H. indica SL0708 hermaphrodite quantification and egg development characterization
The largest hermaphrodite population was obtained at 105 h (31 hermaphrodites) and 115 h (24 hermaphrodites) (Fig. 1) with significant differences post-infection (P = 0.001, df = 5, F = 16.969). Egg stage within hermaphrodite uterus isolated at 105 and 115 h corresponded to stages 6 and 8, respectively (Figs. 2 and 3f, h).
Regarding egg maturation within the hermaphrodite uterus, significant differences were observed at the six different post-infection times evaluated, with eggs at stage 9 in the vicinity of the vagina at 95 h post-infection (Figs. 2 and 3i). For the six time points, evaluated eggs were between developmental stages 5 and 9 (Fig. 2).
The nine stages of egg cleavage were observed within H. indica SL0708 uterus. For the first stage, maternal pronucleus migrated to the posterior pole, forming a pseudoclivage furrow (Fig. 3a). Subsequently, the maternal pronucleus met the paternal pronucleus and jointly migrated to the anterior pole (3B), where pronuclei fusion occurred (Fig. 3c). Forty minutes post-fertilization, first division took place forming two cells: AB and P1 (Fig. 3d). Later AB and P1 initiated their division to form 4 cells: ABa, ABp, EMS, and P2, respectively (Fig. 3e–f). Subsequent divisions occurred in the proximity of the vulva, where gastrulation took place (Fig. 3g), producing approximately 30 cells. These cells elongated and invaginated, resulting in J1 development (Fig. 3h). At 95 h post-fertilization, H. indica SL0708 J1 was observed moving within the egg (Fig. 3i).
For the six post-infection time points evaluated, in addition to observing hermaphrodites with their eggs at different stages developmental stages (1–9) (Fig. 3), individuals in Endotokia matricida were found. The greatest number of hermaphrodites was isolated at (105 and 115 h) post-infection.
The presence of hermaphrodites in Endotokia matricida have been possibly related to hermaphrodite’s incapability of laying adult eggs, since no muscular contraction of the vulva occurred, allowing for opening and egg release. Additionally, many eggs were laid at the same time, approximately 22 to 24 h post-fertilization. However, there was a time-lapse, where no egg laying occurred. Therefore, some eggs could have remained within the hermaphrodite and hatched within the uterus (Johnigk and Ehlers 1999a, b).
On the other hand, H. indica SL0708 eggs must be extracted at stage 9, given if they are extracted at earlier times the egg does not fully develop and no J1 hatches. After the eggs are fertilized, they locate themselves in the center of the uterus, where they undergo development of their first six stages. Later, they move close to the vulva, where gastrulation, morphogenesis, and J1 take place within the egg. This is a biologically relevant condition when entomopathogenic nematodes are produced in vitro. Furthermore, in some cases, not all fertilized eggs hatch, given the force vulva contraction exerts, when eggs are laid, which can result in egg rupture and J1 death (Lunau et al. 1993; Bucher and Seydoux 1994 and Johnigk and Ehlers 1999a, b).
The huge amount of hermaphrodites found at 105 and 115 h could be related to IJ invasion percentage, which was greatest at these hours (61.9 and 48.3%), respectively. Henceforth, not all IJs can enter G. mellonella larvae, and those that can achieve it at different times, even if they are exposed to the host at the same time and under the same conditions. These differences are due to signals released from the host, such as feces and CO2 that must be detected by H. indica SL0708 IJ. The IJ can then move until it localizes the host.
H. indica SL0708 hermaphrodite at 95 h post-infection had in its uterus 835 eggs (641–1116). Only 9.7% of them were in stage 9, i.e., 81 eggs (58–108) (Fig. 3i). Therefore, 81.3% were at earlier stages, before J1 was completely formed (Fig. 3). Variability in egg number and developmental stage was likely associated with uterus size and hermaphrodite physiological maturation (Han and Ehlers 1998).
In the uterus of a single H. indica SL0708 hermaphrodite, it is possible to find more than one egg developmental stage related to the process of fertilization. For fertilization to take place, first sperm has to be generated, which is stored in the receptaculum seminis. Germ cells that do not differentiate into sperm start oogenesis during the second meiotic division. Eggs mobilize from the ovotestis to the center of the uterus, going through the receptaculum seminis, where fertilization takes place. Thus, not all eggs are fertilized at the same time. Moreover, less mature eggs cannot be fertilized, since sperm is limited (Barlow et al. 2007).
H. indica SL0708 J1 axenic viable egg isolation
After performing hermaphrodite rupture and sterilization protocols, eggs were counted. Significant differences for viable J1 s were observed among treatments with 0.1 M NaOH and 1% NaOCl (P = 0.001, df = 2 F = 9.515) and incubation times (P = 0.001, df = 1, F = 16.866) (Fig. 4). After 48 h of egg incubation, the largest number of J1 was obtained at 10 min (598 J1 s), followed by 15 min (582 J1s) of exposure. Hatching percentage ranged between 23.9 and 24.6% of total eggs obtained from 30 H. indica SL0708 hermaphrodites at 95 h post-infection.
Hermaphrodite rupture and egg sterilization were achieved since NaOH hydrolyses collagen. On the other hand, NaOCl breaks down sulfhydryl bonds in the cuticle, responsible for stabilization and resistance. In addition, it eliminates bacteria associated with the chorion of the egg, since it is an alkaline solution and disrupts bacterial cytoplasmic membrane stability. Furthermore, it inhibits enzyme activity associated with trans-membrane proteins, resulting in biosynthesis alterations and phospholipid degradation (Lee 1966; Estrella et al. 2002 and Quintero and Zapata 2017).
Regarding bacterial growth, significant differences were observed for the three exposure times evaluated (P = 0.008, df = 2; F = 5.133). However, no differences were detected for incubation time (P = 0.110, df = 1, F = 2.619). The least growth for P. luminescens subsp. akhurstii SL0708 was determined at 15 min. Therefore, the time with the most viable and axenic J1 was at 15 min treatment with 0.1 M NaOH and 1% NaOCl solution, evidenced in TSA media.