Selection of reference genes for RT-qPCR analysis in Trichogramma chilonis (Hymenoptera: Trichogrammatidae)

Trichogramma chilonis is an important natural enemy for control of various Lepidoperan crop pests. The biology of T. chilonis is well-studied, but the molecular mechanisms of this biology require further study. Screening suitable reference genes is a vital step for use of RT-qPCR to understand underlying molecular physiology. In the present study, nine candidate reference genes including elongation factor 2 (EF2), ribosomal proteins (RPS23, RPL13, and RPL44), malate dehydrogenase (MDH), eukaryotic translation initiation factor 3 subunit F (EIF3F), zinc finger protein 268 (ZFP268), muscle specific protein 20 (MP20), and ATP synthase subunit alpha (ATP5F1A) were evaluated at different conditions including development stage, diet, temperature, and insecticide treatments. Four common algorithms (the Delta Ct method, geNorm, BestKeeper, and NormFinder) and RefFinder were used to analyze gene expression stability. Our results indicated that two reference genes used for normalization were sufficient, and the optimal combinations were: RPS23 and EF2 for developmental stages, ZFP268 and EF2 for feeding with different diets, ZFP268 and RPL13 for temperature treatments, and EF2 and RPL44 for insecticide treatments. The results provide preliminary determination of suitable reference gene for standard RT-qPCR analyses in T. chilonis, which might establish the foundation for further molecular biology research.     

Isolation and characterisation of 26 polymorphic microsatellite markers for the parasitoid wasp Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Polymorphic microsatellite markers were developed for the parasitoid wasp Trichogramma dendrolimi. Forty-four microsatellites were isolated from a (TCG)₈-enriched genomic library and characterised. Twenty-six of these loci were polymorphic and could be used for evaluation of the genetic variation of T. dendrolimi.     

Continuous rearing on Ephestia kuehniella reshaped quality of the parasitoid wasp Trichogramma brassicae (Hymenoptera: Trichogrammatidae)

Continuous mass rearing of Trichogramma brassicae (Bezdenko) at commercial mass-rearing insectaries may affect both quality and performance of natural enemies. In the present study, we studied the quality and performance of a colony of T. brassicae reared for over 45 generations (G) on Ephestia kuehniella Zeller using two-sex life table parameters and parasitism capacity. Our results revealed that although different generations showed no significant difference in terms of female longevity or total life span until G35, G5 and G10 had the highest values of fecundity, gross reproductive rate (GRR), net reproductive rate (R₀), intrinsic rate of natural increase (r), and finite rate of increase (λ). No significant difference in male adult longevity was found among different generations. The longest and shortest mean generation times (T) were found in G10 (13.65 ± 2.31 d) and G45 (13.25 ± 3.37 d), respectively. The finite rate of parasitism (ω) ranged from 0.355 ± 2.332 host/parasitoid/day in G5 to 0.242 ± 0.017 host/parasitoid/day in G45. However, ω did not show any significant difference until G20. These results indicate that T. brassicae wasps held under continuous laboratory rearing declined in quality after 20 generations, and therefore periodical rejuvenation of the colony by adding feral parasitoids is strongly recommended.     

Strategic ejaculation in the egg parasitoid Trichogramma turkestanica (Hymenoptera: Trichogrammatidae)

Abstract.  1. Several parameters influence sperm allocation by males, including their size and sperm stock, intra-specific variability, quality of females', as well as the risk and intensity of sperm competition.
2. Models predict that males should invest the maximum ejaculate size when sperm competition intensity is low. As sperm competition intensity increases, males should decrease the number of sperm transferred during mating.
3. This decrease in sperm transfer to females occurs because the benefits gained by males with each extra unit of expenditure on sperm decrease. When sperm supply is not unlimited, males could expect a better return by keeping some or all sperm for mating under lower competition intensity.
4. In this study, the ejaculate size of males that were kept in groups of one, five or 10 males prior to mating, has been investigated in the haplodiploid egg parasitoid Trichogramma turkestanica Meyer (Hymenoptera: Trichogrammatidae).
5. As predicted by theory, the number of sperm transferred decreased significantly with an increase in the number of rivals.
6. This is the first study showing strategic sperm allocation depending on sperm competition intensity in a parasitoid.     

Effects of various insecticides on the development of the egg parasitoid Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae).

The toxicity of six insecticides, acephate, methomyl, ethofenprox, cartap, chlorfluazuron, and Bacillus thuringiensis (Bt) was tested on different developmental stages of the egg parasitoid, Trichogramma dendrolimi (Matsumura). Each of the insecticides tested showed different degrees of toxicity to the parasitoid, Ethofenprox showed the highest toxicity and cartap showed relatively higher toxicity compared with the other insecticides. The development of the parasitoids treated with these two insecticides was normal, similar to that of the control group. Only the emergence of adult wasps from host eggs was disturbed. Emergence of the host, Mamestra brassicae larva was reduced following treatment with ethofenprox, cartap and methomyl. However, adult female wasps, which emerged from host eggs treated with the insecticides had the ability to oviposit normally.     

Pollen as a source of food for egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae)

The value of corn pollen as a food source for Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) was assessed in the laboratory by testing the effect of different pollen diets on the longevity, capacity of parasitism and other life-table parameters. T. brassicae females that fed on corn pollen and water (wet filter paper dusted with pollen) lived significantly longer (4.97 days) than females that fed on water alone (2.67 days), but significantly shorter than those feeding on honey alone (8.37 days) or corn pollen and honey (8.23 days), respectively, in the absence of host eggs. The presence of European corn borer eggs (Ostrinia nubilalis Huebner, Lepidoptera: Pyralidae) significantly increased longevity. With host eggs available, female T. brassicae that fed on corn pollen and water lived significantly longer (4.9 days) than on water alone (2.60 days), but significantly shorter than on honey alone (12.33 days) or corn pollen and honey (12.17 days), respectively. The cumulative lifetime fecundity was 82.53 offspring pupae when fed on pollen and water, which was significantly higher than 61.70 for water alone but lower than feeding on honey alone (95.70) or corn pollen and honey (99.97), respectively. Compared to water alone, corn pollen and water increased the net reproduction rate (R₀), intrinsic rate of increase (r_m), and finite rate of increase (λ), and reduced the doubling time (T₂), but had no effects on the emergence rate, sex ratio, and mean generation time (T_c). In another set of experiments, 13 out of 30 females reduced the number of pollen grains offered to them. The average number of pollen grains utilized per female in this 48-h test 2.46±0.40 (mean±SE). Hence, T. brassicae used corn pollen as food that significantly increased longevity and fecundity of the tested females. Pollen and honey supplied a complete diet for T. brassicae, compared to pollen and water or water alone.     

Biological studies on two neotropical egg parasitoid species: Trichogramma nerudai and Trichogramma sp. (Hymenoptera: Trichogrammatidae)

The main biological attributes of two Neotropical egg parasitoids, the arrhenotokous Trichogramma nerudai and the thelytokous Trichogramma sp., were assessed under controlled laboratory conditions. Developmental time from egg to adult, and parasitoid survival, fecundity and fertility were studied using life tables. Results showed that T. nerudai had a faster developmental time than Trichogramma sp. (13.014±0.4019 and 13.595±0.4931 days, respectively). Both species showed similar life table statistics, r_m was 0.222 and 0.225 for T. nerudai and Trichogramma sp., respectively. Parasitoid survival averaged 95% for both species. The results obtained are discussed in the context of selecting one of these natural enemies as a potential biological control agent for the European pine shoot moth Rhyacionia buoliana (Lepidoptera: Tortricidae) in pine forests and the codling moth Cydia pomonella (Lepidoptera: Tortricidae) in apple orchards in Argentina.     

The impact of cold storage durations on Trichogramma evanescens (Westwood) (Hymenoptera: Trichogrammatidae) during their pupal stage

This experimental study was done at the Biological Control Laboratory, Sakha Agricultural Research Station, Sakha, Kafrelsheikh, Egypt. We aimed to estimate the impact of different cold (10 °C) storage durations [0 (non-cold-stored parasitized eggs), 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days], on Trichogramma evanescens (Westwood) (Hymenoptera: Trichogrammatidae) during the pupal stage using the eggs of Sitotroga cerealella after exposing to T. evanescens. The emergence percentage of non-cold-stored eggs of S. cerealella was higher than all cold-stored durations. Also, the female’s percentages of T. evanescens in the cold storage durations were lower than the non-cold storage one, and they were influenced by extended cold storage durations. There were non-significant differences in the female’s longevity of T. evanescens obtained from 0, 3, and 6 days cold-stored parasitized eggs of S. cerealella at 10 °C, but it began to decrease from those produced after 9 days of cold-stored eggs. In addition, the emergence percentage in F1 progeny of T. evanescens was greater than 50% until 21 days of cold storage. It could be concluded that cold storage reduced the % emergence, % females, female’s longevity, and emergence percentage in F1 progeny of T. evanescens. For a successful biological control program, the decrease of T. evanescens performance after cold storage durations should be considered in mass production, and the release percentage should be increased by the equivalent of a lack of % emergence. Also, the economic importance of using cold storage periods in commercial mass rearing should be assessed in the biological control program.     

Acceptability and suitability of Tuta absoluta eggs from irradiated parents to parasitism by Trichogramma nerudai and Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)

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The dynamics of egg production, oviposition and resorption in a parasitoid wasp

1. The extent to which parasitoid wasps are limited by their egg supply is very important in understanding their reproductive strategies. Egg reserves are dynamic, with most wasps maturing new eggs throughout their life (synovigeny) and many species resorbing eggs that are not used in oviposition. We investigated the extent to which a parasitoid modulates its egg reserves in the light of its experience in finding hosts.
2. The egg dynamics of the Encyrtid Wasp Leptomastix dactylopii , a solitary parasitoid of mealybugs, were studied in the laboratory. This species is synovigenic and practises egg resorption.
3. We allowed newly emerged wasps to experience one of four environments of increasing value in terms of reproductive opportunities. We proposed that wasps that experienced good quality environments would maintain more mature eggs ready for oviposition. Dissection of wasps subject to different periods of host deprivation after the experimental treatment failed to confirm the hypothesis: egg load was independent of experience.
4. We also proposed that any adjustment of egg supply to make up for eggs oviposited would be effected through a reduction in egg resorption. Instead, we found that the wasp quickly made up for eggs oviposited by increased egg production.     

Costs and benefits of polyandry in the egg parasitoid Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae)

Polyandry implies costs (i.e., time, energy, predation risk, etc.) especially in short-lived parasitoid species but females of several hymenopteran parasitoid species, mostly gregarious, do mate with multiple males. Several hypotheses have been proposed to explain the benefits of polyandry but controversy remains, especially in facultative gregarious species that bridge the gap between solitary and gregarious development. In this study, we investigated the possibility that polyandry may bring material benefits to Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) females, a short-lived and facultative gregarious egg parasitoid. Females mated several times with different males both at emergence and throughout their life. No significant difference was found in the offspring sex ratio and the fecundity of multiple mated and single mated females and pre-mating duration increased with the female’s age. The longevity of females did vary significantly with the number of matings but only in the presence of hosts. Female T. evanescens received enough sperm from one mating to allocate an optimal offspring sex ratio and we found no evidence of either nutritional resources or convenience polyandry in this species. Polyandry in facultative gregarious parasitoids might be an adaptive strategy to minimize the risk of mating with males that have already emptied their sperm bank or to accumulate sperm from several partially sperm-depleted males. Polyandry may also increase the probability of non-sib mating in patches exploited by several females.     

短时极端高温对松毛虫赤眼蜂成虫致死作用及子代适合度的影响

极端高温天气事件在高产作物种植区频繁发生,潜在影响了寄生蜂对害虫的防控能力.为明确短时极端高温对松毛虫赤眼蜂Trichogramma dendrolimi Matsumura成虫致死作用及子代发育适合度的影响,本研究模拟夏季田间最高气温,以42℃高温处理松毛虫赤眼蜂成蜂0.5h和1 h,统计并观测了各处理条件下母代蜂致...     

Acute and sublethal effects of neonicotinoids and pymetrozine on an important egg parasitoid,Trichogramma ostriniae (Hymenoptera: Trichogrammatidae)

Both neonicotinoids and pymetrozine are important insecticides and may have the potential to be used in combination with natural enemies in integrated pest management (IPM). However, to determine their compatibility with biological control, it is necessary to examine the side effects of these chemicals on natural enemies. We examined the acute and sublethal effects of four commonly used neonicotinoid insecticides, imidacloprid, dinotefuran, nitenpyram and thiamethoxam, and the pyridine azomethine, pymetrozine, on adults of Trichogramma ostriniae. By analysing acute toxicity and evaluating the safety quotient of these chemicals, we found that thiamethoxam and dinotefuran pose extremely high risks to T. ostriniae, imidacloprid and nitenpyram posed high risks, but pymetrozine was found to have a low risk. In terms of sublethal effects, longevities of female wasps were significantly shortened when they were exposed to residues of dinotefuran. Percentages of ovipositing females decreased significantly after exposure to pymetrozine, thiamethoxam and dinotefuran. Numbers of offspring per female were significantly lower when females were exposed to pymetrozine, dinotefuran or thiamethoxam, compared with control females. Percentages of offspring that emerged as adults were lower when parental females were exposed to either pymetrozine or imidacloprid. Based on the results of the sublethal concentration assay and the acute toxicity bioassay, nitenpyram would be safe for IPM programmes utilising T. ostriniae, and we suggest that T. ostriniae could be released safely after foliar applications of nitenpyram. However, foliar application of imidacloprid, thiamethoxam, dinotefuran or pymetrozine in an agro-ecosystem where T. ostriniae is a predominant biocontrol agent should be carefully evaluated.     

Long-term storage of sexual and asexual Trichogramma brassicae (Hymenoptera: Trichogrammatidae)

The effect of exposure duration to pre-storage (representing fall conditions) and storage (representing winter conditions) temperatures on the emergence rate in sexual and asexual Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) was studied under controlled laboratory conditions. Asexual T. brassicae was infected by Wolbachia, an endosymbiont bacterium. We considered 0, 30, 40, 50 and 60 days as exposure duration at 10°C and 0, 30, 60, 90 followed by 180 days as exposure duration at 4°C for both T. brassicae strains. Emergence rate was significantly affected by the presence of Wolbachia, the duration of exposure to 10°C, the duration of storage at 4°C and their interactions, but not by the interaction between strain × storage duration at 4°C. Emergence rate of adults was positively correlated with exposure duration at 10°C, but it gradually decreased as the cold storage duration (4°C) increased in both strains. In conclusion, we are able to store both asexual and sexual strains for longer periods of time at 4°C, but the emergence rate of sexual T. brassicae was greater than asexual in all the treatments. The presence of Wolbachia could have a negative effect on fitness of T. brassicae and reduces its emergence rate after long-term storage.     

Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae)

A strain of T. chilonis, an egg parasitoid of lepidopteran pests tolerant to the most commonly used cyclodiene insecticide--endosulfan was developed in the laboratory. Tolerance to endosulfan was induced by exposing adult parasitoids sequentially from a sub-lethal concentration (0.004%) to the field recommended concentration (0.09%). The strain acquired tolerance to the insecticide after 341 generation of continuous exposure with LC50 values of 1074.96 ppm as compared to LC50 of (70.91 ppm) in susceptible strain. The genetical study showed that F1 crosses exhibited a semi-dominant response to endosulfan with degree of dominance value (D) of 0.58. The resistant factor of tolerant strain was 15.1 folds and of F1 cross were 8.53 folds over susceptible strain. Under net house conditions, the tolerant strain parasitised 56% Helicoverpa armigera eggs on potted cotton plants immediately after an insecticide spray, compared to 3% by the susceptible strain. High percentage survival of the immature stages of the tolerant strain proved their ability to withstand the insecticide load. Breakdown of insecticide tolerance in the strain occurred after four generations in absence of insecticide load. Use of the tolerant strain as a component of bio-intensive IPM in various crops where insecticide use is higher is discussed.     

Development of a continuous cell line from the insect egg parasitoid,Trichogramma pretiosum (Hymenoptera; Trichogrammatidae)

Summary A cell line (IPLB-TpE1) was established from embryos of the hymenopteran parasitoid,Trichogramma pretiosum Riley. Cultures contain a mixture of attached, elongate spindle-shaped cells and large aggregates of suspended cells. Chromosomes of the cells were typical ofTrichogramma species and isozyme characterization showed patterns similar toT. pretiosum adults, but distinctly different fromHeliothis zea, the lepidopteran host from which parasite eggs were obtained. The cells are capable of growth over a wide range of osmotic pressures with equal growth between 350 and 600 mOsm/kg. Optimal growth was obtained with a pH of 6.5. Doubling time at the 40th passage was 72 h and cultures are currently subcultured at weekly intervals. The mention of a commercial product does not constitute an endorsement by the U. S. Department of Agriculture.     

Sequence data reflect the introduction pathways of the Sirex woodwasp parasitoid,Ibalia leucospoides (Ibaliidae,Hymenoptera)

1. The parasitoid wasp Ibalia leucospoides is native to the northern hemisphere and has been introduced to the southern hemisphere as a biological control agent for the invasive woodwasp Sirex noctilio. Two subspecies of the parasitoid, Ibalia leucospoides leucospoides (Palearctic distribution) and Ibalia leucospoides ensiger (Nearctic distribution), were introduced and are reported to have hybridized.
2. Despite extensive records of the numbers and origins of the wasps imported into the southern hemisphere, nothing is known regarding their current population diversity. We investigated the genetic variation of I. leucospoides in its native and introduced ranges using mitochondrial (COI) and nuclear (ITS) markers.
3. Mitochondrial DNA diversity in the introduced range was limited, with only five haplotypes, although sequence divergence between these haplotypes was high. Similarly, the ITS rDNA sequences revealed multiple clades present in the introduced range.
4. These results reflect introductions from a wide geographical range but where genetic bottlenecks have possibly reduced the genetic diversity. The data further reflect the origin of the I. leucospoides populations in South America and South Africa from New Zealand or Australia. We found no evidence of hybridization between the two subspecies of the parasitoid in its introduced range, and no evidence that I. leucospoides ensiger has established outside its native range.