The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is a major rice pest in East and Southeast Asia regions. The pheromone trapping method has been used worldwide for monitoring and controlling C. suppressalis populations in the field (Chen et al. 2014; Witzgall et al. 2010; Vacas et al. 2016), but the success of this method relies on removing unmated males from the population in order to reduce female mating opportunities. However, assessment of the effectiveness of this management method does not rely on counting the number of unmated males trapped in pheromone traps but rather on indirect methods such as estimating numbers of fertile eggs oviposited by females, determining the damage level of the next generation, or simply counting the number of trapped males. A technique to determine the mating status of male C. suppressalis is needed to estimate the number and proportion of unmated males captured.

Techniques for determining the mating status of male moths of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Snow and Carlyse 1967), the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval) (Haines 1981), the tobacco budworm, Heliothis virescens (F.) (Henneberry and Clayton 1984), the spruce budworm, Choristoneura fumiferana (Clemens) (Bergh and Seabrook 1986), the oblique-banded leafroller, Choristoneura rosaceana (Harris) (Evenden et al. 2003), and the European corn borer, Ostrinia nubilalis (Hübner) (Hu and Andow 2009) have been developed. These techniques capitalize on the dynamics of the secretion inside the male primary simplex before and after copulation using the secretion as a reliable indicator of mating status of male moths.

The objective of this study was to develop a technique for determining the mating status of male C. suppressalis using the aforementioned studies. We used the method of Hu and Andow (2009) because the internal reproductive system of male C. suppressalis (Song et al. 2012) is similar to that of O. nubilalis (Hu and Andow 2009), and the last segment of the primary simplex of both species contains a similar white secretion. Consequently, the secretions inside the primary simplex before and after copulation were compared for use as an indicator to determine the mating status of C. suppressalis as per Hu and Andow (2009).

Materials and Methods

The C. suppressalis specimens used in this study were obtained from a colony that was initially established by collecting >100 overwintering larvae from rice fields on the China National Rice Research Institute (CNRRI) Farm (Fuyang, Zhejiang Province, China, N30°04′50″, E119°55′08″). Larvae were fed an artificial diet described by Hu et al. (2013) at 27°C under constant light. Pupae and adults were held in a mating and oviposition room under a daily regime of 27:21°C, 16:8 (L:D) and 60–90% relative humidity. Adults from the third generation of the colony were used for study. Moths targeted for dissection were killed by submergence in a 60% ethanol for <1 min. Dissections were conducted in 0.9% saline solution. Mated males were derived by combining unmated males with unmated females (12–24 h after emergence) in a mating cage (55 × 25 × 27 cm) at 7:00 p.m. For the next 7 h, the mating cage was checked every half hour, and any copulating pairs were carefully collected in a 50-ml vial and removed from the mating cage. Any copulating pair that separated within 30 min after collecting was discarded to insure the transfer of secretion during the mating process. Previous observations showed that copulation ranged from 30–90 min (Y.H. pers. obs.). The success of copulation was confirmed by presence of a rounded spermatophore inside the female bursa copulatrix. Twenty mated males were killed and dissected at 0–2, 12–14, 34–36, and 46–48 h postcopulation to characterize the dynamics of the gradually regenerated and refilled secretion of mated males from newly mated (0–2 h) up to 2 ds (46–48 h) after copulation. Unmated males used for comparisons were obtained by determining the gender during the pupal stage and keeping those separate from females as pupae and adults. Twenty unmated males were killed at 0–2, 46–48, and 94–96 h after emergence to characterize the dynamics of the secretion within males over time.

In order to determine the mating status of males captured in pheromone traps, we placed four traps (Newcon Co. Ltd, Ningbo, China; water-type trap and rubber-septa lure) at a distance of 40 m from each other in each of two sites located 800 m apart on the CNRRI Farm. Traps were placed adjacent to rice fields 5 d after rice was transplanted. The first collection of captured males began 1 d after trap placement and continued daily up to 88 d after placement. Collections were made at 8:00 a.m. each day; septa lures were replaced weekly.

Numbers of captured males were pooled over 4-d periods for each trap to stabilize the variation of collected numbers and the unmated proportions because no males were captured on some days. The captured males between two sites were compared with an independent samples t-test with unequal variance using Data Processing System software (Tang and Zhang 2013).

Results

Mating status of C. suppressalis males could be determined by characterizing the form of the white secretion inside the 8th segment of the primary simplex. In unmated males, the white substance fully filled and was evenly distributed in the segment. Furthermore, the secretion remained unchanged in each of the intervals observed (Table 1).

Table 1 Characteristics of the white secretion in the 8th segment of simplex of different ages of unmated and mated Chilo suppressalis males.

View Fullscreen

In mated males, the white secretion inside the segment was not observed immediately after copulation (0–2 h postcopulation). Then, the substance reappeared in the segment and gradually refilled the segment as time elapsed after copulation (Fig. 1; Table 1). However, the form and appearance of the secretion differed and was no longer evenly distributed as observed in unmated males (Table 1).

View Figure

• Download as Powerpoint
• Download Figure

Each pheromone trap captured >150 males, on average, at both sites throughout the observation period (Table 2). Of the total males captured at two sites, only a mean of 22.75 and 24.00 unmated males were captured per trap at Site 1 and Site 2, respectively, with a great proportion (∼85%) of captured males having already mated.

Table 2 Chilo suppressalis males captured by pheromone traps.

View Fullscreen

Based on the pheromone trap captures, we observed two male flights at these sites (Fig. 2). The first flight was seen from 1–4 to 21–24 d after trap placement. The second flight was roughly from 33–36 to 77–80 d after trap placement.

View Figure

• Download as Powerpoint
• Download Figure

Discussion

Our results demonstrate that the technique for determining the mating status of O. nubilalis described by Hu and Andow (2009) can be adapted for C. suppressalis. We found that the white substance within the last segment of primary simplex in C. suppressalis did not vary with ages of unmated males. Absence of the substance in the segment indicated that males had recently mated. As time elapsed after mating, the substance would reappear in the segment, but it did not appear the same and did not fill the entire segment as was seen in unmated males. Thus, we concluded that the character of this secretion is a reliable feature for determining the mating status of C. suppressalis males.

The success of the pheromone trap method to manage C. suppressalis population depends on limiting female mating opportunities by removing unmated males from the population. Currently, assessments of the effectiveness of pheromone traps for this management tactic does not rely on captures of unmated males but rather on counting numbers of trapped males, estimating the fertile eggs laid by females, and/or the damage caused by the next generation (Alfaro et al. 2009; Chen et al. 2014; Kondo and Tanaka 1991; Vacas et al. 2016). The technique developed here could provide an alternative assessment by determining the mating status of trapped males.

In our field collection of males in pheromone-baited traps, we found that most of the trapped males had already mated, thus suggesting that pheromone traps contribute little in reducing female mating opportunities. Furthermore, C. suppressalis males can mate multiple times (Jiao et al. 2006). Pheromone trap density and pheromone chemistry might impact these results and should be further investigated (Alfaro et al. 2009; Chen et al. 2014).

Like other lepidopterans, C. suppressalis males are able to mate multiple times while females normally mate only once. Given C. suppressalis adults have a 1:1 sex ratio and males are able to mate 2.7 times and females mate only once (Jiao et al. 2006), a 50% reduction of female mating can be theoretically achieved if 81.5% of unmated males are removed from the population. Furthermore, to reduce plant damage by 50%, even more unmated males must be removed from its population because mating activity of C. suppressalis males remains at a relatively high level for up to 8 d (Kanno and Sato 1978). Meanwhile, a female had similar fecundity when mated with either an unmated male or a mated male (Jiao et al. 2006).