Effect of some environmental and biological factors on reproductive characters of Trichogramma spp

Many studies have demonstrated that many species of parasitoid wasps can control offspring sex ratio in response to environmental variables. This article tests the effects of temperature, relative humidity, photoperiod, foundress number, foundress age, host number, interval time of host supplying, species of food, species of host and symbiotic bacterium (Wolbachia) on sex ratio of Trichogramma ostriniae. Results showed that the sex ratio of T. ostriniae was affected significantly by temperature (35°C), number of foundress (>2 number), age of foundress (3-day-old) and Wolbachia. Through transmitted Wolbachia from Trichogramma embryophagum to Trichogramma dendrolimi, even the reproductive mode of T. dendrolimi was changed from gamogenesis to thelytoky. Our work verifies the sex ratio theory and implies that the potential effectiveness of Trichogramma spp. as a biological control agent can be further improved.


INTRODUCTION
Most hymenopteran parasitoids are haplodiploid (White, 1973;Cook, 1993) and have the potential for controlling offspring sex ratio by regulating sperm access to eggs (Flanders, 1956).In fact, many studies have demonstrated that many species of parasitoid wasps can control offspring sex ratio in response to environmental variables.It has been reported that the sex ratio of parasitoid was affected by the superparasitism (Kanungo, 1955;Salt, 1936;Walker, 1967;Abram et al., 2012;Montoya et al., 2010), the host size (Ueno, 1998), the photoperiod (Bouletreau, 1976;Hoelscher and Vinson, 1971), the relative humidity (Legner, 1977), the number of wasp foundresses (Somjee et al., 2011) and the body size of male and female wasps (Kant et al., 2012) and so on.
Symbiotic microorganism has notable influence on the sex ratio of wasp.It was found that the thelytoky of Muscidifurax uniraptor and Trichogramma deion was caused by the existence of rickettsia-like bacteria (Wolbachia), one endosymbiont of the wasps (Stouthamer and Luck, 1993).It is estimated that 16% of the insect species have this kind of microorganism (Werren, 1997).In arthropods, Wolbachia are generally facultative endosymbionts not involved in host survival and act as a key manipulator of host reproduction by inducing feminization, parthenogenesis, male killing, or cytoplasmic incompatibility (Duron and Gavotte, 2007).It can be vertically transmitted to progeny through eggs or be horizontally transmitted to other insects.Based on the amplification of part of wsp gene which encodes the outer membrane protein of Wolbachia, the presence of Wolbachia has been successfully detected in A. gossypii Glover.The parasitizing and bionomics behavior of Trichogramma dendrolimi have been studied in detail, but it lack the knowledge of Wolbachia affecting the sex ratio in this kind of parasitoid wasp.Through horizontal *Corresponding author.E-mail: bin1956@163.com,qianht1975@163.com.The corn borner is a major pest of corn worldwide.Infestation rates have been reported to reach 30 to 50% when no adequate control methods were applied.T. ostriniae was the dominance parasite of corn borner eggs in field all over the world.At present, T. ostriniae can be mass produced and be used for biocontrol for the first and the second generation of Ostrinia.furnacalis (Guenee) in China.So it is important for us to investigate the factors which affect the sex ratio of T. ostriniae in order to enhance the efficiency of mass production and biocontrol.In this study, the effects of several environmental and biological factors on sex ratio of T. ostriniae was examined.

The rearing of O. furnacalis
O. furnacalis, which were kept at 28 to 30°C, relative humidity (RH) 70 to 80% and a photoperiod of 16 light: 8 dark (L:D), were reared in the laboratory.Half of the eggs were used to maintain the population of O. furnacalis (come from Shenyang, Liaoning province, China) and the rest, which was sanitized in 2 to 5% formaldehyde for 15 to 20 min followed by three time distilled water washes, were used for experiment.

Investigation of factors effect on reproductive characters of T. ostriniae
Mass mating was done an hour after eclosion.Then single female (The female had no access to host egg) was reared in a glass tube (13.3 mm in length × 10 mm in diameter) with a piece of cotton soaked with 10% honey for two hour at 23°C, RH 80% , 14:10 (L:D) (There no food were provided in experiment of the species of food at this two hour).After reared the female wasp was introduced into tube for experiments immediately.The factors affected on the reproductive characters of T. ostriniae were studied as follows: Temperature, relative humidity, photoperiod, foundress number, foundress age, host number, interval time of egg supplying, species of food and species of host.After the host was parasitized 24 h, the female was removed out.The parasitized host eggs were reared at 23°C , RH 80% and 14:10 (L:D) in constant temperature incubator.When the hatched wasp died naturally, the male and female were counted under a stereomicroscope.All dead hosts were dissected immediately to determine whether or not they were parasitized.Each treatment was replicated three times.

Temperature
The mated female wasp was provided by a egg-mass of O. furnacalis (about 80 eggs) and treated at five temperature conditions: 15, 20, 25, 30 and 35°C (14:10 (L:D), RH 60 to 80%).The temperature was controlled within±0.5°C of the set value.

Relative humidity
Super saturation aqueous salt solution placing in desiccators was used to maintain the relative humidity (Rockland, 1960).The tube with the female put in the desiccators.In order to retain the same humidity with the desiccator, the nozzle of tube was covered with mesh gauze.Seven levels of relative humidity were carried out (Table 1) (14:10 (L:D), 23°C).The variation range of relative humidity (RH) was ±3%.

Species of food
The foundress was fed on water, 10% glucose, 10% honey and no food for experiment respectively.Sufficient food were provided until the end of experiment (14:10 (L:D), 23°C, RH 80%).

Cytological analysis of Wolbachia
The Macchiavello method was used to dye the cell of T. ostriniae and T. embryophagum.The Rickettsia-like organism was stained with the modified Macchiavello's stain: Using a counterstain of 0.1% methylene blue in distilled water without phenol and by staining deparaffinized histological sections instead of air-dried tissue smears.Macchiavello stain-organisms were bright red against the blue background of the tissue.

Horizontal transmission of Wolbachia Interspecific transfer between Trichogramma
Wolbachia was transferred between T. dendrolim and T. embryophagum, T. ostrinia and T. cacoeciae, T. ostrinia and T. embryophagum.

Interspecific transfer between order and family in insecta
For the suspension of Wolbachia: T. embryophagum infected by Wolbachia (check by PCR) was collected in an eppendorf tube and was placed in freezer for 5 min and then was added phosphatebuffered saline (PBS) and crushed with a pestle on ice.The suspension mixture was centrifuged for 15 min at 3100 rpm; then the supernatant was transferred to a clean eppendorf tube and centrifuged at 8300 rpm for 5 min.After discarding the supernatant, the deposition was suspended in 100 µl of PBS buffer, which was the suspension of Wolbachia.To prepare the capillary for injection, an injection needle was made by using glass capillary which was burnt and pulled on alcohol burner.For microinjection of Braconidae, A. pernyi pupa and G. mellonella larva, the Wolbachia suspension was injected with the glass-needles and microinjected in the pupa abdomen of Braconidae, in the third leg of G. mellonella old larva and in the first segment of A. pernyi pupa abdomen, and then developed to eclosion at 24°C and conserved at -20°C for test.
Test for horizontal transfer of Wolbachia by wsp gen: The presence of Wolbachia was verified using a wsp gene specific reaction by PCR.A total of 30 to 50 adult females were washed in double distilled water and then put into tube with 200 uL extraction buffer (100 mmlo/L Tris-HCl pH 7; 1.4 mol/L NaCl; 20 mmol/L EDTA; 2% hexadecyl trimethyl ammonium bromide (CTAB)).The tube was placed in -20℃ icebox for five minutes and then taken out.The sample was crashed and incubated at 65°C for 1 h.Extracting with chloroform-isoarnylic alcohol (24:1), the DNA was washed with 70% ethanol, dried in air, resuspended in 50 µl double distilled water and preserved at -20°C.The primers used to amplify the wsp gene were described by Zhou et al. (1998).81FOR: 5′-TGG TCC AAT AAG TGA TGA AGA AAC；691 REV: 5′-AAA AAT TAA ACG CTA CTC CA。 The PCR solution contained 2 µl DNA template, 2 µl 10×buffer, 2 µl, 25bmmol/ L MgCl2, 0.5 µl 10mmol/ L dNTPs, 0.5 µl 20 µmol/L of each primer, 1 Unit of Taq DNA polymerase and 12.3 µl ddH2O.PCR reactions were performed in a GeneAmp PCR System PTC-200 thermal cycler.Cycle parameters were 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min.After amplification, the product was analyzed on a 1.2% agarose gel to determine the presence and size of the amplified DNA.

Data analysis
All analysis were performed with statistical package SAS, version 8.2 (SAS Institute Inc.).The effect of factors on the sex ratio was compared using one-way analysis of variance (ANOVA) and means were separated using LSD test when the ANOVA was significant (P < 0.05).The percentage data were transformed to arcsine square root before analysis.

Temperature
Significant differences in sex ratio among the various temperatures were detected.From 15 to 30°C, the sex ratio had no significant change (about 20%).At 35°C, the sex ratio increased to 67.7% (Figure 1).
The number of female, male and the total T. ostranine also could be remarkably influenced by temperature.The number increased linearly with temperature in the range of 15≤T≤30°C, but droped at 35°C (30.33).The number was highest at 30°C (57.67).

Relative humidity
The sex ratio of did not differ significantly in all relative   humidity.At 80%, the sex ratio was highest (35.7%) (Figure 2).Humidity had a significant impact on the number of female, male and total of T. ostranine.At 75% relative humidity, the number of offspring reached the highest values (53.33).

Number of foundress
Significant differences were found among the sex ratio of T. ostriniae at the foundress numbers studied (Figure 3).There was a significant difference between one and two foundresses.The sex ratio was 17.5 and 65.2% when only one foundress laid eggs and two foundresses laid eggs in the same environment, respectively.The proportion of female was also significantly lower than one foundress when four, six or eight foundresses laid eggs in the same environment.It was meant that the foundress could adjust the sex ratio of offspring according to the quantity of the foundress in the same environment.
The increasing of parasitism rate was not linear with the increasing of the quantity of foundress.The total quantity offspring increased with the increasing of the quantity of foundress, but the average effective fecundity was about 20 when eight foundresses laid eggs in the same environment.It reduced double than only one foundress laid eggs.

Age of foundress
The sex ratio of T. ostriniae differed singnificantly in response to foundress age (Figure 4).The sex ratio of offspring increased with the increasing of foundress age, highest on 3-day-old foundress (83.6%).
The fecundity was inversely to the foundress age.The fecundity of one-day-old foundress was at most (49), three-day-old at least (29).

Photoperiod
The sex ratio of T. ostriniae did not differ singnificantly at all photoperiod (Figure 5).The male proportion of offspring was lowest (12.1%) at treatment 0L:24D and highest (34.8%) at 20L:4D, ranged between 20 and 30% at other treatments.Photoperiod had not significant impact on fecundity.The fecundity was highest at treatments 14L:16D and lowest at 24L:0D .

Quantity of host
Quantity of host had not significant impact on the sex ratio of T. ostriniae (Figure 6).The sex ratio was about   80% no matter whether 10 or 80 corn borer eggs were supplied.

Interval of host supply
The interval of offering eggs had not significant impact on the sex ratio of T. ostriniae (Figure 7).The change of sex ratio was "V" type.The sex ratio was lowest (8.4%) at the treatment of zero hour interval and highest (44.5%) at one hour interval.The fecundity could be influenced significantly by the interval of offering eggs.The fecundity  reduced with the increasing of the interval.

Species of food
Food had no significant impact on the sex ratio of T. ostriniae (Figure 8).The sex ratio had no significant difference between feeding with 10% honey, 10% glucose, water or no food.The fecundity reached the highest values when the water of honey was fed.

Species of host
The species of host had no significant impact on T. ostriniae (Figure 9).The sex ratio and the fecundity showed no significant change whether the eggs of corn borer or the eggs of rice moth were supplied.

Cytological analysis of Wolbachia
Dyeing with the method of Macchiavello (Agricultural entomology research group, Department of plant protection, 1975), microorganisms (Wolbachia) could be found in the cell of T. embryophagum (Figure 10) and  nothing in the cell of T. ostriniae (Figure 11).

Horizontal transmission
Amplification of specific genes by the polymerase chain reaction has been the usual route taken to circumvent the problems that arise in identifying bacterial parasites of insects.By using the general primers (81F, 691R), a 500 to 750 bp fragment of the wsp gene was amplified from genomic DNA of Wolbachia harbored in T. embryophagum (Figure 12).Transmission between Trichogramma spp.
DNA fragments of the wsp gene were amplified from the treated T. dendrolimi (lane 5) but not in the treated T. ostriniae (lane 4) (Figure 13).This demonstrated that T. dendrolimi can be infected by Wolbachia (it had proven to become a stable thelytokous lines after multi-generation feeding), but T. ostriniae could not be infected by Wolbachia through the way of sharing food.DNA fragments of the wsp gene were amplified from the treated Braconidae spp.(lane 4 and lane 5), but not in untreated Braconidae spp.(lane 3) (Figure 14).The result show that Wolbachia could transmit between families in Hymenoptera.

Transmission between orders (from Hymenoptera to Lepidoptera)
DNA fragments of the wsp gene were not amplified from the treated Galleria mellonella (lane 4) and tussah ovarian tissue (lane 5).This demonstrated that Wolbachia could not transmit from Hymenoptera to Lepidoptera (Figure 15).

DISSCUSION
Temperature is one of the most important abiotic factors affecting the sex ratio of Trichogramma spp.According to Al-Ahmed and Kheir (2003), temperature is considered an important factor affecting the duration of the preoviposition period.Results that is similar to ours.We found that the sex ratio of T. ostriniae could be affected significantly by temperature.The proportion of male increased when the temperature was over high (>30°C) or over low (<20°C).As the temperature increased, the activity of female strengthened and the duration of development decreased.So the oviposition or parasitization was affected.Crozier (1977) suggested that lower temperatures could promote fusion of nuclei and increase the proportion of diploid offspring, which could explain the high sex ratio at 15°C in all strains studied.However, we found the sex ratio of T. ostriniae could not be affected when the temperature was over low.But the oviposition amount decreased, resulting in decreasing of reproduce efficiency.Between 15 and 30°C, the sex ratio had no significant change.But the fecundity increased significantly as temperature increased.The optimal temperature for development and survival was 25°C.
The development and reproductive of many species of parasitoids could be affected significantly by relative humidity.According to Legner (1977), the sex ratio of many species of Braconidae and Pteromalidae could be affected by relative humidity.Kajita (1979) noticed that the parasitic quantity of Encarsia formosa Gahan could not be significantly affect by relative humidity of 43 to 90%, but affected significantly in 30%.The relative humidity of 74% was favorable to the reproductive of E. formosa Gahan.The results that are similar to ours, we found that the progeny sex ratio of T. ostriniae could be affected by relative humidity, especially in low relative humidity (RH<55%).So the low relative humidity should be avoided for reproduction of T. osbriniae.The high relative humidity had no significant effect on the sex ratio, but decreased the quantity of oviposition.In addition, the host would be moldy and degenerative in high relative humidity, and the progeny could not enclose successfully.In conclusion, the female proportion and the oviposition of T. ostriniae were max at 75% RH.
The sex ratio of T. ostriniae could be affected by photoperiod.But the degree of effect by photoperiod was weak than by temperature or relative humidity.The proportion of male was higher on the 8 and 20 h illumination time than others.The reason that the sex ratio could not be affected by photoperiod significantly maybe was the short longevity of the T. ostriniae adult, so the modulation mechanism of sex ratio in response to the photoperiod did not form.
The local mate competition (LMC) theory (Hamilton, 1967) predicts that the foundresses should adjust their sex ratios in response to the variations in foundress numbers.The sex ratio is predicted to increase toward 50% with increasing numbers of foundresses.The theoretical predictions empirically supported by gregarious parasitoid wasps and fig wasps with a variable number of foundresses in local oviposition patches (Guo, 1992;Herre et al., 1997;King, 1996;Wang, 1990;Werren, 1983).We found that the female of T. ostriniae could adjust the sex ratio of the offspring themselves based on the foundress quantity of ovipositing in the same environment.The proportion of the male progeny increased and the parasitic efficiency of every foundress declined, as the increasing of the ovipositing foundress in the same environment.
The physical condition of foundress was directly related to their age.The female progeny proportion of young foundress was high; conversely, the proportion was low.For example, the female progeny proportion of Pseudanastatus albitarsis was more than 80% within 18 days after eclosion, below 70% after 20 days, and all drones after 27 days (Wang, 1990).It was found that the sex ratio of T. ostriniae was related to the age of foundress.The proportion of male increased with the increasing of foundress age.Because the longevity of T. ostriniae was short, the physical condition of foundress decreased rapidly with the increasing of foundress age.On the other hand, the sperm in the female were exhausted, so the ovum could not be fertilized.Thus, the male offspring proportion of three days foundress was very high.
The progeny sex ratios of gregarious parasitoids were affected by various environmental factors (Flanders, 1965).A most prevalent factor was the host/parasitoid ratio.The decrease of this ratio results in the increase of the progeny sex ratio (Kanungo, 1955;Salt, 1936;Walker, 1967).It was found that the progeny sex ratios of T. ostriniae could not be affected by host quantity, but the fecundity could be affected by it.The fecundity of every foundress increased with the increase of host.
It was that if the T. ostriniae was supplied for sufficient host at twice and there were some interval time between the two supplies.The sex ratio of T. ostriniae would be affected by the length of interval.In conclusion, with the prolonging of interval, the proportion of male increased.The sex ratio reached the highest values at 1h interval, then the male ratio trended to decrease.
The offspring sex ratio could be directly affected by food sort supplied to foundress.When Telenomus enproctidis was fed on 10% hydromel or water, the proportion of female offspring accounted for 69.1 and 56.9% respectively (Wang, 1991).For Bracon hebetor, the proportion of female offspring was increased to a great extent if foundress has been fed on host larvae's body fluids (Wang et al., 1992).We found that although the offspring sex ratio of T. ostriniae could not be affected significantly by food sort, the proportion of male was the lowest when glucose water was supplied.This reduction could be due to the fact that glucose could be quickly and effectively used.
Trichogramma had the ability to identify host.T. dendrolimi could change the sex ratio of offspring in one host according to the host species or the size of eggs (Guo and Zhang, 1993).Qiu et al. (1999) found that the host reception behavior of T. dendrolimi was flexibility and had adaptation process to new host.For example, when the T. dendrolimi from the tussah eggs was supplied by corn borer eggs as host, the contemporary foundress was not suited to the next host, so the parasite rate and fecundity reduced.The parasite rate and fecundity increased after continuous supplying corn borer eggs.However, we found that there were no differences in sex ratio and fecundity no matter corn borer eggs or rice moth eggs were supplied to T. ostriniae as host.This meant T. ostriniae had a strong adaptability to different host eggs.Huigens and Luck (2000) noticed that Wolbachia could transmit from strains (infected) to others of the same species (not infected).In our study, Wolbachia transmitted from T. embryophagum (from Germany) to T. dendrolimi (from China) successfully, indicating that Wolbachia could transmit between species in Trichogramma.The possible transmission ways of Wolbachia between two Trichogramma: (a) The larvae of T. Dendrolimi were infected after eating dead larvae of T. embryophagum; (b) This two species of larvae fought in the host egg, then Wolbachia spread through blood contact; (c) Wolbachia was transmitted into host with the ovipositing of foundress, then the larvae of Trichogramma were infected by oral or wounds.It was found that the arrhenotoky T. Dendrolimi was successfully changed to the thelytoky T. Dendrolimi through the food sharing, but failed for T. ostriniae using the same method.This result indicating that many factors, such as the contents of Wolbachia in the host, the adaptation of Wolbachia to the host and the immune ability of host to Wolbachia, could affect the ability of Wolbachia horizontal transmission between species of Trichogramma.Boyle et al. (1993) suggested that cytoplasmic incompatibility expression was affected by the density of Wolbachia.Our PCR results showed that Braconidae was successfully infected by Wolbachia through microinjection, but could not be change to arrhenotoky.It was the reason that the density of Wolbachia in Braconidae was too low to change the reproduction mode or Wolbachia was not adapting to the new host.The injection of G. mellonella and tussah defeated, due to there were many differences between orders, such as nutrition, immune system and so on, so Wolbachia could not adapt to new host.In short, the Wolbachia can transmite between species of Trichogramma and affect the sex ratio of hosts significantly, but the success ratio of transfection was decided by the similarity degree of two hosts.The higher the degree of similarity, the higher the success ratio of transfection and the change chance of the reproduction mode after transfection, and vice versa.
In conclusion, the sex ratio of T. ostriniae could be affected by many factors (such as temperature, relative humidity, photoperiod, biological factors).The foundress of T. ostriniae could adjust offspring sex ratio responding to these factors through a complex mechanism.Which verifies the sex ratio theory and implies that the potential effectiveness of T. ostriniae as a biological control agent can be further improved.In addition.Only the independent effect of various factors to sex ratio was studied; the combined effects need to be confirmed further.

Figure 1 .
Figure 1.Effect of temperature upon the sex ratio of Trichogramma ostriniae.

Figure 2 .
Figure 2. Effect of relative humidity upon the sex ratio of T. ostriniae.

Figure 3 .
Figure 3.Effect of maternal wasp numbers upon the sex ratio of T. ostriniae.

Figure 4 .
Figure 4. Effect of female wasp age upon the sex ratio of T. ostriniae.

Figure 5 .
Figure 5.Effect of photoperiod upon the sex ratio of T. ostriniae.

Figure 6 .
Figure 6.Effect of host numbers upon the sex ratio of T. ostriniae.

Figure 7 .
Figure 7. Effect of the interval of offering eggs upon the sex ratio of T. ostriniae.

Figure 8 .
Figure 8.Effect of varieties of food upon the sex ratio of T. ostriniae.

Figure 9 .
Figure 9.Effect of the alteration of host upon the sex ratio of T. ostriniae.

Figure 10 .
Figure 10.The dyeing result of T. embryophagum celling result of T. embryophagum cell.

Table 1 .
The relationship of saturated salt aqueous solution with the relative humidity.