Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

The effect of Achyranthes japonica extract on larval survival and development and oviposition behavior of Plutella xylostella L. (Lepidoptera: Plutellidae)

The extract of Achyranthes japonica was tested for effects on larval survival and development and the oviposition behavior of the diamondback moth, Plutella xylostella L. Chinese cabbage dipped in A. japonica extract solution showed 51–80% antifeedant activity for 5 days against P. xylostella larvae, and more larvae were also on untreated cabbage leaves 24 h after release. The mortality of P. xylostella larvae increased proportionally to the duration of dipping time in the extract, and both pupation and emergence rates of larvae feeding only on treated cabbage were lower than those for larvae raised on untreated or with a choice of cabbage. The 20-hydroxyecdysone (20E) concentration in leaves was approximately 549, 1232, 1275, and 1426 μg/g at 6, 12, 24, and 48 h after dipping treatment, respectively. Notably, naive females laid more eggs on untreated cabbage than on treated cabbage, and females from larvae raised on treated Chinese cabbage also preferred the non-treated leaves. Our results are in contrast to those from earlier studies using various insect models that confirmed most females prefer to lay eggs on the host type that was eaten in the larval stage (Hopkins host selection principle). Cabbage dipped in the A. japonica solution for 24 h caused 59% larval mortality and inhibited both pupation and emergence rates of the larvae when exposed to plants 15 and 22 days after planting in the field, with the 20E concentration in the treated cabbage leaves at 1600.9 ± 122.36 and 1386.8 ± 24.69 μg/g, respectively. Therefore, the biological effectiveness could be attributed to the 20E in the treated cabbage leaves.     

Molecular identification of glucose-regulated protein 78 (grp78) gene from the Indian meal moth,Plodia interpunctella,and its regulation by nutrient uptake

Glucose-regulated protein 78 (GRP78) is a member of the HSP70 family of proteins and is localized in the endoplasmic reticulum (ER) within cells. GRP78 and its gene has been identified in only a few species of insects, and its role is not clear. Here, we identified full-length grp78 cDNA from the Indian meal moth, Plodia interpunctella, and demonstrated the role of grp78 in developmental and physiological processes of the insect. The deduced amino acid sequence of GRP78 contained highly conserved functional motifs of the HSP70 family and the C-terminal motif of KDEL, which is characteristic of ER-localized HSP70. It also showed high identity (93–94%) with GRP78 and related HSP70 proteins of lepidopteran species. Gene expression analysis showed that grp78 mRNA levels were high in the egg, feeding larval, and adult stages, but low in the molting, wandering larval, and pupal stages of development. In a tissue comparison of fifth instar P. interpunctella, grp78 level was higher in the gut than in the integument or fat bodies. Grp78 level decreased greatly when fifth-instar larvae were starved for 48 h, but recovered within 3–6 h after re-feeding. Our data suggest that grp78 is highly associated with dietary energy conditions during development and may play an important role in the nutritional physiology of insects.     

Enhanced expression of stress-responsive cytokine-like gene retards insect larval growth

Growth rates of immature animals are governed by their feeding activities. A reduction in feeding sometimes causes serious growth retardation in insects; a typical case is often seen in host insects parasitized by a solitary endoparasitoid wasp. However, understanding of the mechanisms underlying the physiological repression of parasitized insects is fragmentary. Here we analyzed brain gene expression of the host common cutworm, Spodoptera litura, parasitized by a solitary endoparasitoid, Microplitis manilae, and identified a novel gene whose expression was significantly enhanced by parasitization. The gene encoded a pre-pro-peptide of a cytokine-like molecule and its expression was observed mainly in nervous tissues, hemocytes, and integuments. The 25 amino acid cytokine-like peptide encoded by the C-terminus of this gene was demonstrated to exist in the hemolymph of S. litura larvae and to change hemocytes from non-adhesive to adhesive in vitro. Further, injection of the active peptide reduced feeding activities of test larvae and consequently delayed their growth. The enhanced gene expression was also observed in larvae under severe stress conditions: abdominal ligature, proleg cutting, mechanical vibration, low temperature, and heat shock at 45 °C. Elevated gene expression was maintained only in seriously growth-retarded larvae but not in recovered larvae at 24 h or 48 h after heat treatment. Thus, it is reasonable to conclude that stress-induced elevation of the peptide gene expression highly correlates with reduced feeding activities and growth retardation of the host larvae parasitized by M. manilae. Based on the conclusion, we named this peptide stress-responsive peptide (SRP).     

Evaluating the safety of Rodolia cardinalis to two species of Galapagos finch; Camarhynchus parvulus and Geospiza fuliginosa

Some species of coccinellids exude a fluid that contains an alkaloid which can be toxic to vertebrates that ingest them. Because of this, before introducing Rodolia cardinalis to the Galapagos Islands as a biological control agent for the invasive scale insect, Icerya purchasi, it was considered necessary to determine whether it is toxic to birds. In this study, experiments were carried out with two common, insectivorous finches that are endemic to the Galapagos Islands; Camarhynchus parvulus (12 individuals) and Geospiza fuliginosa (16 individuals). Finches were maintained in captivity for 12–18 days. This period was divided into three phases: adaptation, experimental, and observation. During the experimental phase, test individuals, in addition to their regular diet, were fed 0.15 g portions of larvae or adults of R. cardinalis on alternate days over four days. The control group were fed 0.15 g of Asciodes quietalis abdomens in place of R. cardinalis. Symptoms of toxicity (mortality, deterioration, critical weight loss) from feeding on R. cardinalis were not observed in either species while in captivity, but birds avoided or rejected beetles indicating that they were distasteful. Finches regurgitated a higher number of larvae than adult beetles suggesting that larvae were less palatable than adults. Although long-term monitoring was not possible, results suggest that at least two species of finch will be unaffected by the release of R. cardinalis into the Galapagos Islands.     

Influence of host plant nitrogen fertilization on hemolymph protein profiles of herbivore Spodoptera exigua and development of its endoparasitoid Cotesia marginiventris

Nitrogen has complex effects on plant–herbivore–parasitoid tritrophic interactions. The negative effects of low nitrogen fertilization in host plants on insect herbivores can be amplified to the higher trophic levels. In the present study, we examined the impact of varying nitrogen fertilization (42, 112, 196, and 280 ppm) of cotton plants (Gossypium hirsutum L.) on the interactions between the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), and the hymenopteran endoparasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). We predicted that the development and fitness of C. marginiventris would be adversely affected by low host plant nitrogen fertilization through the herbivore S. exigua. The percentage of C. marginiventris offspring developing to emerge and spin a cocoon, and total mortality of parasitized S. exigua larvae were unaffected by nitrogen level. The developmental time of C. marginiventris larvae in S. exigua larvae feeding on low (42 ppm) nitrogen cotton plants was approximately 30% longer than that of those feeding on higher (112, 196, and 280 ppm) nitrogen plants. Parasitoid size (length of right metathoracic tibia), a proxy for fitness, of C. marginiventris males was positively affected by nitrogen level. Total amounts of S. exigua hemolymph proteins were not affected by nitrogen level, but were reduced by parasitism by C. marginiventris. Two proteins with molecular weights of ca. 84 and 170 kDa dominated the S. exigua larval hemolymph proteins. Concentrations of the 170 kDa hemolymph protein were unaffected by nitrogen treatment, but parasitism reduced concentrations of the 170 kDa protein. Concentrations of the 84 kDa protein, on the other hand, were interactively affected by parasitism and nitrogen treatment: higher nitrogen fertilization (112, 196, and 280 ppm) increased protein concentrations relative to the 42 ppm treatment for unparasitized S. exigua larvae, whereas nitrogen treatment had no effects on parasitized larvae. For S. exigua larvae feeding on 42 ppm nitrogen plants, parasitism increased concentration of the 84 kDa protein, while for those feeding on 112, 196, and 280 ppm nitrogen plants, parasitism decreased concentrations of the protein. Possible mechanisms and ecological consequences for the extended development of C. marginiventris on S. exigua hosts grown on low-nitrogen plants are discussed.     

An adjustable action threshold using larval parasitism of Helicoverpa armigera (Lepidoptera: Noctuidae) in IPM for processing tomatoes

An adjustable action threshold that uses estimates of larval parasitism of Helicoverpa armigera (Hübner) in individual fields was assessed over three consecutive years in processing tomatoes in Hawke’s Bay, New Zealand. Tomato fields were monitored weekly for levels of infestation by H. armigera larvae. When infestation levels became of concern, either approaching or exceeding the standard action threshold of one larva per plant, collections of 30+ representative larvae were made. These larvae were measured and reared individually at 30 °C, and after 4 days rates of parasitism were estimated visually. From these data, spray recommendations were made using a formula that adjusted the action threshold to allow for mortality from parasitism. At harvest, damage assessments were made to validate these recommendations. Results showed that estimates of parasitism after 4 days were accurate predictors of final assessments of parasitism. Overall parasitism during the three seasons was 71%, confirming that the original threshold, which relies on about 50% parasitism, needed revision. The dominant parasitoid was Cotesia kazak, reared from 91% of all parasitized larvae over the 3 years. Microplitis croceipes and the self introduced polyphagous parasitoid Meteorus pulchricornis were reared from 4% and 5% of the parasitized larvae respectively. In 16 of 17 fields examined, the adjustable threshold kept fruit damage at harvest below the tolerated level of 5%. The single crop with excessive damage had only 0.5% fruit damage above this level. This adjustable threshold, which varied in this study from 1–8.3 larvae per plant, has been incorporated into an updated IPM programme and contributed to a 95% reduction in insecticide use.     

Effect of leaf toughness and temperature on development in the lilac pyralid,Palpita nigropunctalis (Bremer) (Lepidoptera: Crambidae)

This study focuses on three factors that affect the survival of the lilac pyralid, Palpita nigropunctalis (Lepidoptera:Crambidae): (1) the effect of leaf toughness on survival rate to clarify the availability of leaves as food, (2) the effect of temperature on immature development to determine the lower thermal threshold, and (3) the effect of temperature on head capsule width to clarify whether head capsule width can be used to discriminate among field-collected larval instars. Larvae could develop on Osmanthus fragrans var. aurantiacus leaves collected in April, but not on leaves collected in June or September which were too tough to eat. More than 80% of the larvae on the leaves of Ligustrum lucidum, Ligustrum japonicum, Ligustrum obtusifolium and Syringa vulgaris completed development, regardless of the collection time. P. nigropunctalis completed development on L. lucidum at temperatures from 15 to 27.5 °C with a photoperiod of either 15 L:9D or 16 L:8D, but not at 30 °C, at which temperature no eggs hatched. The lower thermal threshold and thermal constant for total development from egg to adult were estimated at about 7 °C and 450–460 degree-days. Most of the larvae were 5-instar type larvae (passed through 5 instars) regardless of the temperature, but a few 6-instar type larvae (4 of 355) were noted at temperatures of 22.5 °C and higher. No overlap of the ranges of head capsule widths was detected for the 5-instar type larvae, indicating that head capsule width can be used to discriminate among field-collected larval instars.     

Sugar concentration and timing of feeding affect feeding characteristics and survival of a parasitic wasp

The availability of food sources is important for parasitoid survival, especially for those that inhabit ecosystems where nectar and honeydew are spatially or temporally scarce. Therefore, the value of even a single meal can be crucial for survival. Psyttalia lounsburyi is a parasitoid, and biological control agent, of the olive fruit fly, Bactrocera oleae. In order to improve our understanding of the basic nutritional ecology of P. lounsburyi and its role in survival we evaluated the effect of a single sucrose meal on the longevity of female and male wasps. We measured the duration of feeding, volume ingested, sucrose consumption, energy content, and longevity of wasps provided with different concentrations of sucrose (0.5, 1, and 2 M) at different times after emergence (0, 1, 2 or 3 days after emergence). Our results showed that longevity was significantly influenced by sucrose concentration and timing of feeding. For females, feeding on sucrose increased the likelihood of survival to varying degrees, ranging from 32.3% to 95.4%, compared to water-only controls. The longest duration of feeding was observed for the highest sucrose concentrations and oldest wasps. The amount of sugar ingested and energy uptake increased, up to a point, as sugar concentration increased. Our results suggest that P. lounsburyi derived greatest benefit from the intermediate concentration (1 M) of sucrose provided 2 or 3 days after emergence. Our study emphasizes the importance of finding balance between increasing longevity and limiting the duration of feeding, and concomitant uptake of nutrients, that is fundamental for survival of the wasp in nature.     

Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells

According to the World Health Organization, 2015 registered more than 1.206.172 cases of Dengue in the Americas. Recently, the Aedes aegypti has been not only related to Dengue, but also with cases of Zika virus and Chikungunya. Due to its epidemiological importance, this study characterized the morphology of the embryonated eggs of A. aegypti and provided a protocol to culture stem cells from eggs and digestive tract of fourth instar larvae in order to examine cell biology and expression of markers in these vectors. Cells were isolated and cultured in DMEM-High at 28 °C, and their morphology, cell cycle and immunophenotyping were examined. Morphologically, embryos were at the end of the embryonic period and showed: head, thorax, and abdomen with eight abdominal segments. The embryonic tissues expressed markers related to cell proliferation (PCNA), pluripotency (Sox2 and OCT3/4), neural cells (Nestin), mesenchymal cells (Vimentin and Stro-1), and endosomal cells (GM130 and RAB5). In culture, cells from both tissues (eggs and larvae gut) were composed by a heterogeneous population. The cells had a globoid shape and small size. Cell cycle analysis on passage 1 (P1) showed 27.5% ± 2.0% of cell debris, 68% of cells on G0-G1 phase, 30.2% on S phase, 1.9% ± 0.5% on G2-M phase. In addition, cells on passage 2 showed: 10% of cell debris, 92.4% of cells on G0-G1 phase, 6.8% on S phase, 0.6% on G2-M phase. Embryonated eggs expressed markers involved with pluripotency (Sox2 and Oct 3/4), mesenchymal cells (vimentin and Stro-1), neural cells (Nestin), and cellular death by apoptosis (Caspase 3). Specific endosomal markers for insect cells (GM130 and RAB5) were also highly expressed. In cell culture of A. aegypti larvae gut the same labeling pattern was observed, with a small decrease in the expression of mesenchymal (vimentin and Stro-1) and neural (Nestin) markers. In summary, we were able to establish a protocol to culture embryonated eggs and larvae gut of A. aegypti, describing the characteristics of undifferentiated cells, as well as the cell cycle and expression of markers, which can be used for biotechnology studies for the biological control of this vector.     

Effect of chronic low body temperature on feeding and gut passage in a plethodontid salamander

Although feeding in some plethodontid salamander species, such as Dusky Salamanders (Desmognathus, family Plethodontidae), occurs at short-term (acute) low temperature below 5 °C, it is unknown whether feeding, digestion, and gut passage continue to occur during periods of long-term (chronic) low temperature. We performed a controlled laboratory experiment to examine the effect of several chronic low environmental temperatures on both feeding and gut passage in semiaquatic Spotted Dusky Salamanders (D. conanti). We quantified salamander feeding and defecation for different experimental groups maintained for many weeks at a constant temperature of 4, 7, 10, or 13 °C. Although feeding frequency, number of prey items consumed per feeding, and defecation frequency were significantly less for individuals at 4 °C than for individuals at 10 or 13 °C, salamanders continued to feed, defecate, and maintain body mass for 12 weeks at 4 °C. The ratio of the number of fecal pellets produced to the number of prey items consumed each week by individuals did not significantly decrease at 4 °C, which indicates gut passage was sustained at this temperature. Because both time between feeding and time between defecation were similarly affected by prolonged low temperature, the significant decrease in feeding frequency at 4 °C may depend, in part, on a decrease in digestive function and an extended time for gut passage at low temperature. We conclude that most individuals of D. conanti can feed, digest, and maintain body mass for several months at constant low temperature down to 4 °C. Our results support a growing body of data that indicate some plethodontid salamanders may acquire energy at environmental temperatures only a few degrees above freezing.     

Specific dynamic action and postprandial thermophily in juvenile northern water snakes,Nerodia sipedon

1. We measured oxygen consumption of seven juvenile Northern water snakes, Nerodia sipedon, at 3, 19, 41, and 89 h after feeding. 2. Oxygen consumption 19 h after eating was significantly higher than at 3, 41, and 89 h after feeding and the values at 3 h post-feeding were significantly higher than those at 41 and 89 h. 3. We monitored substrate temperature preference in nine juvenile N. sipedon at 2000, 0000, 0800, 1200, and 1600 h for 3 days. 4. After feeding and at night snakes selected warmer substrate temperatures.     

Antifeedant activity of numb and salty taste compounds against the larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Helicoverpa armigera, an important polyphagous insect pest in agriculture, attacks more than 200 plant species of more than 30 families. Our previous study showed that the choice feeding percentages of H. armigera larvae to tobacco leaf discs treated with sweet, bitter, and hot taste substances were higher than the control leaf discs, while numb and salty substances could significantly inhibit their feeding. To quantitatively determine the synergistic effect of numb and salty substances, in this paper, the antifeeding activities of numb and salty substances and their mixtures blended in different doses or volume ratios were assayed on H. armigera larvae. The first bioassay was designed to elucidate the relative feeding preference of the larvae to the leaves from several common host species, each paired with tobacco leaf discs, and the result indicated that the most preferred host leaf by the larvae was tobacco leaf, followed by cotton and peanut leaves, suggesting that tobacco leaf was the most suitable matrix for the antifeeding bioassay, and the larval consumption of maize, pepper, or tomato leaves were significantly lower than that of tobacco leaf. The second bioassay was to test the choice feeding response of H. armigera larvae to tobacco leaf discs treated with Zanthoxylum bungeanum extracts obtained with different solvents, and the result showed that the antifeeding activity of the alcohol extracts was the strongest (93.38%), and the leaf consumption in the treatment and the control showed extremely significant difference (t = 4.23, t_0.01 = 3.25, P = 0.0022), followed by the dichloromethane extracts (47.64%), while the other three solvents (water, acetone, and n-hexane) could not extract the active antifeeding components from Z. bungeanum. The larval consumption of tobacco leaf discs treated with the alcohol extracts of Z. bungeanum and NaCl solution were significantly less than their corresponding controls. The mean larval consumption of the treated leaf discs decreased with ever-increasing dosage, and the consumption of tobacco leaf discs coated with different doses of alcohol extracts of Z. bungeanum or NaCl solution showed extremely significant difference (F_{alcohol extract of Z. bungeanum} = 3.88, F_0.01 = 3.58, P = 0.0064; F_{NaCl solution} = 54.29, F_0.01 = 3.58, P = 0.0000), with maximum antifeeding effects at a dosage of 30 μL per 1.5 cm ID leaf disc. We further tested the larval consumption of tobacco leaf discs treated with alcohol extracts of Z. bungeanum in saturated NaCl solution mixed in different volume ratios, and the result showed that the choice antifeeding percentages of the treatments with 15 μL or more Z. bungeanum alcohol extracts were higher than 90%, among which the mixture with 25:15 volume ratio of Z. bungeanum alcohol extracts and saturated NaCl solution exhibited the strongest antifeeding activity, and the mean consumed leaf area of tobacco leaf discs coated with this blend was only 0.10 mm². In the further test on feeding dose-response of the 25:15 mixture, the mean leaf consumption decreased linearly with ever-increasing dosage, with a regression equation y = ?3.9356x + 120.78(R² = 0.9998), and the 30 μL dose could completely inhibit H. armigera feeding.     

Fitness cost of malaria vector Anopheles stephensi induced with larvicidal and nutritional stress

Our study focused on how externally applied single or multiple stressors alter the fitness of early IV instar Anopheles stephensi larvae by inducing various larval stressors such as starvation and sublethal doses (LC₁₀, LC₂₅ and LC₅₀ for 24, 48 and 72 h) of various conventional and biorational larvicides. Larval stress specific response was observed in terms of their nutritional (glycogen, sugar, lipid and protein) and biochemical (DNA) status compared with respective control group which were found to be significantly (P < 0.05) altered. Nutrition depletion index was found to be concentration dependent depicting maximum reduction at LC₅₀ concentration with all applied larvicides. Significant (P < 0.05) reduction in DNA level was observed only with neem oil (10–23%) and Bti (21–23%) treatments. DNA damage was further evidenced by generating RAPD profiles that revealed variations in band intensities along with addition or deletion of few band in stress induced larvae. Overall, our results depicted that An. stephensi larvae may possibly tolerate the induced stress within certain limits by modifying their nutritional and biochemical levels, which may occur at a significant fitness cost.     

Development of Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans) feeding on four tetranychid mites (Acari: Phytoseiidae,Tetranychidae) and pollen

The development of the predatory mites, Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans), feeding on four tetranychid mites (Tetranychus urticae, Tetranychus kanzawai, Oligonychus mangiferus, Panonychus citri), maize pollen or Chinese loofah pollen was studied at 25 °C. Immature stages of N. womersleyi feeding on T. urticae and T. kanzawai had shorter developmental duration (4.71 and 5.02 days for females, 4.77 and 5.19 days for males, respectively) than those feeding on other food sources. Immature stages of E. ovalis females feeding on O. mangiferus and T. urticae developed in 4.99 and 5.13 days, respectively, the shortest developmental duration measured. Immature stages of E. ovalis males feeding on O. mangiferus and T. urticae developed in 5.12 and 5.37 days, respectively. The longevity of N. womersleyi males (13.31 to 14.51 days) and females (17.67 to 21.81 days) feeding on T. urticae, T. kanzawai or maize pollen was longer than the longevity of N. womersleyi feeding on O. mangiferus, P. citri or loofah pollen. E. ovalis males (12.91 to 16.74 days) and females (16.24 to 23.77 days) feeding on O. mangiferus, T. urticae or maize pollen lived longer than E. ovalis males and females feeding on T. kanzawai, P. citri or loofah pollen.     

Pinus koraiensis twigs affect Monochamus saltuarius (Coleoptera: Cerambycidae) longevity and reproduction

This study was conducted to investigate the longevity, reproduction and ovarian development of M. saltuarius adults feeding on Pinus koraiensis twigs of three different age classes (current-year, 1-year and 2-year). Longevity and fecundity of female adults decreased with increasing twig age class (58.4, 41.9 and 35.9 days, respectively, for longevity and 43.0, 13.2 and 6.6 eggs, respectively, for fecundity). In ovarian development, the average follicle size after 1 week of feeding on current-year twigs was 3.1–3.5 times greater than those of beetles fed 1- and 2-year-old twigs. After 2 weeks of feeding, follicle size was 2.3–2.4 times greater than those of beetles fed 1- and 2-year-old twigs. These results showed that current-year twigs of P. koraiensis increase longevity and reproduction of M. saltuarius. By analyzing sugars using high performance liquid chromatography (HPLC), the quantity of fructose was found to be highest in current-year twigs and lowest in 2-year-old twigs. A sugar feeding bioassay indicated that fructose was preferred by M. saltuarius. Therefore, fructose is important to adult feeding and ovariole development in M. saltuarius.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Nutritional supplementation improves the sexual response of bucks exposed to long days in semi-extensive management and their ability to stimulate reproduction in goats

In confined management systems, well-nourished bucks rendered sexually active by exposure to long days are efficient in fertilizing out-of-season goats. However, underfeeding is common in semi-extensive management systems and may reduce the reproductive efficiency of bucks. The objective of the present study was to determine whether nutritional supplementation improved the sexual activity of bucks submitted to long days in semi-extensive management systems and their ability to stimulate the reproduction of goats in semi-extensive or confined conditions. In experiment 1, three groups of bucks were placed in different flocks and grazed daily with females for 7 h. Each day after grazing, males were separated from females and moved into open pens. One group did not receive any treatment (control group; n = 6). Two other groups were submitted to artificially long days from 15 November to 15 January. From 16 January, one group did not receive nutritional supplementation (long-day group; n = 5), whereas bucks from the other group each received 600 g of a commercial concentrate (long-day + supplementation group; n = 5). The fourth group was kept in confined conditions, exposed to long days and fed alfalfa hay (long-day confined group; n = 6). On 26 March, anovulatory goats from other flocks were assigned to four groups (n = 27 each) and confined separately in open pens. Three bucks of each group were housed with the females. Pregnancy rates were greater in the goats housed with the long-day group than those housed with the control group (P < 0.01). However, pregnancy rates did not differ between the long-day confined group (89%) and long-day + supplementation group (70%; P = 0.09), but these rates were greater than those from the long-day (37%) and control groups (0%; P < 0.05). In experiment 2, two groups of males (n = 3 each) were incorporated into two flocks under semi-extensive management and grazed daily with females for 7 h. One group of males did not receive any treatment (control group). The other group was submitted to long days and nutritional supplementation as in experiment 1 (long-day + supplementation group). Males remained with females during the whole study. The pregnancy rate was greater in the goats joined by males of the long-day + supplementation group (78%) than in those from the control group (0%; P < 0.001). We conclude that long days and nutritional supplementation improve the ability of bucks kept in semi-extensive management to stimulate reproduction of out-of-season goats in confined or semi-extensive management systems.     

Supplementary feeding of horses with processed sorghum grains and oats

Two feeding experiments and in vitro hind gut fermentation tests were carried out to study the effect of processing sorghum grain on digestion of starch and on the gastrointestinal (GI) tract environment of the horse. In experiment 1, 12 yearling Australian stock horses were blocked on the basis of sex then randomly divided into four equal groups, each containing one castrated male and two females of approximately the same age and weight. Horses were offered at 0800 and 1500 h, 3 kg medium quality liverseed grass (Urochloa panicoides) hay and 2 kg of either oats (O), dry rolled sorghum (DRS), steam-flaked sorghum (SFS) or expanded sorghum (ES). Lanthanum was used as external solid marker for the measurements of apparent total tract digestibility. Fresh water was available ad libitum. Horses were allowed 18 days to adapt to the diets followed by a 3-day faecal collection period. Digestibility of dry matter (DM), and acid detergent fibre (ADF) were higher (P<0.01) for dry rolled and expanded sorghum diets. Digestibility of starch was similar across treatments, averaging 0.98 ± 0.009. Faecal pH values were lower (P<0.05) for the sorghum treatments but remained slightly below neutral (≥6.42) for the steam-flaked sorghum. Sub-samples of fresh faeces were supplemented with glucose and used for in vitro measurement of potential lactate production and final pH. The 20 h in vitro pH was also lower (P<0.05) for the steam-flaked sorghum group. All horses maintained good health and completed the experiment successfully.Results of this experiment indicate that digestibility of starch of processed sorghum is comparable to that of oats (0.97 versus 0.98), but processing sorghum grains may produce conditions favourable for lactate production and accumulation.In experiment 2, eight Australian stock horses were used to study changes in pH and accumulation of VFA and lactate in the different parts of the gastrointestinal tract. In this experiment SFS was compared with DRS. Horses were divided into two groups on the basis of sex and body weight. Each group contained two females and two castrated males. The nutritional management of the horses was the same as for the DRS and SFS groups in experiment 1. At the end of the adaptation period horses were slaughtered at intervals of 1.5, 3.0, 4.5, and 6.0 h after morning feeding, one horse per interval per treatment. Samples were collected from the different anatomical regions, and analysed for pH, VFA, and l- and d-lactate. Fermentation in the pre-glandular stomach produced mainly lactic acid and reduced pH to 4.0–4.3 while VFA was the main product in the hind gut. Total and d-lactate were higher (P<0.01) for SFS and a similar trend was observed for total and individual VFA and l-lactate but differences lacked significance (P>0.05). It was concluded that steam flaking of sorghum exacerbates starch fermentation, which may lead to acidosis-related damage to the epithelium of the pre-glandular stomach.