Polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol (ecdysteroids) are common constituents of various plants. In 1965, they were accidentally discovered in the search for the insect moulting hormone. These biologically important natural compounds are neither insect hormones nor inducers of insect ecdysis. Due to their strong anabolic, vitamin D-like effects in insects, domestic animals and humans, I propose the use of the arbitrary term vitamin D₁. The present paper describes the effects of vitamin D₁ on the growth and regeneration of excised epidermal cells of the tobacco hornworm, Manduca sexta (Sphingidae). The periods of programmed cell death and cell proliferation (histolysis and histogenesis, respectively) exactly coincide in insects with endogenous peaks of increased concentration of vitamin D₁. Epidermal cells communicate with each other, creating a mutually integrated tissue, connected by mechanical, chemical, electrical, ionic or other so far incompletely known factors. After natural cell death, or after the artificial removal of some epidermal cells, the neighbouring cells that lose communication integrity, begin to divide mitotically to replace the disconnected part. Cell divisions are arrested as soon as the integrity of the living tissue is established. During insect ontogeny, the application of juvenile hormone causes regenerating epidermal cells to repeat the previous morphogenetic programme (i.e., development of patches of larval tissue on the body of a pupa, or metathetely). Conversely, the application of vitamin D₁ (20-hydroxyecdysone) caused the regenerating cells to prematurely execute a future morphogenetic programme (i.e., development of patches of pupal tissue on the body of a larva, or prothetely). Among the key features of insect regeneration, is the arrest of cell divisions when tissues resume living cell-to-cell integrity. This prevents the formation of aberrant groups of cells, or tumours. It is well established that the main physiological systems of insects (e.g., circulatory, respiratory, neuro-endocrine) are structurally and functionally similar to corresponding systems in humans. Thus the basic principles of cell regeneration and the role of vitamin D₁ in insects may also be valid for humans. The common vitamins D₂ (ergocalciferol) or D₃ (cholecalciferol), are exclusively lipid soluble secosterols, which require activation by UV irradiation and hydroxylation in the liver. By contrast, the neglected vitamin D₁ is a natural derivative of polyhydroxylated 7-dehydrocholesterol of predominantly plant origin, which is both partly a water and partly a lipid soluble vitamin. It neither requires UV irradiation, nor hydroxylation due to 6 or 7 already built-in hydroxylic groups. Like other vitamins, it enters insect or human bodies in plant food or is produced by intestinal symbionts. Vitamin D₁ causes strong anabolic, vitamin D-like effects in domestic animals and in humans. I am convinced that avitaminosis associated with a deficiency of vitamin D₁ in human blood may be responsible for certain hitherto incurable human diseases, especially those related to impaired nerve functions and somatic growth, aberrant cell regeneration

In the sorghum/aphid/ladybeetle ecosystem found in the Texas High Plains Region of the United States, we found that the corn leaf aphid, Rhopalosiphum maidis (Fitch), is a key aphid species that provides a critical early-season food source for native coccinellids. From 1988 to 2000 data on the seasonal abundance of sorghum-infesting aphids and ladybeetles were collected from a total of 21 irrigated and 12 rain-fed grain sorghum fields. The data indicated that successful biological control of the greenbug by coccinellids is normally dependent on early-season colonization of the sorghum field by corn leaf aphids. When corn leaf aphids exceeded 100/plant before sorghum boot stage greenbugs never exceeded 125 aphids/plant. In all cases where greenbugs were found in densities that would cause economic damage to sorghum (>250/plant), corn leaf aphids reached a density of 100 or more per plant after sorghum reached the boot stage. In irrigated fields, the first record of coccinellid eggs and peak coccinellid abundance were positively and significantly (p = 0.05) correlated to the day of the year when corn leaf aphids reached or exceeded a minimum of 100/plant and corn leaf aphid peak abundance in both irrigated and rainfed fields. On the other hand, greenbug peak abundance was significantly correlated only to coccinellid peak abundance in irrigated fields. Regression analyses indicated that in paired analyses of irrigated and rain-fed sorghum fields, an increase of one aphid at time t, resulted in an increase in coccinellid peak abundance from 0.024 to 0.025 per 15 m of row at time t + 2 depending on aphid species, if corn leaf aphids reached a level of 100 or more per plant by sorghum boot stage and irrigation parameters. We concluded that corn leaf aphids are an important early-season food source for predaceous coccinellids, drawing these predators into the fields where they feed on the aphids and deposit eggs, engendering a captive larval population that is present when greenbug first begin to enter the field later in the season.

Chilo partellus Swinhoe 1885 (Lepidoptera: Crambidae) is a serious pest of maize in many parts of the world. It was reported for the first time in 2014 in the Mediterranean part of Turkey. Degree-Day (DD) models were developed to predict seasonal dynamics in maize fields using 3-parameter 'Boltzman' and a 4-parameter Logistic regressions. Larval/plant counts were monitored in pesticide-free maize fields in Adana, Turkey from 2017 to 2019. Depending on the year, the first record of C. partellus was between 03-14 May, corresponding to ~ 9-12 DD accumulated from 01 May. There were two main peaks; one in each of the maize growing seasons. The models accurately predicted the phenology of C. partellus with an Adj. R ² greater than 0.80. The 'Boltzman' regression had lower AICc, ΔAICc and Wі values than the logistic regression. Based on the 'Boltzman' model, 50% of the accumulated larvae/plant count for the first and second generations (peaks) occurred at 80DD and 375DD, 81DD and 413DD, and 54DD and 289DD in 2017, 2018 and 2019, respectively. For the logistic regression model, 50% of the accumulated larvae/plant counts occurred for the first and second generation at 90DD and 362DD, 62DD, and 45DD and 250DD in 2017, 2018, and 2019, respectively. Both these models could play a crucial role in the integrated pest management (IPM) of C. partellus in maize fields in Mediterranean regions.

The aphid Uroleucon ambrosiae (Thomas) is one of the principal pests found on greenhouse lettuce crops, and there is no efficient biological control agent of this pest in Brazil. This work evaluates the aphid U. ambrosiae as a host for the parasitoid Praon volucre (Haliday), aimed at using P. volucre as a potential biological control agent of U. ambrosiae on lettuce. As Macrosiphum euphorbiae (Thomas) is a common host of P. volucre in the field, the development of the parasitoid was compared on these two aphid species. Twenty nymphs of the 2^nd instar were kept with P. volucre for one hour at 22 ± 1°C, 70 ± 10% RH and a 12 h photophase. The size of the aphid's tibiae at the moment of oviposition indicated that there was no significant size difference between U. ambrosiae (0.6 ± 0.02 mm) and M. euphorbiae (0.7 ± 0.03 mm). When mummies were formed, M. euphorbiae had significantly longer tibia (1.5 ± 0.03 mm) than U. ambrosiae (1.4 ± 0.02 mm). No significant differences were detected in the percentage emergence (74.9 ± 7.92 and 87.5% ± 3.31 for U. ambrosiae and M. euphorbiae, respectively), or proportion of female offspring (56.2 ± 7.62 and 44.2 ± 7.14%). The development time from oviposition to adult and longevity of females and males of P. volucre reared on the two host species were not different. High parasitism levels were recorded for both host aphid species, but the percentage parasitism of M. euphorbiae (54.4 ± 4.40) was higher than of U. ambrosiae (35.6 ± 5.30). Female parasitoids reared on M. euphorbiae had longer tibiae (0.78 ± 0.01 mm) than those reared on U. ambrosiae (0.72 ± 0.01 mm). Our results demonstrate that the alternative host species U. ambrosiae, compared to the natural host species M. euphorbiae, affects the female's size, but did not affect parasitoid development time, longevity, emergence or sex ratio. The parasitoid P. volucre seems to be a good candidate for using as a biological control agent of U. ambrosiae on lettuce in Brazil.

Insects can recognize invading pathogens and initiate an immune response. Among them, Drosophila has emerged as an invertebrate model for investigating innate immune responses in which antimicrobial peptides play a crucial role. In the present study, immune-induced antimicrobial peptides were characterized in D. melanogaster and D. ananassae using the agar well diffusion method, HPLC, SDS-PAGE and LC-MS/MS after infection with either S. aureus or E. coli. The HPLC revealed two and three differentially induced components, respectively, in D. melanogaster and D. ananassae flies infected with S. aureus and E. coli. The tricine SDS-PAGE analysis also revealed two and five differentially induced proteins, respectively, in D. melanogaster and D. ananassae infected with E. coli. In E. coli infected flies, the ~6 kDa band was produced at higher level. Based on LCMS/MS and Mascot analysis, the peptide was identified as a putative cecropin A-like peptide, and the data suggested that both species of Drosophila have exhibited a clear immune response. The flies were also able to discriminate between bacteria, as this putative cecropin A-like peptide was produced in flies infected with E. coli but not S. aureus.

Like many insects, aphids (Hemiptera: Aphididae) can host a wide diversity of symbiotic bacteria that can be important drivers of their evolutionary ecology. In addition to the nutritional obligate symbiont Buchnera aphidicola, these phloem-sap feeding insects can host various facultative symbionts whose functional diversity depends on complex interactions with the host genotype and environmental factors. During sampling in citrus orchards in northern Tunisia, we collected aphids on citrus plants. The specimens belonged either to the cotton-melon aphid Aphis gossypii or the green citrus aphid Aphis spiraecola. We analysed the prevalence of Arsenophonus, Serratia symbiotica, Hamiltonella defensa and Regiella insecticola, four facultative symbionts frequently found in the genus Aphis and potentially associated with phenotypic effects related to nutrition, protection against parasites and tolerance of high temperatures. We found that the diversity and prevalence of facultative symbionts differed between these two aphid species that exploit similar ecological niches. In particular, we found a high prevalence of Arsenophonus in A. gossypii populations and that the defensive symbiont H. defensa was only present in A. spiraecola populations. These results are discussed in light of the ecology and life cycles of each of the species of aphid studied.

Intraguild predation (IGP) and cannibalism occur in the field and could affect the dominance structure of guilds of coccinellid species. The exotic biological control agent Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is now well established in most areas of Northern and Central Italy, but it is unclear what effect this species could have on native dominant and non-dominant coccinellids with which it co-occurs. In order to predict the trophic interactions in coccinellid guilds and the likely effect of H. axyridis, the incidence of IGP and cannibalism among the following six species were evaluated under laboratory conditions: H. axyridis, three native dominant species, Adalia bipunctata (L.), Oenopia conglobata (L.) and Coccinella septempunctata L. and two native non-dominant species, Platynaspis luteorubra (Goeze) and Scymnus apetzi (Mulsant). Unfed and fed fourth instar larvae of the above species were paired in an experimental arena and the incidence of predation recorded over a period of 24 h. In absence of aphids, the survival probabilities (SP) of A. bipunctata and O. conglobata were lowest when paired with either C. septempunctata or H. axyridis (< 0.20 SP after 24 h). The SP of C. septempunctata was similar if paired with either a conspecific larva or H. axyridis (< 0.34 SP after 24 h) and that of H. axyridis was reduced similarly if paired with either a conspecific larva or C. septempunctata (> 0.71 SP after 24 h). The SP of P. luteorubra was lower when paired with A. bipunctata and C. septempunctata (< 0.07 SP after 24 h) than with other species and that of S. apetzi was greatly reduced when paired with all the dominant and exotic species (< 0.27 SP after 24h). In presence of aphids no predatory events occurred in most combinations. H. axyridis acted as a strong predator of native dominant and non-dominant coccinellids when the aphids are scarce. We did not find any evidence, however, that the incidence of IGP among exotic and native species is higher than either IGP or cannibalism in native species. The likelihood of IGP occurring in the field is discussed.

To improve the mass production of the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) the effects of supplementary nutrition on its reproduction and mating behaviour were investigated using Plodia interpunctella Hübner (Lepidoptera: Pyralidae) larva as a host. The survival of mated male wasps was markedly prolonged after feeding with five different types of nutrients and that of unmated males was markedly prolonged except when provided with yeast solution. Providing female wasps with supplementary nutrition did not affect their longevity. When H. hebetor reproduced sexually and was supplied with an abundance of hosts, females produced 101.0 and 96.7 female offspring, respectively, when fed 20% sucrose or 20% honey solutions, however, when provided with only ten hosts the provision of supplementary nutrition did not affect the number of female offspring they produced. The 20% maltose, 20% sucrose and 20% honey solutions significantly promoted the courtship behaviour of male wasps, and the 20% yeast and 20% honey solutions increased the mating success of males paired with virgin females.

The cynipoid wasp Gronotoma micromorpha (Perkins) (Hymenoptera: Figitidae) is a parasitoid of the leaf miner Liriomyza trifolii (Burgess) (Diptera: Agromyzidae). The effects of adult age and body size on egg maturation in G. micromorpha were determined. The results showed that its egg load (number of mature eggs per female) increased when offered honey, water, but not hosts for 3 or 6 days after adult emergence. However, there was no significant difference in the egg loads of 3- and 6-day-old wasps. These findings and the results of previous studies on other cynipoid parasitoids suggest that when hosts are not available, females of parasitoid Cynipoidea enhance their reproductive capacity in anticipation of a future improvement in the availability of hosts by using carbohydrates and reserves stored during the larval stage. Moreover, large female wasps had higher egg loads throughout their lifetime. Given that rapid increases in the population density of L. trifolii are commonly reported in greenhouses, the demographic data of 0- and 3-day-old G. micromorpha females fed honey, need to be compared in the future. The effects of body size on the fecundity and longevity of G. micromorpha wasps should also be determined.

The efficacy of natural enemies in controlling pests under field conditions is largely correlated with their capacity to spread within infested crops. In this study the spatial dispersal of the California red scale parasitoid Aphytis melinus DeBach (Hymenoptera: Aphelinidae) was evaluated in the field after augmentative releases. The experiment was conducted in 2007 in six 1-ha plots in a Sicilian citrus orchard under integrated pest management. A total of 180,000 A. melinus adults was released in each of three plots and the other plots were left as untreated control. The flight range of the parasitoid was evaluated, for 35 days after the release, on 16 trees per each plot, located at 20 and 40 m from the central release point using yellow sticky traps activated with Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae) sexual pheromone and by monitoring the percentage parasitism of the scale on fruits and twigs. The effects of the distance from the release point and density of susceptible stages of host on parasitoid dispersal were evaluated. The number of wasps captured during the whole trial was greater in the traps located 20 m from the release point than in those at 40 m and in the control plots. Aphytis melinus dispersed over distances less than 40 m based on both the lower percentage parasitism and numbers captured recorded at distances of 40 m. The results are discussed in the context of the biological control of California red scale in citrus orchards by means of wasp releases. In particular, the release points should be no more than 40 m apart for a quick and homogeneous colonization of the area treated.

Herbivory on liverworts is rarely reported. We studied the effects of feeding by larvae of the sciarid fly Scatopsciara cunicularius on the growth of the thalloid liverwort Marchantia polymorpha at two different constant temperatures, 12°C and 22°C. Larvae reared at the lower temperature fed slower and over a longer period of time, which resulted in more damage and a greater reduction in the growth of the liverwort than that caused by those reared at the higher temperature. The reduction in growth of the liverwort was positively density-dependent in terms of number of larvae at both temperatures. These results indicate that the larvae of S. cunicularius are likely to be an effective means of controlling M. polymorpha, which is a common weed in plant nurseries and greenhouse cultures.

Differences in the time of occurrence of both sexes of adult ladybirds, which reflect differences in the longevity and reproductive strategies of the sexes, is important from the perspective of applied entomology, including biological control, as well as basic entomology. Nevertheless, there is little field census data on this topic. We investigated the seasonal occurrence of both sexes of Cheilomenes sexmaculata (Fabricius) in Osaka City, central Japan, from 2003 to 2011. Female adults tended to appear earlier than males after hibernation or aestivation. That males never appeared earlier than females during the nine year period of this study indicate that protogyny occurs after hibernation and aestivation in this species.

The European spruce bark beetle (Ips typographus) is a highly destructive pest of spruce monocultures. Adult spruce bark beetles are well-adapted to survive over winter however, the ability of sub-adult stages to overwinter has not been clearly established. The increase in average temperature recorded over the last three decades has resulted in an increase in voltinism by one generation, but due to insufficient time the last generation may not complete its development. It is crucial to investigate the survival and development of sub-adult stages at low temperatures in order to predict the effect of increased voltinism on the population dynamics of this species. We measured the development and survival of larvae and pupae (over 12 weeks) in logs kept at winter temperatures outdoors (in shade and exposed to sunlight) and in the laboratory (at 0 and 5°C), with 10°C as a control, at which normal development was expected. Overall, findings revealed that development continued at low temperatures, although it was slower than at high temperatures. Importantly, after 12 weeks significant numbers of spruce bark beetles were present, including newly emerged adults. We demonstrate, for the first time, that sub-adult spruce bark beetles can mature over winter and the percentage survival was significant, indicating that some of the beetles that did not complete their development before the onset of winter can complete their development during winter and potentially adversely affect forests and pose problems for their management.

Agrothereutes lanceolatus is a solitary ectoparasitoid attacking mature larvae, prepupae and pupae of tortricid and pyralid moths, including Homona magnanima, which is an important pest of tea. Adult A. lanceolatus are sexually dimorphic and vary considerably in size in the wild, suggesting they parasitize a variety of hosts of different sizes. Here, the effects of host size on offspring survival, size and sex ratio in A. lanceolatus were investigated. Wild-caught and laboratory-reared parasitoids were used to identify the effects of laboratory rearing on the above three biological parameters. The percentage of hosts producing parasitoid offspring was not dependent on host size regardless of parasitoid generation. Offspring survival was lower in laboratory-reared generations than the F1 generation. Large parasitoids emerged from large hosts. The offspring sex ratio (proportion of females emerging) was positively correlated with host size. The sex ratio at the egg stage (= primary sex ratio) inferred from maternal fertilization act also changed in response to host size, although there was imperfect control of female egg deposition. Thus, the observed sex-ratio resulted from maternal control of offspring sex ratio. A striking difference was detected in the offspring sex ratio in the different generations. Offspring sex ratio was more male-biased in laboratory reared generations. These results suggest that inbreeding depression caused a reduction in offspring survival and the male-biased sex ratio in the laboratory. The sex-ratio difference could result either from the difference in host size in the field and laboratory or from the production of diploid males in the laboratory.

Capsazepine is a competitive antagonist of capsaicin, a TRPV1 agonist responsible for the spicy taste of pepper. TRPV1 agonists and antagonists are known to affect mammalian body temperature, but their action on thermoregulation in insects is poorly known. In this study we evaluated the effect of capsazepine on the thermal preference of the American cockroach, Periplaneta americana using a thermal gradient. Our results revealed that capsazepine in submicromolar concentrations induces a preference for higher ambient temperatures when compared to the control insects. To assess whether capsazepine may act also as an antagonist of capsaicin in insects, we determined this insects' thermal behaviour when capsazepine was applied before capsaicin. The hypothermic response to capsaicin was clearly blocked by pre-treatment with capsazepine only in female American cockroaches. Our results indicate the involvement of structures functionally similar to TRPV1 in insect thermosensation.

Cytochrome P450s (P450s) involved in insecticide resistance reduce the efficacy of insecticide-based vector control by rendering vector control ineffective. They are recorded in many species of vectors and have various constitutive and insecticide induction profiles. In this study, the isolation and prediction of the structure of a P450 from a strain of Aedes aegypti originating from Malaysia is reported. Quantitative mRNA expression of this gene and a previously reported P450, CYP4H28v2, in the developmental stages of the mosquito after exposure to sub-lethal concentrations of insecticides is also reported. The isolated P450, CYP4H31v2, is an allelic variant of CYP4H31 and contains several conserved motifs of P450s. The secondary structure of the protein is mostly made up of alpha helices and random coils. The tertiary structure was generated using homology modeling and was of good quality based on structure validation using protein structure assessment tools. CYP4H28v2 and CYP4H31v2 were differentially expressed in the developmental stages of the vector, with a significantly increased expression in adult males. The genes were significantly over-expressed in larvae exposed to deltamethrin and permethrin for 6 h. In the DDT-treated larvae, only CYP4H31v2 was significantly over-expressed after a 6 h exposure. Under-expression of the genes was predominant in larvae treated with the organophosphates malathion and temephos. Though the functions of these P450s are unknown, their response to induction by exposure to insecticides indicates the likely involvement of these genes in insecticide tolerance.

Galleria mellonella larvae spin protective tubes, which they use until they finish feeding, when they spin cocoons. A feeding choice experiment showed that some of the silk produced by feeding larvae was consumed in addition to the standard diet (STD). To determine the effect of feeding on silk, last instar larvae were fed for 24 h on foods based on STD but modified by replacing the dry milk component (10% of the diet) with equal amounts of different kinds of silk. While each control larva consumed 21 ± 0.5 mg of the STD and produced 4.5 ± 0.1 mg of silk, larvae that ate the food that contained larval silk consumed 10 ± 0.4 mg of food and produced 6.1 ± 0.1 mg silk; the percentage ratio of silk produced to diet consumed was 21% and 61%, respectively. A more pronounced reduction in food consumption occurred when larvae were supplied with Galleria "cocoon" silk or the sericin fraction of such silk, and only 3.8 mg/larva was ingested of the diet containing Bombyx mori cocoon silk or its sericin fraction. Silk production expressed in terms of percentage of diet consumed was always higher than that recorded for larvae fed STD. We conclude that G. mellonella larvae recycle part of the silk that they produce during feeding. Presence of silk in the diet reduces food intake but increases the ratio of silk production to diet consumption. Sericin fraction of the cocoon silk seems to deter feeding.

Duponchelia fovealis Zeller is a polyphagous insect that has been recently reported attacking strawberry plants (Fragaria x ananassa Duchesne). Despite its economic importance there are few studies on this pest because it is difficult to rear it in the laboratory. With a constant supply of insects, studies on alternative methods of pest control can be conducted. This study aimed at developing an artificial diet for rearing D. fovealis with biological characteristics similar to those reared on their natural diet. This study was carried out in a climate-controlled room (25°C ± 2°C, RH 70% ± 10%, and 14L : 10D). The natural diet consisted of 'San Andreas' strawberry leaves (D1), while the artificial diet (D2) was developed in which beans, casein, soy protein, yeast and wheat germ are used as sources of protein. Five instars were identified. D. fovealis completed its life cycle in 38 and 40 days when fed D1 and D2, respectively. Survival was highest for the larvae fed the artificial diet. Females fed D1 lay a mean of 300.2 ± 62.3 eggs, while those fed D2, 220.3 ± 41.8 eggs. The artificial diet is suitable for the continuous rearing of D. fovealis in the laboratory.

Aphis fabae and Myzus persicae (Hemiptera: Aphididae) are insect pests that damage sugar beet and bean crops. Both are responsible for losses in yield and transmission of viral diseases, and may be present on the same host at the same time. Three parasitoid species, Aphidius colemani, Lysiphlebus testaceipes and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae) have the potential to be used as biological control agents against at least one of these species of aphids. As a first step prior to the implementation of a biological control program, our aim was to understand the host selection behaviour of the parasitoids, particularly when both aphids are present. We recorded the host acceptance (number of insertions of the ovipositor / number of antennal contacts), suitability (number of mummies / the number of insertions of the ovipositor) and emergence (number of adults emerging from mummies) of these three aphid parasitoids when parasitizing the two aphids. We also analyzed the effect of the host plant on the host preference of the parasitoid. Females of each parasitoid species (n = 15) were exposed to 20 aphids of A. fabae or M. persicae, or a mixture of these two species of aphids, for 15 min, on a leaf disc of each of the two host plants, sugar beet and bean. Higher host acceptance and suitability were recorded for A. colemani attacking both species of aphid: A. fabae (43 and 46%) and M. persicae (43 and 46%) on beet and bean plants respectively, compared to L. testaceipes and L. fabarum. L. testaceipes and L. fabarum showed a clear preference for A. fabae. L. fabarum accepted M. persicae on both plants only when it was mixed with A. fabae, probably due to a confusion effect. We found that the host plant played a significant role in host acceptance, host suitability. We conclude that A. colemani is the better of the three parasitoids studied for the biological control in bean, and particularly, sugar beet crops.

Stable reference genes (RGs) determine the reliability of quantitative polymerase chain reaction (qPCR) analyses and it is recommended that different reference genes are used for different types of DNA and tissues. The present study aimed to screen for stable RGs for the qPCR analysis of the immune responses of the whitefly Bemisia tabaci to the Wolbachia wMel strain from Drosophila melanogaster. A total of eight candidate RGs were evaluated using five different methods, i.e., Coefficient of Variation analysis, GeNorm, NormFinder, BestKeeper and ΔCt. The stability of these RGs was assessed for both genomic DNA (gDNA) and complementary DNA (cDNA). The results indicate that β-actin (Actin) and elongation factor 1 alpha (EF-1α) were the most stable RGs for gDNA, whereas 18S rRNA (18S) and glyceraldehyde phosphate dehydrogenase (GAPDH) were the least stable; in contrast, Actin and GAPDH were the most stable for cDNA, whereas RPL29 and ATPase were the least stable. The effectiveness of the most stable RGs was then validated against the least stable using qPCR analysis of the titre of wMel (gDNA) and the transcriptional responses of the antimicrobial peptide Alo-3-like and the phosphatidylinositol-bisphosphate 3-kinase catalytic subunit delta isoform (cDNA) to wMel transfection. The results support the notion that reliable RGs are essential for a qPCR analysis of samples of both gDNA and cDNA.

The longhorn crazy ant, Paratrechina longicornis (Latreille), is a ubiquitous agricultural and urban pest that has invaded most tropical and subtropical regions. Although P. longicornis has been found worldwide for more than a century, the genetic structure, origin, and invasion history of this species have not yet been extensively studied, partially because of the limited number of genetic markers currently available. In the present study, we developed 36 polymorphic microsatellite markers for P. longicornis and characterized these markers by genotyping P. longicornis workers from 74 colonies in East and Southeast Asia. All loci were polymorphic, with the number of alleles per locus ranging from 3 to 18 (8.5 on average). Extremely high levels of heterozygosity were found in all populations, suggesting that workers are invariably produced from the mating of divergent queen and male lineages. Queens and males possess non-overlapping allele size ranges at 18 loci, indicating the potential resolving power of the subset of markers in inferring the history of queen and male lineages. Genetic differentiation among three studied populations was low yet significant and may likely reflect their close association with human activities. Overall, the new microsatellite markers developed in the present study serve as a practical tool to reconstruct routes of invasion and assess the population genetics of this invasive ant.

This study of the role the environment in the composition of the communities of carabid beetles was carried out in several types of forests at Obihiro and Furano, Hokkaido, Japan. In addition, we analysed the altitudinal distributions of two forest specialists, Damaster gehinii (Faimaire) and Procrustes kolbei Roeschke, which occur in six mountain forests on Hokkaido. At Obihiro, the forest specialist, P. kolbei was found at only one site in a large area of broadleaf forest. A redundancy analysis indicated that the carabid assemblages were significantly different in the different types of forest, and that two species, Leptocarabus arboreus arboreus (Lewis) and Leptocarabus opaculus opaculus (Putzeys), were associated with broadleaf forests. At Furano, a forest specialist D. gehinii and a forest generalist Cychrus morawitzi Gehin were mainly collected in natural broadleaf and mixed forests, whereas the percentage made up of L. o. opaculus was very high (80.8%) in natural broadleaf forest. Pterostichus thunbergii Morawitz made up the highest percentage of the species in the conifer plantations. In the six mountain forests, D. gehinii was scarce and mainly occurred at low altitudes; however, P. kolbei was relatively abundant and mainly recorded at middle to high altitudes. The management of the forests on Hokkaido needed to maintain the diversity and abundance of carabid beetles is discussed.

The diversity of dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) is affected by urbanization, habitat fragmentation and disappearance of native mammals, which result in changes in the quality and availability of food, and resources for nesting. We evaluated the diversity, distribution of species in different trophic guilds and foraging strategies, and temporal variation in abundance of dung beetles in an urban fragment of Cerrado in a Federal District of Brazil. This study was conducted at the Experimental Station of Biology (EEB) of the University of Brasília (UnB), Federal District, Brazil. Fortnightly, between October 2012 and September 2013, Scarabaeinae were collected using baited (human faeces, pig meat, or banana) and unbaited pitfall traps. Aspects of the dung beetle assemblage were evaluated, including species richness and abundance, feeding habits and foraging strategies. A total of 614 individuals were collected, which included eight species belonging to seven genera and four tribes. Dichotomius carbonarius (Mannerheim, 1829), a generalist species, dominated the assemblage, indicating that it is adapted to living in fragmented and urbanized areas. As regards feeding habits, Eurysternus caribaeus (Jablonsky & Herbst, 1789) was considered to be strictly coprophagous, Coprophanaeus cyanescens (d'Olsoufieff, 1924) copronecrophagous and D. carbonarius a generalist. Most species were classified as paracoprids, which accounted for 75% of the species collected. Regarding the attractiveness of the bait, the highest numbers of individuals and species were recorded in pitfall traps baited with human faeces. A high abundance of Scarabaeinae was recorded in the first half of the rainy season (October-December) with 98.2% of the individuals collected in this period. The urban area studied harboured a low number of species with high abundance of a few species.

Based on research carried out from 2016 to 2018 there are twenty-six species of aphids of the tribe Eulachnini in Lithuania. Seventeen are members of the subgenus Cinara of the genus Cinara, three of the subgenus Cupressobium and two of the subgenus Schizolachnus. In addition, there are four species of the genus Eulachnus. Summarized information on the distribution and host specificity of Eulachnini in Lithuania is presented. Nine species were in all climatic regions of Lithuania: C. (Cinara) brauni, C. (Cinara) hyperophila, C. (Cinara) neubergi, C. (Cinara) piceicola, C. (Cinara) pinea, C. (Cinara) pini, C. (Cinara) pruinosa, C. (Cupressobium) cupressi and C. (Cupressobium) juniperi. Five species of Lithuanian Eulachnini inhabit host plants of the genus Picea, three live on Larix, thirteen on Pinus, one on Abies, one on Thuja and three on Juniperus. Cinara (Cinara) piniphila was recorded on Pinus mugo and C. (Cinara) nuda on Pinus heldreichii for the first time. Partial COI and EF-1α sequences of C. (Cinara) hyperophila, C. (Cinara) pilosa and C. (Cinara) piceicola were obtained for the first time. Partial sequences of mitochondrial (COI) and nuclear (EF-1α) DNA of Lithuanian samples were used to explore molecular diversity using NJ trees, Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). Species delimitation using GMYC (both on COI and EF-1α sequences), PTP (BI on COI) and ABGD (EF-1α) were the most consistent with traditional classifications. Pairwise between-species sample divergences (COI and EF-1α fragments) of the samples of the species complex C. (Cinara) pinea - C. (Cinara) piniphila indicate it is composed of a single species. Of the species of aphids that live on conifers, there are seven species of the tribe Eulachnini that are likely to shortly colonize Lithuania.

Instances of heat waves and cold snaps are becoming more frequent and of increasing duration worldwide. It is well established that short exposure to high or low-temperatures, such as during extreme weather events, often results in adaptive (acclimation/hardening) or maladaptive plastic changes in tolerance of organisms to subsequent thermal stressors. However, little information is available about how the duration of a prior stressful thermal event mediates future organismal thermal responses. Understanding durational effects of thermal conditioning can help predict ectothermic survival in response to novel extreme weather patterns.

Scientists and society are increasingly becoming aware of loss of insect biodiversity and biomass. The level of biodiversity determines the efficiency of ecological communities to capture essential resources, produce biomass, decompose and recycle nutrients. Relationships between plant and insect diversity can be modified by changes in insect abundance. This study determined the associations between microclimate and diversity of forest plants on overall insect abundance and abundances of different insect orders in the canopy of temperate deciduous forests dominated by Fagus sylvatica L. (European beech; Fagaceae) in Central Germany. Following model selection, a linear mixed model was used to analyse the associations between abiotic factors (air temperature and relative humidity) as well as biotic factors (species richness and cover for the ground, shrub and tree layer) and insect abundance and abundances of insect orders. Within similar strength of evidence, best models were chosen as those with a single explanatory parameter. Significant associations were recorded between abundances of Lepidoptera and Neuroptera and relative air humidity, insect abundance and species richness in the shrub layer, and between abundances of Diptera and Psocoptera and percentage cover of shrubs. Trends in associations were detected between the abundance of Hemiptera and species richness recorded in the shrub layer, and between the abundance of Thysanoptera and the species richness in the ground layer. No significant associations with single explanatory parameters were recorded for the abundances of Coleoptera and Hymenoptera. Phytodiversity, especially that in the shrub layer, were more often associated with the abundances of insect orders than microclimatic factors. The potential importance of the association between shrub layer parameters and the abundance of insects might be based on the shrub layer combining the associations with the ground layer (bottom-up processes) and canopy layer (environmental conditions). In addition, associations between phytodiversity and abundance of insects seem to vary most for Coleoptera and Hymenoptera in which there are markedly different functional groups.

Facultative diapause in the wax moth, Galleria mellonella, occurs in the final larval instar. Application of juvenile hormone analogs (JHAs) to the larvae of this species has similar effects to diapause, in terms of prolonged development of the larval stages and the arrest in the metamorphosis of internal organs. Here, we focus on testes development and spermatogenesis at the end of larval development in G. mellonella, how they are affected by diapause induced by an environmental decrease in temperature to 18°C and the application of a JHA (fenoxycarb) to larvae. Because neither testis development nor spermatogenesis are described in detail for this species, we examined them in individuals not in diapause during the period from the last larval instar to the newly emerged adult and present a timetable of changes that occur in the development of testes in this species. These observations have increased the very limited data on the course of spermatogenesis in pyralid insects. We then used these data for comparative analysis of testes in larvae from two experimental groups: individuals in diapause and those treated with fenoxycarb. The results on the general morphology testes revealed obvious degenerative changes caused by fenoxycarb (but not by diapause), including testicular wall hypertrophy and disarrangement of testicular follicles. Moreover, treatment with fenoxycarb finally resulted in the disintegration of nearly all testicular cyst-containing germ cells at different stages of spermatogenesis, a situation never previously described in the literature. In contrast, the main effect of diapause on testes was merely the degeneration of spermatocytes in the proximal regions of the testicular follicles. Finally, the TUNEL analyses, revealed that the degenerative changes in germ cells were apoptotic in character in the testes of both individuals in diapause and fenoxycarb-treated males.

Brackish marshes resulting from embankments are a rare habitat in Europe and for which there is little knowledge of the entomofauna. There is an urgent need to document the insect diversity and its unique properties in the light of ongoing habitat losses. The current study focuses on snail-killing flies in the remaining Belgian brackish marshes, in particular the role of soil salinity in determining their diversity and how it compares with that in freshwater marshes. Snail-killing fly communities in brackish marshland were relatively species poor and clearly different from those in freshwater, yet species richness and abundance increased with decreasing soil salinity. We recorded no habitat-specific species, but Tetanocera arrogans was more abundant in brackish marshes. Malaise traps proved to be a successful and repeatable method of sampling sciomyzid communities, the species composition of which was strongly site-specific. We conclude that extant Belgian brackish marshes have poor to moderately rich snail-killing fly communities that lack unique species. Richness and abundance, however, increased as soil salinity decreased.

The sugarcane borer Diatraea saccharalis is an important pest of sugarcane and ants are one of its main predators. The practice of burning sugarcane straw in situ after harvest has been gradually replaced in Brazil by other practices. However, it is unknown whether ants can control the abundance of this borer in the presence of straw. In this study, we assessed the diversity and species composition of ants attacking different stages of the pest's life cycle. Specifically, we asked whether the species richness and abundance of ants varies during the course of day and a year. We established one-hectare plots at random locations in a sugarcane plantation. Once a month, we collected 20 samples of each stage of the D. saccharalis life cycle and randomly distributed these samples as bait on plants spaced 20 m apart within a plot. Ants were collected daily in the morning and afternoon over a period of 12 months. We identified several aspects of ant feeding behaviour that may affect their biological control of the borer: (1) the greatest number of ants were collected from baits consisting of the immature stages of the sugarcane borer, (2) ants were most active in the morning and (3) their activity varied from month to month. Solenopsis saevissima and morphotypes of Crematogaster sp.7 and Pheidole sp.35 are potentially important predators of borers in sugarcane crops in which the straw is not burnt.

The Chinese pine caterpillar Dendrolimus tabulaeformis is an important destructive leaf borer in boreal coniferous forests in China. This species overwinters in the larval stage. Changes in supercooling capacity and physiological-biochemical parameters of D. tabulaeformis larvae from a natural population were evaluated at different stages during the overwintering period. Cold hardiness of overwintering larvae collected in January was significantly greater than that of larvae collected in other months. January larvae survived for 15 days at -10°C and for approximately 2 days at -15°C. By contrast, larvae collected in September survived for no more than 4 h at -5°C and those in November and March no more than 1 day at -15°C. Supercooling point gradually decreased from -5.9 ± 0.3°C in September to a minimum of -14.1 ± 1.0°C in November, then gradually increased to the original value with the advent of spring. Water content gradually decreased from September to November, remained at approximately 74.5% until March and then gradually increased to levels similar to those in September. The lipid content gradually decreased from September to November, remained stable at approximately 3.2% until March and then gradually increased to levels similar to those in September. Glycogen content increased to a peak in November and then decreased. The concentrations of several metabolites showed significant seasonal changes. The most prominent metabolite was trehalose with a seasonal maximum in November. Glucose levels were highest in January and then gradually decreased until in May they were at levels similar to those in September. Glycerol levels remained relatively stable during winter but increased significantly in May. This study indicates that D. tabulaeformis is a freeze-avoidant insect. Larvae increase their supercooling capacity by regulating physiological-biochemical parameters during overwintering. Our results provide the basis for further research into the mechanism of cold hardiness in this species.