental rate. In the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae), only the first insemination results in fertilization and males guard quiescent deutonymph females. As quiescent individuals take on a silvery appearance before moulting, the period of the quiescent stage can be divided into two parts: from entering the quiescent stage to becoming silvery (1st period) and from becoming silvery to moulting (2nd period). Females may be sensitive to precopulatory mate guarding immediately before moulting (i.e. 2nd period). Thus, I examined whether precopulatory mate guarding during either period affects the total developmental duration of quiescent deutonymph females. When guarded by a male, the whole developmental duration of the quiescent deutonymph females became significantly shorter (by 3-5%) than that of solitary ones, regardless whether the guarding occurred during the 1st period, the 2nd period or both periods. In conclusion, quiescent deutonymph T. urticae females use precopulatory mate guarding by conspecific males as an environmental cue for their developmental rate, although they are sensitive to the mate guarding not only immediately before moulting.

Effect of calcium with and without probiotic, lactose, or both on organ and body weights, immune response and caecal microbiota in moulted laying hens.

A total of 72 laying hens were used to investigate the effect of probiotic and lactose on body weight loss, tibia ash, antibody production against sheep red blood cell (SRBC), heterophile-to-lymphocyte (H/L) ratio and gut microbiota in a common moulting method for 14 d. Hens were randomly allocated to 6 experimental groups consisting of (i) full feed (FF), (ii) feed withdrawal (FW), (iii) FW with calcium (Ca), (iv) FW with Ca and offering 7 g/lit lactose in drinking water (CaL), (v) FW with Ca and offering 1 g/lit probiotic in drinking water (CaP), and (vi) FW with Ca and offering a mixture of lactose and probiotic in drinking water (CaLP). The results showed body weight loss in all FW groups were more than 25% that was significantly higher than FF group (p < 0.05). The relative organ weights of hens in FW groups were lower than FF group; especially, it was significant for liver and ovary (p < 0.05). No significant difference was observed between all groups for tibia ash. The highest H/L ratio was related to FW group and offering Ca and lactose numerically and probiotic alone significantly resulted in decrease of this ratio (p < 0.05). The results also showed no significant difference for antibody production against SRBC among experimental groups. The highest coliform bacteria numeration observed in FW group and lactose could significantly reduce this population (p < 0.05). Lactic acid bacteria (LAB) numeration demonstrated a significant difference among treatments, so that FF hens had higher LAB than others (p < 0.05). In addition, FW moulted hens had significantly lower LAB compared to other moulted hens (p < 0.05), except for Ca group. In conclusion, probiotic and lactose was effective in maintaining caecal microbiota balance and improving immunity in hens exposed to moulting.