Host suitability and fitness-related parameters of Campoletis sonorensis (Hymenoptera: Ichneumonidae) as a parasitoid of the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae)

The seven age-classes of Trichoplusia ni (Hübner) larvae evaluated in this study as hosts of Campoletis sonorensis indicates that early 2nd larval instar (3–5 day-old larvae) of T. ni represents the most suitable host stage for the development of the larval endoparasitoid C. sonorensis. The higher suitability of early 2nd larval instar of T. ni resulted in more parasitised larvae, a higher rate of successful parasitoid emergence, a higher rate of female progeny, and a lower rate of immature parasitoid mortality. The fitness gain of C. sonorensis on late 1st larval instar (2 day-old larvae) and late 2nd larval instar –early 3rd instars (6–8 day-old larvae) stages of T. ni is negatively affected by the trade-offs between the different physiological and behavioral characteristics influencing their suitability as hosts of C. sonorensis.     

Pathology and bioassays of the lesser cornstalk borer (Elasmopalpus lignosellus) entomopoxvirus

The pathology and virulence of a naturally occurring entomopoxvirus of the lesser cornstalk borer (Elasmopalpus lignosellus) were studied in the laboratory. Diseased larvae appeared red and white, as opposed to the normal blue-green and brown color of healthy larvae. Chronic disease protracted the larval life span up to 40 days beyond normal. Infection appeared to be restricted to the hemocytes and the fat body cells. The LC₅₀ for 1st instars was 9 spheroids; for 3rd and 4th instars 93 spheroids; and for 5th and 6th instars, ca. 700 spheroids.     

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.     

Dosage-mortality and time-mortality studies of a granulosis virus in a laboratory strain of the codling moth,Laspeyresia pomonella

Different doses of a granulosis virus were administered to first- and fifth-instar larvae of the codling moth Laspeyresia pomonella. Virus was very pathogenic for both larval instars. The LD₅₀ values for first- and fifth-instar larvae were 5 and 49 capsules/larva, respectively. However, fifth-instar larvae were much more variable in their response to virus than first-instar larvae. Using probit methods it was calculated that 1 capsule could cause death in about 25% of both larval instars but 1578 capsules were required to cause 70% mortality of fifth-instar larvae as compared to 12 capsules for first-instar larvae. This is the first report of a decided difference in variability of response to virus by two larval instars of the same species. A bimodal response by both larval instars was observed in time-mortality studies. Apparently, about 20% of the larvae were very resistant to virus infections.     

Performance of Microplitis tuberculifer (Hymenoptera: Braconidae) parasitizing Mythimna separata (Lepidoptera: Noctuidae) in different larval instars

The solitary parasitoid Microplitis tuberculifer (Wesmael) is an important biological control agent of various lepidopteran pests in Asia. We examined the preference of M. tuberculifer for different instars of its common host, Mythimna separata (Walker), host instar effects on parasitoid development, and the consequences of parasitism in different stages for growth and consumption of host larvae. The wasp successfully parasitized the first four larval instars of M. separata, but not the fifth, which appeared to be behaviorally resistant. First and second instars were parasitized at higher rates compared to thirds and fourths in no-choice situations, ostensibly due to longer handling times for the latter, but second instars were most preferred in a choice test that presented all stages simultaneously. Although later instar hosts yielded heavier cocoons, the fastest parasitoid development was obtained in second instars. Lower sex ratios were obtained from first instars as females appeared to lay a smaller proportion of fertilized eggs in small hosts. Both weight gain and food consumption of parasitized larvae were reduced significantly within 24 h of parasitism, regardless of the stage parasitized, and final body weights were less than 10% those of unparasitized larvae. Thus, M. tuberculifer has good potential as a biological control agent of M. separata, successfully parasitizing the first four larval instars and dramatically reducing plant consumption by the host in all cases.     

Larval development and growth ratios of Odonata of the Azores

To fully understand odonate life cycles, it is vital to analyse the patterns of larval growth, which are tightly associated with variations in environmental factors. However, the identification of larval instars is often difficult, especially for early development stages. We hypothesise that pond hydroperiod influences odonate larval growth, and test this idea with novel information about the environmental characteristics of 12 study ponds in Terceira Island (Azores). This study, which represents the first analysis of larval development of Odonata in the Azores, involved determining the instars of development and growth ratios. We measured the morphological characteristics of 898 Odonata larvae, and found that they significantly differed between temporary and permanent ponds. To estimate the larval instars, we used two methods: frequency distribution methods and correlation diagrams between body characteristics. The first method was the most effective, allowing the estimation of 17 instars for Anax imperator, 13 for Sympetrum fonscolombii and 10 for Ischnura hastata, as well as growth ratios between instars for head width, total length, abdomen length, wings and antenna length. Our results also suggest that a combination of the two methods is the most appropriate strategy for estimating the number of instars and growth ratios during larval development.     

Comparative toxicity,growth inhibitory and biochemical effects of terpenes and phenylpropenes on Spodoptera littoralis (Boisd.)

Eleven monoterpenes, phenylpropenes and sesquiterpenes were evaluated for their insecticidal and growth inhibitory activities against the second and fourth larval instars of Spodoptera littoralis. Among the tested compounds, 1,8-cineole revealed the highest fumigant toxicity against the 2nd and 4th larval instars with LC₅₀ values of 2.32 and 3.13 mg/L air, respectively. The monoterpenes, p-cymene, α-terpinene, (−)α-pinene and (−)-carvone were highly toxic to both larval stages as their LC₅₀ values ranged between 7.35 and 13.79 mg/L air against 2nd larval instar and between 14.66 and 32.02 mg/L air against 4th larval instar. In topical application assay against the 4th larval instar, (−)-carvone (LD₅₀ = 0.15 mg/larva) and cuminaldehyde (LD₅₀ = 0.27 mg/larva) were the most potent contact toxicants. In residual film assay, trans-cinnamaldehyde, (−)-citronellal and p-cymene showed the highest insecticidal activity against the 2nd larval instar, while α-terpinene and (−)-carvone were most effective compounds against the 4th larval instar. Moreover, the tested compounds caused strong growth reduction of both larval stages with growth inhibition higher than 80% in the 2nd larval instar and higher than 70% in the 4th larval instar. On the other hand, (−)-carvone, cuminaldehyde and (Z,E)-nerolidol showed pronounced inhibitory effects on acetylcholinesterase (AChE) and adenosine triphosphatases (ATPases) activity of S. littoralis larvae. Cuminaldehyde (IC₅₀ = 1.04 mM) and (Z,E)-nerolidol (IC₅₀ = 0.02 mM) caused the highest inhibition of AChE and ATPases, respectively. Taken together, the results indicate that monoterpenes, phenylpropenes and phenylpropenes could be used to develop new botanical insecticides for S. littoralis management.     

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18 Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.     

Tipula iridescent virus infection in the developmental stages of Tipula oleracea

Tipula iridescent virus (TIV) is infective to all four larval instars, pupae, and adults of both sexes of Tipula oleracea, and iridescence has been observed in infected insects at all these stages. Third- and fourth-instar larvae were more resistant to ingested TIV than first and second instars. When TIV was injected into the hemocoel, the results suggested a possible decrease in resistance from the third larval instar to the pupa. Incubation periods (times from injection of TIV to appearance of iridescence) were significantly shorter in older fourth-instar larvae than in younger fourth-instar or thirdinstar larvae, but variability in incubation period was significantly greater in younger fourth-instar larvae than in the other two stages. Many insects which were inoculated with TIV in one stage developed iridescence and died in later stages. The amounts of infective TIV in two infected adults were estimated.     

Seasonal history ofMacrocentrus grandii [Hym.: Braconidae] andEriborus terebrans [Hym.: Ichneumonidae], two parasitoids of the European corn borer,Ostrinia nubilalis [Lep.: Pyralidae]

The seasonal histories and phenological relationships of European corn borer,Ostrinia nubilalis (Hübner), and its 2 parasitoids,Macrocentrus grandii (Goidanich) andEriborus terebrans (Gravenhorst) were studied in southcentral Minnesota. Both parasitoids overwintered in mature borer larvae, broke diapause, completed development, and emerged at the same time as did borer adults. Thus the 1st generation parasitoids coincided with the peak abundance of their preferred larval instars of the 1st host generation. Both parasitoids had a 2nd generation, matching the bivoltinism ofO. nubilatis in Minnesota. The activity of 2nd generationE. terebrans was before the peak 2nd generation borer moth flight and was not synchronized with the peak abundance of 2nd generation borer larvae. The peak activity of 2nd generationM. grandii occurred after the peak 2nd generation borer moth flight and was fully synchronized with the peak abundance of 2nd generation borer larvae. Thus,M. grandii has both generations synchronized with the host seasonal history, and was the more effective of the 2 parasitoids.     

The dependence of development and fecundity of Samea multiplicalis on early larval nitrogen intake

Larvae of the moth Samea multiplicalis (Lepidoptera: Pyralidae), developed more rapidly when their food plant, Salvinia molesta was richer in nitrogen. Larvae that fed on plants with less than 1.35% nitrogen during their first instar completed the larval stage in 18.0–24.2 days, after passing through six instars. In contrast, larvae that had fed on food richer in nitrogen during their first instar completed the larval stage in 14.1–17.4 days, and passed through five instars. Experience of nitrogen in later instars had little additional effect on total larval development time. Oöcyte complements of 1-day old adult females was correlated with the mean food nitrogen content over the larval period. Food nitrogen content experienced by larvae in earlier instars was as important as that in the final instars, in determining number of mature oöcytes, which suggests early conditioning of the nitrogen use effciencies of later instars.     

The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)

The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and eggs. Western blot analysis demonstrated that the majority of absorbed vicilins were degraded in the fat body. Tracing the fate of vicilins using FITC revealed that the FITC-vicilin complex was present inside cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus. Labelled vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the insects and eventually are sequestered by the eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these peptides are known to have antimicrobial activity. Quantifications performed on internal organs from larvae of C. maculatus exposed to extremely dry seeds demonstrated that the vicilin concentration in the haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These results suggest that absorbed vicilins may also be involved in the survival of larvae in dry environments.     

The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day-old) fed with C. calcitrans and S. costatum and then starved at 5, 15 and 25 °C temperature were estimated as ultimate recovery hour (URH; denoting the starvation point in hours at the end of which larvae can recover and continue development). Effect of temperature on starvation threshold varied significantly with larval age and food species. The URH declined with larval age at 5 °C, but not at 15 and 25 °C. The URH and grazing rates were high for early instars fed on C. calcitrans, and for advanced instars fed on S. costatum. Carbon gain through feeding was maximum for 2-day-old larvae when fed with C. calcitrans and decreased with larval age. However, when fed with S. costatum carbon gain increased with larval age. This confirms that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior of larvae. Molting was observed at times when larvae were undergoing starvation and this could be viewed as stress-induced molting, and it differed with the larval age and food organisms.     

Instar Susceptibility of Simulium vittatum (Diptera: Simuliidae) to the Entomogenous Nematode Neoaplectana carpocapsae

Laboratory bioassays showed that the susceptibility of Simulium vittatum to Neoaplectana carpocapsae increased with successive larval instars. First, second, and third instar larvae were resistant to infection, while seventh instars were highly susceptible. Significant differences in intra-instar susceptibility were also evident, as mortality ranged from 58% for the smallest seventh instar larvae to 97% for the largest. Dissections revealed that the basis for the resistance of early instars was physical exclusion of the comparatively large nematodes. The principle factor regulating the susceptibility of mid and late instars was injury to nematodes caused by larval mouthparts during ingestion. Differences in intra-instar susceptibility were similarly related to nematode injury.     

Age-specific dose-mortality effects of Beauveria bassiana (Deuteromycotina: Hyphomycetes) on the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Three isolates of Beauveria bassiana were evaluated under laboratory conditions for pathogenicity against the European corn borer, Ostrinia nubilalis. Dose-mortality regressions and nonparametric statistical analyses revealed significant differences in pathogenicity between isolates and across O. nubilalis instars. First instars were found to be the most susceptible of the five larval stages. Little difference was noted for instars 2, 3, and 5 within any of the three B. bassiana isolates. Fourth instar larvae were found to be the most tolerant in all cases.     

Effects of oxygen deficiency on survival and glycogen content of Chironomus anthracinus (Diptera,Chironomidae) under laboratory and field conditions

Growth and glycogen content of Chironomus anthracinus in Lake Esrom, Denmark was examined during summer stratification in 1992 and 1993. Simultaneously, effects of oxygen deficiency on glycogen utilization and survival were experimentally studied. The population consisted of almost fullgrown 4th instar larvae in 1992 and 2nd and 3rd instar larvae in 1993. Growth rate and glycogen content changed as hypolimnetic oxygen deficiency increased. During a 1st phase of stratification dry weight and glycogen content increased (2nd and 3rd instars) or was almost constant (4th instar) but decreased significantly during the following 2nd phase. This change from growth to degrowth and utilization of endogenous glycogen reserves correlated with a change in the thickness of the microxic layer (<0.2 mg O₂ 1^–1) above the sediment surface. The layer increased from 2–3 m in phase 1 to 4–5 m in phase 2, and we suggest that this deteriorated the oxygen conditions and resulted in a change in larval energy metabolism from fully aerobic during the 1st phase to partly anaerobic in the 2nd phase. During the 2nd phase larval metabolism was estimated at less than 20% of normoxic rate. Experimental exposure of the larvae to anoxia indicated highly different survival of young larvae (2nd and 3rd instars) and older larvae (large 4th instars). The morality of young larvae was 50% after three days in anoxia at 10 °C, whereas only 25% of the older larvae had died after 3–4 weeks under similar conditions. Extending the treatment, however, resulted in increased death rate of the 4th instar larvae with only 10% surviving after seven weeks. The anaerobic metabolism of 4th instar larvae as estimated from glycogen degradation at 10 °C was 5% of normoxia in the interval from 0–5 days but 1.5% in the interval from 20–25 days. It is concluded that survival of C. anthracinus in anoxia is very limited, but traces of oxygen in the environment allowing for faint aerobic metabolism prolong the survival time of the larvae from a few days (2nd and 3rd instars) or a few weeks (4th instar) to probably 3–4 months.     

Behavioral Response to Larval Tracks and the Influence of Tracks on Intraguild Scavenging by Coccinellid Larvae

A paired design was used to determine that Harmonia axyridis 4th instars were not influenced by the presence of conspecific larval tracks, but well-fed H. axyridis 4th instars spent less time on plants that contained tracks left by Coleomegilla maculata 4th instars. To determine if the presence of larval tracks influences intraguild scavenging by H. axyridis 4th instars, dead 4th instars were placed in Petri dishes that contained or did not contain larval tracks. The presence of larval tracks did not influence the feeding frequency or the amount of time before feeding. However, larvae dragged their pygopod on dish surfaces more frequently if the dish contained larval tracks. In addition, starved H. axyridis larvae were more likely to feed on the prey and dragged their pygopod less frequently than well-fed larvae.