Facultative diapause behavior of pink bollworm, PectinophoragossypiellaSaunders (Lepidoptera: Gelechiidae) in South Indian populations

doi:10.21203/rs.3.rs-5022371/v1

Background

The pink bollworm (PBW) of cotton, Pectinophora gossypiella Saunders is one of the serious pests in the Bt cotton era of India. Development of resistance to insecticides and behavioral adaptations are the main factors attributed for its severity. The North Indian populations of PBW are reported to be undergoing facultative diapause, whereas, no clear reports are available on the diapause pattern of South Indian populations. Hence, the diapause behavior of south Indian populations was studied at the UAS, Raichur, Karnataka.

Results

Populations collected from different parts of Karnataka, South India exhibited diapause behavior though the intensity varied. A significant variation in the morphometry of larval, pupal and adult populations of diapause and non-diapausing group was observed. Similar variation was also noticed in the biochemical parameters. Higher levels of amino acids, fats, proteins, and nitrogen were recorded in diapausing population compared to non-diapause population. Enzyme activities such as LDH, AP, GPT, and GOT varied between diapause and non-diapause population. The diapause behavior in South Indian PBW population was initiated from February, peaked in March, and ceased by May. Most larvae terminated diapause in November, coinciding with peak boll formation in cotton.

Conclusion

South Indian population of cotton pink bollworm exhibit facultative diapasue behavior. A clear cut morphological and physiological differentiation was recorded between diapusing and non-diapasuing individuals of the species. An in-depth study on the factors influencing the initiation, regulation and termination of diapasue in PBW need to be carried out.

Diapause

Behavior

Pink bollworm

Bt cotton

Bio-chemical

In the present Bt cotton cultivation scenario in India, Pink bollworm, Pectinophora gossypiella (PBW) has emerged as one of the prominent pests inflicting huge economic loss on cotton production. The mean infestation ranges from 13–36% in major cotton growing states of Central India, 7–31% in Southern states and 25% in North India (Nagrare et al., 2023). Besides causing direct yield loss, quality of seed cotton also gets affected to the tune of 40%. The reason for its severe damage lies with its nature of feeding. The early instar larvae penetrate the ovaries of flowers or young bolls and later instars stay within the bolls and feed on developing seeds. Maximum pupation occurs within the bolls. On some occasions, larva leaves a circular hole upon its exit from the boll, which is the characteristic of PBW damage (Sarwar et al., 2017). Symptoms like double seed formation, premature opening of the bolls and rotting are observed in infested bolls (Kranthi, 2015). Additionally, the feeding damage contributes to the entry of fungal pathogens and secondary pests inside the boll.

A key factor in this pest’s persistence and rapid spread is its ability to enter a state of diapause in periods that are not conducive to continued development. During unfavorable environmental conditions the late instar larvae diapause and undergo pupation once the conditions get congenial. The diapause in pink bollworm lasts for about 56 to 120 days in the cold winter and may remain quiescent up to 2.5 years (Metcalf and Metcalf, 1993). At this stage, neither feeding nor movement takes place. Larvae make loose silken webs or cocoons generally referred as ‘hibernaculum’ in which they spend the whole winter and spring (Pradhan et al., 2022a). It is well known that diapause responses in insects are largely genetically deterministic, yet environmental factors play a significant role (Emerson et al., 2009). Diapause is often preceded by biochemical adjustments, including the accumulation of lipids, carbohydrate reserves and behavioral adaptations such as seeking the location of a protective site (Rostom, 1960). Survival of diapausing insects is due to decreased metabolism and the reserves of lipids, carbohydrates, proteins, and amino acids (Denlinger, 1985).

The diapause behavior in PBW is tricky to understand. As per the reports it is known to undergo either obligatory diapause (Sreenivas et al., 2021), or facultative diapause (Adkisson et al., 1963; Sri and Maheswari, 2020) whereas some populations are non-diapause strain (Ashok and Robert, 1974). Populations of PBW are known to overwinter in North and Central India (Singh and Singh, 1991; Rao, 2022). However, reports on diapause behavior of South Indian populations are contradictory. Earlier reports recorded non-diapausing behavior in PBW populations of South India (Ashok and Robert, 1974; Raina et al., 1981). Later, summer diapause in South Indian populations of PBW and its emergence pattern was reported in 2000 (Suresh and Patil, 2004). In one of the recent studies (Pradhan et al., 2022b), the amino acid profiling of diapause and non-diapause larva was carried out, but failed to mention the source of diapause culture. In other similar study conducted by them (Pradhan et al., 2022a), the biology of the diapausing and non-diapausing PBW was studied without clear indication on the source of the culture. This triggered the initiation of a detailed study with an objective of generating concrete evidence on the existence of diapausing nature in the South Indian populations of PBW and its emergence pattern. Further, the present work gives special significance in understanding the physiological strategy adopted by

P. gossypiella to overcome harsh climatic conditions. We tried to spot on the enzymatic activities, amino acid composition and morphometrical parameters in males and females that undergo diapause.

Sampling of PBW populations

Green cotton bolls were collected from four different cotton growing locations of Karnataka viz. Mysore (12º 24’ 2.969” N, 76º 40’ 49.908” E), Dharwad (15º 30’ 8.978” N, 75º 5’ 9.657” E), Raichur (16º 11’ 45.804” N, 77º 19’ 0.749” E) and Kalaburagi (17º 21’ 49.301” N, 76º 48’ 57.747” E) during the months of November and December. In each location 250–300 bolls were collected and brought to the laboratory for destructive sampling. The collected bolls were cut open and observed for the presence of pink bollworm larva. The larvae thus obtained were reared individually (location wise) on southland diet (Southland Pink Bollworm Diet Premix; Southland Products, Inc., Lake village, Arkansas, USA) under controlled environmental condition of 25 ± 1º C with 65 ± 5% RH and 12 h photoperiod and observed regularly for its growth and development.

After the completion of larval development, some larvae entered into pre-pupal stage and then into pupal stage. Such larvae were considered as non-diapausing individuals, whereas, some larvae did not enter pre-pupal stage, instead remained in larval stage and constructed a typical thin layered silken web called hibernaculum around them. Such individuals were considered as diapause stage (Pradhan et al., 2022a). Based on the number of larvae exhibiting diapausing or non-diapausing behavior, per cent diapause was recorded in populations collected from different locations. The per cent diapause in each location was quantified. Further, the length, width and weight of the ten individual diapausing larvae was recorded under the stereo zoom binocular microscope with image analyzer (Nikon SMZ745).The larvae were sexed based on the presence or absence of the gonads (testis) which are visible through the dorsal integument on either side of the midline of the abdominal segments (Ramya et al., 2020).

The morphometrics of the pupae and adults emerging either from the diapausing or non-diapausing stages were recorded. Length and width of forewing, hindwing and adult body of ten each of male and female adult moths of diapause and non-diapause moths was recorded.

The pattern of diapause and emergence

To study the diapause and emergence pattern of PBW, 50 larvae were collected through destructive sampling of cotton bolls at weekly interval from cotton farms of Raichur from September, 2016 (peak boll formation stage) till May, 2017 (end of cotton cropping season). Cultures collected at weekly intervals were reared under similar conditions as mentioned above till the emergence of the last adult. The number of individuals entering into diapause/ non-diapause stage, emergence of adults from diapause/ non-diapause stage was recorded at 24 h interval.

Biochemical estimation

Different bio-chemical constituents which play a significant role in initiating, maintenance and termination of diapause were estimated in diapause and non-diapause stages of PBW. Thus, amino acids and their concentration (Lafage et al., 1974), total proteins and nitrogen (Salama and Miller, 1992)and total fats (Adkisson et al., 1963)in diapausing and non-diapausinglarvae were estimated as per the standard methods. The enzymatic activity of Lactic dehydrogenase (LDH) (Kitto and briggs, 1962), Alkaline phosphatise(AP)(Yan et al., 2011), Glutamic oxaloacetic transaminase (GOT) and Glutamic pyruvic transaminase (GPT) (Reitman and Frankel, 1957) which are essential for protein synthesis in animal tissues were analysed in penultimate instar of non-diapause and 90, 120 and 150 days old larvae of diapausing PBW.

Statistical analysis

The experimental data were analysed using IBM SPSS software (Version 22.0). Independent samples t-test was used to compute the morphometrical parameters, amino acid composition, total protein, lipid, fats between diapause and non-diapause individuals. The data on activity of enzymes was subjected to Chi-square test to analyse the test of independence among the treatments. The data on PBW entering into diapause was subjected to simple ‘t’ test to find out the level of significance. Graphical representations with regards to per cent diapause occurring across different sampling locations were made using GraphPad prism 8.0.2 software. Histograms depicting diapause pattern and emergence pattern were made using Microsoft excel 2019.

Diapause behaviour in P. gossypiella collected across different geographical regions

Populations of PBW collected from different locations of South India exhibited diapause behavior. However, the degree of diapause varied from 23.33 to 60.95%. Raichur population exhibited the highest diapause (60.95%) followed by Kalaburagi (37.98%), Dharwad (31.67%) and Mysore (23.33%) populations (Fig. 1).Irrespective of the locations from South India, PBW population exhibited facultative diapauses behavior. This report strengthens the earlier report on occurrence of diapause in PBW of South India. The behavior was further confirmed by various morphological and physiological variations between the diapauses and non-diapause individuals of male and female.

The diapause larva recorded greater length and width in both the male and female than the non-diapause larva (Male: length- F_{1, 18}= 1.66, P < 0.01; Width- F_{1, 18}= 0.003, P < 0.05; Female: length- F_{1, 18}= 2.34, P < 0.001; Width- F_{1, 18}=18.65, P < 0.01) (Fig. 2). Further, the average weight of male and female diapause larva was significantly higher than their non-diapausing counterparts(Male- F_{1, 18}=0.004, P < 0.001; Female- F_{1, 18}=0.29, P < 0.001). Similarly in the pupal stage also, significant variation existed between the diapause and non-diapause individuals. Contrast to the larval stage, male and female pupae from diapause larvae were smaller recording lower length and width than male and female pupae of non-diapause larvae (Male: length- F_{1, 18}= 3.40, P = 0.40; Width- F_{1, 18}=0.19, P < 0.001; Female: length- F_{1, 18}=0.03, P < 0.01; width-F_{1, 18}=4.57, P < 0.05) (Fig. 3). The weight of the pupae from diapause larvae were also lower in both male and female than the pupae of non-diapause individuals (Male: F_{1, 18}=2.91, P < 0.001; Female: F_{1, 18}=1.50, P < 0.01).

A significant variation was observed in the wing and body measurements in male and female adult moths emerging from diapause condition compared to adults emerging from non-diapause condition. The average length and width of both forewings and hind wings of male (FW Length- F_{1, 18}=0.12, P < 0.05; FW Width- F_{1, 18}=0.61, P < 0.05; HW Length- F_{1, 18}=1.00, P < 0.001; HW Width- F_{1, 18}=0.58, P < 0.001) and female adult moths (FW Length- F_{1, 18}=1.00, P < 0.001; FW Width- F_{1, 18}=0.58, P < 0.001; HW Length- F_{1, 18}=7.42, P < 0.001; HW Width- F_{1, 18}=4.18, P < 0.01) of diapause individuals was smaller than that of non-diapause individuals (Fig. 4). Similarly, the average body length of both the sexes emerging from diapause condition was smaller than that of adults emerging from non-diapause conditions (Male: length- F_{1, 18} =0.002, P < 0.05; Width- F_{1, 18} =0.059, P < 0.05; Female: length- F_{1, 18} =0.15, P = 0.10; Width- F_{1, 18} =1.43, P < 0.01) (Fig. 4). The overall larval, pupal and adult morphometrics clearly indicates a significant variation among the diapause and non-diapause populations.

A similar trend was noticed in the biochemical profiles also. Though a total of 17 amino acids were detected in both diapause and non-diapause larvae, irrespective of the sexes, their concentrations varied significantly. In general, all the 17 amino acid concentrations were more in diapause larvae of both males and females compared to the non-diapause larvae(Table 1) Amino acids viz. Lysine, Alanine and Serin were predominantly more in male and female diapauses larva compared to non-diapause larva of both the sexes.

Table 1

Comparison of amino acid concentration using UHPLC (Detector A Ex: 350nm, Em: 450nm)
Sl. no	Amino acid	Non- diapause female (µgg^− 1)	diapause female (µgg^− 1)	Non- diapause male (µgg^− 1)	diapause male (µgg^− 1)
1	Aspartic Acid	0.02	0.92***	0.10	0.19NS
2	Serine	6.79	17.12***	12.30	23.43***
3	Glutamine	2.73	8.91***	2.85	5.01***
4	Histidine	4.01	7.45***	5.63	6.11*
5	Glycine	0.51	2.09***	0.39	1.03***
6	Threonine	0.72	5.31***	0.76	1.24***
7	Arginine	3.36	5.62***	4.30	5.05***
8	Alanine	12.29	26.54***	9.47	17.73***
9	Tyrosine	5.84	15.23***	3.49	9.13***
10	Cystine	6.90	16.12***	8.38	11.30***
11	Valine	0.64	3.52***	0.85	1.94***
12	Methionine	0.77	4.05***	1.53	2.19***
13	Phenylalanine	0.21	0.89***	0.30	0.55***
14	Isoleucine	1.39	5.39***	1.33	3.06***
15	Leucine	4.27	15.35***	4.76	9.82***
16	Lysine	64.63	85.62***	112.10	114.41**
17	Proline	5.25	16.86***	6.47	9.95***
Individual amino acids were quantified using independent samples t-test. Levels of significance: NS = non-significant; = P < 0.05; = P < 0.01; **= P < 0.001

Further, both male and female diapause larvae recorded highest per cent of total proteins (Male:F_{1, 18} =0.003, P < 0.001; Female:F_{1, 18} =0.17, P < 0.001), total nitrogen (Male:F_{1, 18} =1.828, P < 0.001; Female: F_{1, 18} =0.99, P < 0.001) and total fats (Male:F_{1, 18} =6.44, P < 0.001; Female: F_{1, 18} =1.99, P < 0.001) compared to non-diapause larvae (Fig. 5). The enzymatic activity varied significantly between the different aged diapause larvae compared to penultimate instar of non-diapause larvae. LDH concentration was higher in diapause larvae compared to non-diapause but gradually decreased as the age of diapause larvae increases. AP enzyme was also recorded highest in 90 days old diapauses larva compared to non-diapause larva but started decreasing as the diapause age increases. However, GOT activity increased in diapause larva as the age of the larva increases and was higher than the non-diapause larva. The GPT activity was lower in diapause larva compared to non-diapause larva and drastically reduced as the age of diapause increased (Table 2).

Table 2

Changes in activity of some enzymes in diapause and non-diapause larvae of pink bollworm, *Pectinophoragossypiella*
Sl. No	Larvae	Lactic Dehydrogenase (L.D.H) (µ mole/min/g)	Alkaline phosphatase (AP) (µ mole/mg)	Glutamic oxaloacetic transaminase (GOT) (µ mole pyruvate/min)	Glutamic Pyruvic transaminase (GPT) (µ mole pyruvate/min)	Chi-Square	P value
1	L1	49.90	31	14.16	8.50	62.28**	0.044
2	L2	50.54	33	14.50	8.16
3	L3	49.30	28	16.33	4.18
4	L4	48.15	26	16.83	4.00
Where, L1: Fourth instar active larva; L2: Diapause larva 90 days old; L3: Diapause larva 120 days old; L4: Diapause larva 150 days old;
**Means significantly different 1% level of probability.

Diapause behavior and its pattern of emergence

None of the larvae sampled from September to first week of February exhibited diapause behavior. However, the larvae collected from second week of February started entering diapause and continued till end of May. The proportion of larvae entering diapause varied significantly among the samples collected during different weeks (t test = 0.005 @ 0.01%). The diapausing rate increased gradually from second week of February (12%) and reached peak on third week of March with 76% and there upon decreased with a minimum of 12% during third week of May. Among the different months of observation, March recorded highest diapause with an average of 24.75% followed by April (22.75%) and May (9.25%). February month (8.25%) recorded lowest diapause(Fig. 6).

Interestingly, a staggered emergence of moths from diapause larvae was observed from first week of May to second week of January. A total of 708 diapause larvae were observed during this study period. However, the per cent emergence varied across the observation period. The maximum number of larvae (49) terminated the diapause during fourth week of November. Among the different months of observation, the highest number of larvae (151) terminated the diapause in the month of November followed by October (149), August (110), September (100) and December (90). The lowest number of larvae terminated the diapause in the month June (11), May (13), Jan (21) and July (36). The duration of the diapause varied with a shortest period of 78 days to the longest period of 323 days (Fig. 7).

Diapause is one of the adaptive behaviors exhibited by an insect species to overcome extreme environmental conditions and food shortage. Many of the species in Lepidoptera exhibited this behavior in temperate and tropical ecosystems. Pink bollworm, P. gossypiella exhibited varied diapauses behavior across the globe i.e. Obligate (Sreenivas et al., 2021)and facultative (Suresh and Patil, 2004; Hanchinal et al., 2008). Though detailed diapausing behavior of North Indian populations of PBW was reported earlier, similar such studies were lacking in South Indian populations. Earlier reports indicate that South Indian populations of PBW do not undergo diapause (Ashok and Robert, 1974; Raina et al., 1981). Much later, the diapausing behavior in one of the South Indian populations, from Raichur was reported (Suresh and Patil, 2004). In the present study, PBW populations from four different locations have reported diapause behavior though the per cent diapause varied among the observed populations. The variation in the diapause behavior is greatly influenced by the external environmental factors although linked to the genetics (Langston and Watson, 1975; Raina et al., 1981). In the present study the population of Raichur has recorded higher diapause per cent followed by Kalaburagi, Dharwad and Mysore populations. The average annual temperature and the photoperiod vary in Raichur (27.6ºC: 11.86 h), Kalaburagi (26.9ºC: 11.92 h), Dharwad (24.1ºC: 7.48 h) and Mysore (24.1ºC; 11.62 h) which could have resulted in the variation in the diapauses pattern in P. gossypiella. Geographical races that differ in their diapause behavior are quite common among the insect species, where, some populations might exhibit the higher per cent diapause compared to the other populations which might have lower diapausing or no diapausing nature (Danilevskii, 1965).

The pink bollworm being a monophagous with limited immigration/emigration probability has a minimum chance of over lapping with the populations of distinct region as a result the generations of these populations are greatly influenced by the prevailing environmental conditions. Raichur and Kalaburagi populations being exposed to higher temperature and longer photoperiod are quite naturally exhibiting higher diapause per cent compared to the populations of other regions. This study is contrary to earlier report which stated that South Indian population collected from Mandya region did not exhibit diapausing behavior (Ashok and Robert, 1974). However, in the present study, population of Mysore region (which includes Mandya) recorded 23.33% diapause.

Since the diapausing larva of PBW do not feed during its entire diapause period, it accumulates more food reserves in the form of amino acids, protein, fat and nitrogen compared to non-diapause members(Squire, 1940; Adkisson et al., 1963; Lafage et al., 1974; Pradhan et al., 2022b). Correspondingly, the body morphometrics of the diapause larva differ from the non-diapause larva. In the present study, irrespective of the sexes, the diapausing stage accumulated more of amino acids, protein, fat and nitrogen than the corresponding non-diapausing stage. As a result, the diapause larvae recorded more length and width in both the sexes compared to non-diapause larvae. However, the cost of the diapause will always be reflected in inferior post-diapause stages with reduced body size (Matsuo, 2006), longevity and reproduction (Jiang et al., 2010). Since the diapausing larva utilizes maximum food reservoirs during the diapause period, the subsequent pupal size reduces considerably recording lower length and width compared to normal pupa (Win and Ishikawa, 2015). Further, post-diapause adult moths also recorded smaller wing dimensions and body size than the normal adults(Win and Ishikawa, 2015).

In the present study, the initiation of diapause in PBW started from second week of February and last upto end of May (Fig. 6). Similar diapause pattern was observed in earlier reports also (Suresh and Patil, 2004). Two plausible reasons can be attributed. One, an increase in temperature from February onwards due to end of winter in Southern parts of India coupled with increased photoperiod (Nanda et al., 2014). The second reason is non-availability of cotton bolls due to end of the cropping season. The peak diapause was noticed during March and April months which is contrary to the reports of Suresh and Patil(2004) who observed peak diapause in the month of May. This variation could be due to change in the cotton genotypes used then and now. Before 2002, non-Bt hybrid such as NHH-44 was the predominantly grown in South India along with many regional cotton verities. These are long duration cotton genotypes with more than 150 days of cropping period which could have extended the availability of food to PBW. However, after 2002, long duration non-Bt cotton genotypes are replaced by Bt cotton genotypes which are medium duration with a maximum of 150 days of cropping period. This could have shifted the diapause peak in the present PBW populations.

Further, in the present study, the maximum diapause duration of 323 days was recorded contrary to the 116 days observed earlier (Suresh and Patil, 2004). The emergence pattern of diapause induced adults is also differed with the earlier report. In the present study, the emergence of diapause induced adults started from May onwards and continued upto second week of January of subsequent year (Fig. 7). The present findings are contrary to the earlier report who recorded emergence of diapause induced adults from first week of June to second week of August. The reason for variation in the emergence pattern is not clear and need to be investigated.

Diapause in pink bollworm is greatly influenced by the external environmental factors though it is linked to the genetics. Diapause larvae have recorded higher length, width and weight compared to non-diapause larvae in both the sexes. Diapause larva found to contain higher amounts of biochemicals such as amino acids, total protein, nitrogen and fat content. The activity of enzymes viz.LDH, AP and GPT were observed to be higher in the initial period of diapause larva and later decreased as the age of the diapause advanced. However, the activity of GOT increased as diapause extends.

PBW – Pink boll worm

LDH – Lactic Dehydrogenase

AP - Alkaline phosphatise

GOT - Glutamic oxaloacetic transaminase

GPT - Glutamic pyruvic transaminase

Ethics approval and consent to participate - Not applicable

Consent for publication – All authors consent for the publication

Availability of data and materials - The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Conflict of interest: The authors declare that there is no conflict of interest.

Funding – Not applicable

Acknowledgements: Authors acknowledge the Department of Entomology, College of Agriculture for providing facilities to conduct the research.

Author’s contributions – BM, AP conceived, designed the experiment and reviewed manuscript. NM executed the experiment, recorded the data and drafted the paper. SG analyzed the data and revised the draft. All the authors read and approved the final manuscript.

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Facultative diapause behavior of pink bollworm, PectinophoragossypiellaSaunders (Lepidoptera: Gelechiidae) in South Indian populations

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Material and methods

Sampling of PBW populations

The pattern of diapause and emergence

Biochemical estimation

Statistical analysis

Results

Diapause behaviour in P. gossypiella collected across different geographical regions

Diapause behavior and its pattern of emergence

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1