a) Life history studies in laboratory setups

A preliminary study was carried out under the outdoor shelter and in the laboratory during 2004–05 to determine the feasibility of 1) rearing L. tristis in clay vessels and plastic trays filled with field collected soil and litter, 2) maintaining dormant beetles in clay vessels and wooden boxes, 3) inducing dormancy by spraying of water, and 4) identifying the period of arousal from dormancy. These trials revealed that arousal from dormancy and the post-dormancy return of beetles coincide with the onset of annual rubber tree litter fall in early December. Hence, activities of dormant beetles in the dormitory building were monitored until their exodus to rubber plantations ahead of the onset of annual litter fall in rubber plantations in the second week of December. Approximately 300 person hours were spent in plantations and in dormitory buildings recording the behaviour of L. tristis during dormancy and return to rubber plantations.

Twenty randomly selected 1 m² quadrants of rubber litter from the plantation were sifted on alternate days with the onset of annual litter fall in December to determine the return of beetles to the field. 100 males and females were randomly sorted from the collections. Sexing was based on the presence or absence of the sternal notch on 8th sternite (Vinod et al. in press). Females were marked with white acrylic paint on elytra.

Sorted males and females were kept in four mesh-topped large clay vessels (Twenty-five males and twenty-five females in each vessel) (13 × 35 cm) half-filled with soil and freshly fallen rubber plantation litter. Clay vessels were transferred to a wooden growth chamber (1.3 × 1.6 × 0.6 m) with fine meshed side windows, front glass panel and provisions for shading. The exterior of clay vessels and litter inside were sprayed with water to simulate the wet litter conditions prevailing in rubber plantations from the settling of dewdrops in December and January.

As soon as mating pairs were observed in the litter floor, paired males and females were transferred to labeled plastic vials (5.6 × 7.2 cm) with a thin layer of charcoal and plaster of Paris mixture at the bottom. Vials were kept together in tall rectangular plastic trays (43 × 32 × 8 cm) and placed in the wooden chamber. Thirty replicate pairs were maintained. For each pair, mating, duration of pre-oviposition, frequency of oviposition and fecundity were recorded.

One hundred eggs laid during a 12-hr period were transferred to 5 petri dishes (9 × 1.5 cm) (20/dish) and monitored at 6-hr intervals. The number of eggs hatched and the duration of egg development were recorded. Pairs of neonate larvae hatched within a 6-hr period were transferred to labeled plastic vials (5.6 × 7.2 cm) with a moist fine hairbrush and provided fresh litter as food source. Two larvae per container were used, to make it possible to follow the development of successive larval instars. This procedure also avoided the inhibitory effect of crowding on larval development in tenebrionids (Tschinkel and Wilson 1971). Development of paired larvae up to adult eclosion was monitored. Groups of 10 vials (i.e. 20 individuals) were treated as a single sample. Five such groups were maintained. Thus, a total of 100 larvae in 50 vials were available for analysis. A parallel stock of 100 individuals was maintained to replace the individuals lost by mortality in different stages. Larvae were monitored at every 12-hr interval, with the development time of instars determined from the exuviae left behind. The duration of larval, pre-pupal and pupal stages was recorded. Larval and pupal survival was measured as the percentage of emerging larvae or pupal stages.

All newly eclosed (teneral) adults that emerged during a 3 day period were pooled into a single emergence class. Those that eclosed during a 31–33 day developmental period were labeled as emergence class I, 34–36 day period as emergence class II, 37–39 day period as class III and 40–45 day period as class IV. Two individuals that eclosed on 44 and 45 day were added to the emergence class IV to limit the number of emergence classes.

Beetles from each emergence class were transferred to a wide mouthed circular clay vessel (35×13 cm) arranged with a layer of top soil, and rubber litter as detailed above, and closed with fine meshed plastic net. Rainfall inside the experimental chambers was simulated by spraying water until the leaves and upper layer of soil inside the clay vessels were as wet as in plantations (about 80% humidity). Humidity inside the vessels was assessed with a thermo-hygrometer (Barigo,  www.barigo.de). Spraying was performed synchronously with the onset of summer showers and the mass migration of beetles to the dormitory buildings on April 4th in 2005. A rectangular wooden box (15 × 7 × 3 cm) was placed inside the clay vessel as potential shelter. It was half filled with hay and had a hinged door that was kept half open. Spraying continued until beetles moved into the dry wooden shelter. To prevent clustering of beetles on mesh nets, wet cotton cloth was spread over the mesh net. Clay vessels and the litter inside were kept moist for a week to ensure that aggregated beetles did not return to dry litter in the clay vessels. Subsequently, leaf litter was removed from the vessels, as plantations are devoid of leaf litter until following season. Activities of aggregated dormant beetles in each setup were observed. Mortality was recorded by counting the number of dead beetles at bimonthly intervals. Cages were refilled with thin layer of fresh litter in synchrony with the onset of litter fall in December. After dormancy, beetles were returned to the litter and were individually marked with dots of marker paint on elytra and pronotum (Johnson 2004) using a coding system enabling identification of up to 24 individuals. This enabled estimation of the post dormancy duration of each individual. For each beetle, the date of emergence from dormancy and return to fresh litter was noted. Dead beetles were removed daily and post dormancy duration was recorded. All newly emerged larvae (new generation) inside the setups were removed. After the dormancy period, the beetles were observed until 100% mortality was reached. Egg hatchability, duration and mortality of larval, pupal and adult stages were recorded. Longevity was estimated as the sum of the means of larval, pupal and adult durations.

b) Life history studies in natural setups

Twenty eggs of uniform age were collected as described above. Eggs were transferred to a Petri dish in a clay vessel (7 × 30 cm) with fine drainage holes and half-filled with soil and litter. The vessel was buried in the plantation floor with the rim about 1 cm above the soil surface to limit the entry of running rainwater, and covered with muslin cloth supported by iron wires to contain first and second larval instars. Fresh litter was spread over the muslin cloth. We assumed that this design enabled the maintenance of similar insulating conditions for the eggs and larvae placed inside the vessels. Five replicates were thus maintained in the shelter covered with coconut fronds described above.

The exterior of the trays and the litter inside was sprayed with water each morning (7–8 AM). Wetting the litter inside was necessary as the cloth covering prevented entry of dew. The number of eggs hatched was checked at 12-hr intervals. Unhatched eggs were replaced with first instar larvae from the stock culture. When 4th and 5 instar larvae appeared, the cloth topping was replaced with a mesh net. Emergence of pupae was checked at 12-hr intervals, and newly eclosed pupae were transferred to litter in a Petri dish placed in the clay vessel. Adult emergence was checked at 6-hr intervals. Duration of pupation and survival rates were determined. Dead pupae were replaced with field-collected new adults. All newly-eclosed adults emerging within a 3 day period were pooled into a single emergence class as detailed above. Five emergence classes were maintained: adults emerged during 27^th–29^th day after hatching was taken as emergence class I, 30^th–32^nd day period as emergence class II, 33^rd–35^th day period as emergence class III, 36^th–38^th day period as emergence class IV and 39^th–41^th day as emergence class V.

A small clay shelter pot (7 × 20 cm) was laid on its side to prevent water entry, and a thin layer of hay was provided as shelter in each setup. Following the entry of L. tristis into the shelter pots, the mouths of the pots were closed with mesh net and kept closer to natural aggregations of beetles in dormitory buildings. When beetles inside the shelter pots displayed signs of arousal from dormancy by nocturnal movements and attraction to light, the mesh net was removed. The shelter pots were returned to clay vessels in the field and half filled with fresh litter to simulate movement out of the buildings. Beetles that returned to litter in clay vessels were marked with paint as detailed above. Activities of the beetles after dormancy were observed. All newly emerged larvae that developed inside the setups were removed. Activities of the old generation were observed until 100% mortality was reached. Egg hatchability, duration and mortality of larval, pupal and adult stages were recorded. Longevity was estimated as the sum of means of larval, pupal and adult durations.

Figure 3.

Early developmental stages of Luprops (a) egg mass (b) 1^st instar larva (c) 2^nd instar (d) 3^rd instar (e) 4^th instar (f) 5^th instar (g) pupa and (h) teneral adult.

Both laboratory and field culture of eggs were started on the same day to ensure uniformity in both setups. Neonate larvae, intermediate larval stages and pupae that died in the course of the experiment were replaced with individuals from the parallel stock culture. During the entire experimental period, fallen green leaves collected from the plantation were made available for the various stages. Exhausted leaf bits and faecal pellets were removed by upturning the vials topped with mesh net to prevent fungal contamination. Two dry folded leaves were provided in each vial as a resting and hiding substrate. A circle of filter paper moistened with drops of distilled water was placed in the petri dishes and vials used for culturing larvae and pupae.

1. Pre-dormancy phase:

Newly eclosed yellowish brown (teneral) adults were inactive and did not feed until they attained a dark colour 3–4 hours of after eclosion. Subsequently, they fed preferentially on wilted tender leaves. In the absence of visible sexual dimorphic characters and overlapping male and female size ranges (6.7–8.0 mm and 7.0–8.5 mm in male and female respectively, n=30) makes the dimorphism present on the 8th sternal notch (Vinod et al. in press) the sole mechanism to differentiate sexes. The pre-dormancy period lasted for 28.83 ± 3.33 days in laboratory setups (n = 99) and 31.46 ± 3.35 (n = 100) days in field setups with significant variation (t = -5.50, df = 195, P<0.01) (Table 1). Mortality during the phase was low in both setups (no mortality in field setups and 1% mortality in laboratory setups). Based on duration of the pre-dormancy phase, adults in field setups were categorized under 5 emergence classes and under 4 emergence classes in laboratory setups (Table 2). 64% of the adults in the laboratory and 60% of adults in field setups belonged to emergence class II and III. Intrapopulation variation in prediapause period arises from the variation in larval duration and leads to differences in the active feeding time available prior to dormancy. Emergence class I in laboratory setups had an average of 9 days more feeding time in comparison to class IV, and class I in the field setup had an average of 11 days more feeding time than class V (Table 2). Abdominal cavities of L. tristis in emergence class I and II were more fully loaded with fat body reserves during the last phase of dormancy than in class IV and V.

2. Dormancy phase:

L. tristis in field setups entered the shelters (small wooden boxes and pots) in response to the first short duration summer rain itself (April 4, 2005) and those in laboratory setups in response to simulated wet conditions. Within the shelters, the beetles were nocturnally surface active for 8–11 days in field and laboratory placed setups, with a strong attraction towards light and fresh leaf materials, as observed by their aggregation on nets and attempts to crawl out through the mesh sieves in laboratory setups. Subsequently they clustered and entered into a prolonged inactive state for 9 months (Table 1). The first indication that dormancy is coming to an end is nocturnal dispersal within the shelters, which is initiated by a few individuals and is followed by the rest within 2–3 days. Nocturnal dispersal lasted for 15–17 days (December 29^th 2005- January 15^th 2006). Diurnally, they remained inactive in the corners of the shelter in small groups. No beetles from the shelters returned to the litter at this stage.

This prolonged period of inactivity and aggregation is categorized as the active state of dormancy. The periods of short duration nocturnal dispersal and diurnal passivity prior to and after dormancy is characterized as the refractory and termination periods respectively (Mansingh 1971; Danks 1987). During the refractory period, beetles were attracted to food materials while they were not during the active dormancy phase. Dormancy lasted for 277.51 ± 4.05 days in laboratory setups and 278.69 ± 3.34 days in field setups with significant variation between the setups (t = 2.02, df = 149, P = 0.04). The refractory period lasted for 8 to 10 days in the laboratory and 8 to 11 days in the field and the termination phase lasted for 6 to 15 days in the laboratory and 6 to 17 days in the field. Mortality during dormancy was 15% in the field and 21.2% in laboratory setups with no significant variation (χ² = 1.71, df = 1 , P = 0.19). Emergence class variation in mortality was significant (χ² = 22.57 and 21.72, P< 0.01 in the laboratory and field respectively) with low mortality in long duration emergence class (classes I–III; 8.3% and 0 respectively in laboratory and field setups) and higher mortality in short duration emergence classes (class IV in both setups and V in field setups; 66.7% and 54.5% respectively in laboratory and field setups) in both the experiment setups (Table 2). Those that attained adulthood early had a longer feeding duration and, hence, may have had a higher fitness than adults that emerged later. Generally, mortality during dormancy is very low in natural populations (Wolda and Denlinger 1984; Danks 1987). High mortality in the short emergence classes in experimental setups and the high number of dead beetles on the floor of wooden ceilings in residential buildings indicates high mortality during dormancy in L. tristis. At no stage during the analysis did we observe any insectivores such as wall lizards (Hemidactylus sp.), spiders or ants especially the aggressive Oecophylla smaragdina (Fabricius), feeding either on adults nor larvae. Hence, we attribute the high mortality in natural setups to the presence of higher number of beetles in shorter emergence class (shorter pre-dormancy period) with low feeding duration and not to predation.

The long duration dormancy shown by L. tristis involving adults of various emergence classes does not fall under either quiescence or diapause. The term quiescence is used in situations with a shorter period and no induction phase and with active feeding throughout the stage (Mansingh 1971; Danks 1987). L. tristis does not diapause either, as diapause is preceded by a long preparatory phase during which energy reserves are accumulated at a specific stage in life cycle. Entry of beetles with both short and long durations of pre-dormancy into active dormancy, irrespective of the active feeding time available indicates that preparations for diapause by accumulation of energy reserves prior to active dormancy is not mandatory in L. tristis. In classical dormancy, the preparatory phase is succeeded by a typically long refractive phase and the insects cannot terminate diapause and continue morphological development even if exposed to optimal environmental conditions during this stage (Mansingh 1971; Tauber and Tauber 1976; Denlinger 1986, 2002; Danks 1987). However, in the present case, beetles were readily attracted to food resources and water during the short refractory phase. Adults of all emergence classes irrespective of the active feeding duration available entered into dormancy without a preparatory phase nor an induction period in response to an unexpected environmental token stimulus, the short mid summer rainfall that mediated wetness in the rubber plantation litter floor. During the short refractory period, beetles were readily attracted to tender leaves and dry litter conditions. These observations weighed in favor of our categorization of dormancy in L. tristis as oligopause and not as diapause, which is intermediate between quiescence and diapause (Mansingh 1971; Danks 1987; Leather et al. 1995).

Recognizing the environmental stimuli leading to and arousal from dormancy is a difficult task for many tropical insects (Denlinger 1986). Moisture, photoperiod, temperature, food availability and food quality (Mansingh 1971; Tauber and Tauber 1976; Masaki 1980; Hunter and McNeil 1997; Tanaka 2000; Danks 2003; Sato 2003; Nahrung and Allen 2004; Wang et al. 2006; Nakamura and Numata 2006) are the recognized dormancy induction cues. Sudden annual mass entry of huge swarms of L. tristis into residential buildings following the first summer rains is a widely recognized and familiar event in the rubber plantations belts of the region. The absence of beetles in the litter floor of plantations following the first summer rains and the entry of beetles into dry litter conditions in the experiment setups, together with the general affinity of tenebrionids toward dry conditions (Watt 1992; Endrody-Younga and Tschinkel 1993) indicates that wetness in litter habitat leads to the departure of beetles and migration towards shelters. Additionally, no beetles in stock cultures maintained in laboratory setups and away from rainfall-mediated wetness enter into dormancy (unpublished data) even after the onset of monsoon rains later in the month of June. Our current data strongly suggest that rainfall mediated wetness from summer showers is the oligopause inducing cues for L. tristis. However further empirical studies considering the above stated environmental factors recognised as the cues for diapause in insects are necessary.

Table 3.

Emergence class wise mortality of L. tristis during post dormancy phase in laboratory and natural set ups.

Synchrony of diapause arousal and subsequent field return of beetles with the beginning of annual leaf fall in the rubber plantations in the region points towards the following probabilities. Deciduous trees shed leaves well in advance of the onset of the dry season as a mechanism to prevent water loss (Santiago et al. 2004, Santiago and Mulkey 2005, Sundarapandian et al. 2005). Subsequent dry environmental conditions in the region during this phase, along with substantial leaf fall provide a suitable habitat and food source for the beetles. We suggest that the return of dry conditions following the prolonged monsoon season, and the related variation in moisture, photoperiod and temperature in the region are the environmental cues leading to the arousal of L. tristis from oligopause. The seasonal coincidence of a thick layer of freshly fallen litter in rubber plantations both as a food resource and as a habitat facilitates the return of post-dormant beetles to the field.

3.Post-dormancy phase:

Following the termination phase, adults returned to the litter in laboratory setups in batches during the first and second weeks of January (December 29^th —January 15^th), which lasted for 6 to 17 days. The size of aggregations in residential buildings decreased from the week of December 4^th onwards and no beetles were present in the buildings after the 3^rd week of January (January 20). Beetles were less common on the litter floor during the first week of December and their number steadily increased in the following weeks and peaked in the 4^th week of January (200 ± 25.6/m²; n = 30) (unpublished data). Post-dormancy beetles that returned to the litter habitat were attracted to water drops on leaves (dew drops in plantations) and fed on the outer edges of dry leaves until tender leaves were available that emerged synchronous with the premature fall of tender leaves in plantations. Following an 18–25 day period of intensive feeding in laboratory setups, mating pairs appeared with the male climbing on the back of the female and pressing the abdomen down on the abdomen of the female to engage his genitalia with hers. Neither mating pairs nor egg production by the newly emerged adults were recorded either in the laboratory setups or from natural conditions prior to or during dormancy. Total absence of reproductive activities during pre-diapause and diapause periods, and the initiation of mating activities after a brief period of intense feeding following post diapause arousal, suggest the following probabilities. Either the reproductive organs in L. tristis do not mature until the field return or the beetles are undergoing reproductive diapause (Danks 1987; Mansingh 1971), similar to what is reported during the dormancy in S. rotundus (Wolda and Denlinger 1984).

Post-dormancy duration was 34.31 ± 4.72 days (n = 85) in laboratory and 32 ± 4.71 days (n = 78) in field setups with significant variation in the post-dormancy period (t = -3.12, df = 159, P = 0.002) (Table 1). No mortality was recorded in the first two weeks of post-dormancy phase in either experiment setup, and up to the third week in field setups. Subsequently, significantly high mortality was recorded during the 5 week in laboratory setups (χ² = 4.64, df = 1, P = 0.03) and 100% mortality in both setups (Table 3). The adult stage duration was 338.34 days with a pre-dormancy period of 28.83 ± 3.33 days, a post-dormancy period of 32.00 ± 4.71 days and an intermediate dormancy period of 277.51±4.05days in laboratory setups. In field setups, the adult stage duration was 344.46 days with a pre-dormancy period of 31.46 ± 3.35 days, a post-dormancy period of 34.31 ± 4.72 days and an intermediate dormancy period of 278.69 ± 3.34 days. Longevity of L. tristis was 374.34 days in laboratory setups and 378.2 days in the field setups.

Figure 7.

Aggregated dormant beetles settled on the wall and gaps of a residential building.

We attribute the comparatively higher mortality and shorter post dormancy duration in laboratory setups to the more favourable conditions available in natural setups during the dry summer conditions and to the shorter larval phase and the additional 2.63 days of active feeding time available to the adults in field setups during the prediapause phase. Future studies involving laboratory culture of Luprops should make note of the effect of the duration of larval and pre-dormancy phase on mortality, longevity and fitness in subsequent phases.