Interaction Among the Multi-Trophic Lac Insect Complex of Flora and Fauna: Impact on Quantity and Quality of the Resin Secreted

Abstract

Lac insects are a specialized group of phytosuccivorous insects (Coccoidea: Tachardiidae) that secret resin of industrial importance having diverse applications. Due to unique biology, host preference and dispersal mechanisms, lac insects are expected to differentiate locally, forming geographic and host races without adequate morphological differentiation. 101 species of lac-insects and over 400 species of lac host plants have been reported but insects belonging to sub-family Tachardiinae are considered important for laksha-culture (lac insect farming). With a wide host-plant range and diverse habitat, the insects have developed a specialized ecosystem with multi-trophic complex of flora and fauna. Not only the lac insect but also the host plants and various biotic associations play a significant role in determining the quantity and quality of the produce. This insect being an obligate phloem sap sucker completes its life cycle on host plant species. Phloem sap is nutritionally unbalanced, as it is rich in carbohydrates but deficient in essential amino acids. Due to the scarcity of essential elements in phloem sap, endosymbionts are likely to co-evolve within the insect cell, while fulfilling their nutritional requirement. Implication of these intricate biotic associations on quantity and quality of the lac resin produced merits thorough understanding for sustained lac production.

1. Introduction

Lac, reputed as the only resin of animal origin is secreted mainly by the Indian lac insect, Kerria lacca (Kerr) (Hemiptera: Tachardiidae), which thrives on the tender twigs of specific trees called lac hosts (kusum, palas, ber, Flemingia etc.). Since time immemorial, lac farming has been practised for the products of commerce viz., lac, a resinous or non-resinous covering substance over their body, dye – a natural crimson/yellow color in the body fluid and the lac wax − present within and above the lac resin. These products find application in diverse areas such as food, pharmaceuticals, cosmetics, paints and varnish industries [1]. Lac insects (Hemiptera: Coccomorpha) specialized coccids (scale insects) belonging to the family Tachardiidae (=Kerriidae) [2, 3] are sap-sucking insects thriving on certain plant species called lac host plants. Almost all the stages of lac insect are sedentary and attached to the host plants except for neonate nymphs (crawlers) and adult male insects. Lac insects can be found in tropical and subtropical regions (between the latitudes 40⁰ N and 40⁰ S) due to their preference for a warm climate. Lac production is mainly done in some South, East and Southeast Asian countries like India, Thailand, China, Indonesia, Bangladesh, Myanmar, Laos and Vietnam but the product is in demand all over the world. India is the global leader in lac production followed by Thailand.

The family comprises of nine genera and 99 species worldwide [4]; Kerria Targioni Tozzetti is the largest genus in the family. Recently two more species K. destructor [5] and K. canalis [6] have been described taking the total number of species to 101. The family is characterized by sclerotized features of the adult female. The outer lac encrustation, called lac cell, does not always help in recognition, although it provides indicators in some cases, e.g., lac is resinous and alcohol soluble in sub-family Tachardiinae, but hard, horny and insoluble in Tachardiininae.

India is endowed with a rich wealth of lac insect resources. The genus Kerria includes 28 species worldwide, 21 of which are recorded from India [7]. 27.7% of lac insect biodiversity reported from the world is found in our country under two genera i.e., Kerria (23 species) and Paratachardina (5 species). Species of Paratachardina do not produce true lac and are considered pests of economically important plants but have been utilized as bio-control agents for controlling weeds. Other minor species include K. chinensis and K. sharda. K. chinensis is the principal insect in East and Southeast Asian countries. The kusmi form of Kerria lacca, known for its superior quality of lac and higher productivity is unique to India.

Strains of lac insects: Indian lac insect is known to comprise two distinct infra sub-specific forms (commonly termed strains), ‘Kusmi’ and ‘Rangeeni’ [8]. Kusmi strain is grown on Kusum or on other alternate host plants and the kusmi crops are (i) Jethwi – summer season (maturing in June/July) and (ii) Aghani – winter season (maturing in Jan./Feb.). Rangeeni strain thrives on host plants like palas but not on kusum and it has also two crops; they are (i) Katki – rainy season (maturing in Oct. /Nov.) and (ii) Baisakhi – summer season (maturing in June/July).

2. Factors affecting lac production

Production of lac per unit area and time depends on various biotic and abiotic factors impinging upon lac insect ecosystem. Important biotic factors, which affect lac productivity are:

2.1 Lac insects

Species mainly belonging to Kerria are exploited for commercial production of lac. Yield of lac insect varies significantly depending upon the lac insect species and its strain. Differences in lac yield also exist between the two strains. Average resin secreted by kusmi cells and rangeeni cells is 16.96 mg and 8.07 mg, respectively. Similarly, kusmi strain produced twice the quantity of resin (4.99 mg/mm) per unit size of the cell compared to rangeeni strain (2.55 mg/mm) [9].

2.1.1 Initial density of settlement and mortality

Lac insects are gregarious in nature and settle in close proximity. Hence, density of settlement has a bearing on lac yield. Different lac insects showed a varied density of settlement (average 80−192 per sq. cm in rangeeni, 186−242 per sq. cm in kusmi and 160–264 per sq. cm in Meghalaya stock) based on the broodlac quantity used [10]. Under the optimal broodlac conditions, kusmi strain tends to settle closer compared to rangeeni strain, which is evident from their mean density of settlement. However, under excess brood condition ‘crowding effect’ is exhibited, which is more marked in rangeeni. On the other hand, crawlers of K. chinensis (Meghalaya collection) tend to settle closer during both the seasons. Settlement of larvae in variance with the desired number affects the production adversely. Higher density of settlement results either in increased mortality due to insufficient availability of space and nutrition or a higher male population, which ultimately affects lac yield. Mortality up to 21 days of inoculation is attributed to non-feeding of the larvae at the time of initial settlement. Higher initial mortality is indicative of non-suitability of the host plant and/or of unfavorable environmental conditions.

2.1.2 Sex-ratio

Although the contribution of male lac insects to commercial lac production is very little, they are vital for good lac crop and vis-à-vis broodlac production since the rate of lac secretion increases vigorously in the females after fertilization. However, female lac insects are the sole commercial lac producers. Progeny size and sex ratio vary widely in different lac insect stocks as well as when same lac insect is reared on different host plants. Variation in sex ratio in different crops of lac insect vis-à-vis host plant is more pronounced in rangeeni strain than in kusmi, especially during the summer season. It varied between 18.07–64.39 and 25.59–31.48%, respectively, for summer and rainy season crops of rangeeni and between 22.02–29.33 and 25.49–35.44%, respectively, for summer and winter season crops of kusmi strain [10].

Average male population is lower in smaller progenies and higher in larger progenies; suggesting that larger progeny size increases the male population. In general, sex ratio ranges between 20 and 50% depending on various biotic and abiotic factors. However, in smaller colonies, it may vary between 0 and 100 per cent. Sex ratio is found to vary with (i) season [11]; (ii) sequence of emergence [12]; (iii) site of colonization [12]; iv) density of settlement [13, 14]; v) plant-host [15] and plant-host variety [16]. However, the exact reasons for the wide fluctuation of sex ratio in lac insect population need to be investigated further.

2.2 Host-plant

Although more than 400 species of plants have been reported to support lac insects on them, various biological attributes such as survival, resin production and fecundity differ greatly from the host species used [17]. Coccoids have been found to be very specific not only to different host species but also to specific varieties and even individual phenotypes of host plants due to inter-specific and intra-specific variation in the host plant defense. Srinivasan [18] has indicated preference of lac insects to specific phenotypes − kariya over charka − the later has a lighter colored bark than the former in palas (Butea monosperma) and Kusum (Schleichera oleosa); however, the two are botanically inseparable.

Food quality: The quality and quantity of food available to the insect are important in determining its survival and reproduction rate. This is particularly true for phloem sap-feeding insects. Passive exudation of sap of phloem bundles has a substantial role in supply of phloem sap to the insects. Quantitative and qualitative differences in the nutrition available from different host plants, cause variation in biological attributes of the lac insect. Variability studies in case of four species of Flemingia viz. macrophylla, semialata, stricta and bractiata with regard to various attributes of lac insects have shown significant differences [19].

Sap condition: Lac insect feeds on the phloem sap of the host plant. The insect inserts its proboscis and feeds on exudation of sap by i) turgor pressure and (ii) capillary action. Turgor pressure of the host changes with the season and phenotypic activity of the plant. The sap pressure is higher during rainy season and considered favorable for the growth of the insect and inverse is true for summer season. ‘Sap reaction’ and ‘sap density’ are possibly among the factors, which influence the suitability of the host plant for lac infection. Good host plants have phloem sap of pH ranging between 5.8 and 6.2 [20]. Similarly, sap density of good hosts ranges between 0.14−0.1728.

2.2.1 Initial density of settlement and mortality

Initial settlement of crawlers is affected mainly by host plants and the physical characteristics of the twigs where they settle down. Density of settlement on S. oleosa (kusum) is higher than on Albizia lucida (Galwang). Some lac host plants support a certain species or strain in a better way and vice-versa. Although lac insect crawlers can be made to settle on any plant twigs, they would be able to survive and complete their life cycle only on good hosts. Rangeeni lac insect cannot survive on kusum and it exhibits very high mortality on F. semialata; similarly, kusmi strain cannot survive on B. monosperma (palas), while Ziziphus mauritiana (ber) supports both strain up to maturity. There is a significant decrease in survival when lac insect was reared on pumpkin fruits (Cucurbita moschata) in comparison to Flemingia macrophylla. The increase in mortality observed was 67.2% and 104.9% for kusmi and rangeeni strains, respectively [21].

2.2.2 Sex-ratio

Chauhan [15] has reported that the Meghalaya lac insect stock showed significant difference in sex ratio on different host plants. It was observed that 72%, 82% and 98% were in favor of males on F. macrophylla, Cajanus cajan and Z. mauritiana, respectively. Similarly, Sharma and Ramani [21] have also observed that male percentage of rangeeni and kusmi strains of K. lacca on F. macrophylla was 39.76 and 37.28%, which increased to 70.05 and 62.65% when they were reared on C. moschata fruits.

2.2.3 Effect of host-plant on resin production

Average mean cell diameter of a kusmi female cell was 3.02, 3.16, 3.50 and 3.54 mm on C. moschata, F. macrophylla, Acacia auriculiformis and S. oleosa, respectively [9]. Resin produced by individual female lac cells varied significantly. It ranged from 6.11 mg on C. moschata fruits to 22.84 mg on S. oleosa. Resin production by individual female lac insects was the highest on S. oleosa followed by A. auriculiformis, F. macrophylla and C. moschata fruits (Figure 1). Very high intra-strain variations were observed in resin-producing efficiency of lac insect even when cultured on the same host plant.

Figure 1.

Resin productivity and cell size relationship in Kerria lacca (kusmi strain) on different host plants during winter season crop.

Similarly, the average mean diameter of a rangeeni female cell grown on A. auriculiformis, C. moschata, F. macrophylla and B. monosperma was 3.10, 3.17, 3.19 and 3.22 mm, respectively. Average resin secreted by an individual rangeeni female cell ranged between 6.00 mg (on C. moschata fruits) and 9.09 mg (on A. auriculiformis). Resin production by single rangeeni female lac insect was found to be the highest on A. auriculiformis followed by B. monosperma, F. macrophylla and the lowest on C. moschata fruits (Figure 2). Higher values of coefficient of regression in good hosts S. oleosa and A. auriculiformis for kusmi strain and B. monosperma in rangeeni strain corroborate the fact that a good lac host allows full manifestation of the resin-producing potential of the lac insect. Resin productivity is higher on tree hosts in comparison to F. macrophylla (a shrub) and the pumpkin fruit. Though variations in cell size were less prominent, weight of the cell and resin output per female recorded greater variations showing the effect of host on resin productivity of the insect. Lac insect – host plant interaction in terms of lac production and host suitability is reflected in the data provided in Table 1.

Figure 2.

Resin productivity and cell size relationship in Kerria lacca (rangeeni strain) on different host plants during rainy season crop.

Host-plant	Initial host preference (%)	% Survival at crop maturity	Diameter of the cell (mm)	Weight diameter ratio	Host suitability index
A. Kusmi strain
Acacia auriculiformis	26.67	13	3.50	5.404	65.53
Cucurbita moschata	10.00	8	3.02	2.023	4.89
Flemingia macrophylla	80.00	19	3.16	3.617	143.34
Schleichera oleosa	86.67	22	3.54	6.452	435.50
B. Rangeeni strain
A. auriculiformis	20.00	15	3.10	2.932	27.27
Butea monosperma	93.33	18	3.22	2.720	147.16
C. moschata	13.33	9	3.19	1.881	7.20
Flemingia macrophylla	86.67	18	3.17	2.363	116.85

Table 1.

Host suitability index of different lac host plants for rearing Indian lac insect, Kerria lacca (Kerr).

2.2.4 Host suitability index

Host preference, length of settlement at crop maturity, lac insect survival at crop maturity and resin production by the insect are the most important attributes affecting the production of lac. Host Suitability Index is calculated for identifying an ideal host plant by using the following formula. By taking the lowest value of Host Suitability Index for a particular host plant as 1.00, relative suitability indices are calculated for the other hosts.

$$\text{Host Suitability}=\text{Index}$$

$$\begin{array}{l}\text{\%Host Preference}\times \text{\%Length of settlement}\times \mathrm{\%lac}\text{insect survival}\\ \times \text{Mean resin production}\left(\mathrm{mg}\right)\times 100\end{array}$$

where % host preference − (% of host-plants on which lac insect survived till crop maturity), % length of settlement at crop maturity − (% length of encrustation of available shoot length), % survival of lac insect (per square cm) at crop maturity − (% surviving lac insect number of the initial density of settlement) and resin produced (mg) by lac insect − (average weight of resin produced by fifty randomly collected individual female lac insects).

2.3 Local environment

Abiotic and biotic components also affect the suitability of the host plant to lac insect. Ziziphus xylopyra (Ghont) is a good host in Madhya Pradesh but not in Ranchi (India). Similarly, C. cajan and Grewia spp. are used as lac hosts in North Eastern states but attempts to cultivate lac Grewia at Ranchi proved futile. Analysis of weather data of 1984−2012 of Ranchi (India) revealed that the winter months (December and January) have become colder and pre and post-winter months (November and February) the warmer [22]. These changes in climatic parameters have implications in lac cultivation as it is a critical period of lac insect development (pre-sexual maturity) during the summer season crop. Monsoon and winter rainfall spells and magnitude were also found to affect lac crop performance. Effect of abiotic factors (temperature, rainfall and relative humidity) was correlated with lac production of rangeeni crop during 2006–2007 to 2012–2013. It was observed that maximum temperature had a significantly negative (−0.911* and − 0.837*) and RH a positive significant (0.850* and 0.800*) correlation with lac production during the critical crop period (March and April) of development in the summer season (baisakhi) crop whereas, during rainy season (katki) crop, minimum temperature had a significant negative (−0.765*) correlation with lac production. The vulnerability level of lac insect is high during and prior to sexual maturity stage in the summer crop, thus post-winter season is the critical period for lac insect survival and any undesired variability in weather parameters in this stage can impact adversely on lac productivity.

3. Factors affecting lac quality

Lac insects have been designated as crimson, yellow, cream or white depending upon the quality and intensity of the water-soluble pigment (laccaic acids) present in their body. Resin pigment (erythrolaccin) is not soluble in water and is not desirable in some of its application areas. Quality of the secreted resin due to the presence of dye differed significantly among various stocks of lac insects. Kusmi strain of K. lacca produced the lightest colored resin whereas that produced by K. chinensis and K. sharda insects was the darkest. Saha [23] has also reported significant differences in physicochemical properties of lac resin secreted by three different lac insect species. Lac insect species and their host plants also affect the quantity of lac dye (erythrolaccin) present in the particular lac insect stock.

Biochemical parameters of the lac host plants get altered after lac insect inoculation. Fengshu et al. [24] have analyzed phenolics, sugars, amino acids and some inorganic elements of the host plants of different ages and strains with and without lac inoculation. The biochemical parameters showed variations after lac inoculation and correlated with the seedlac quality and quantity in different seasons. Liu et al. [25] have analyzed tannins and phenolics in relation to the quality and size of the winter generation K. lacca on different lac hosts such as Dalbergia szemaoensis, D. hupeana, D. obtusifolia, Pueraria thunbergiana, Zenia insignis, Cassia siamea and C. cajan. Yang et al., [26] have reported that the host tree root secretion and the quality of the lac produced are the indexes to the inter-adaptability between the lac insect and its host tree such as Acacia suma, Hibiscus syriacus, Moghania macrophylla and S. oleosa. Various studies indicated a decrease in the amount yet increase in the variety of amino acids in the host trees on lac inoculation. Lac composition also differed significantly from the lac insects grown on different host trees.

4. Associated fauna

A number of major and minor pests/diseases at times almost destroy the lac crop, thereby, not only reducing the yield drastically but also affecting the quality of the lac. Sessile nature of lac insects makes them more prone to predators and parasitoids.

4.1 Predators

The losses in lac cultivation due to various insect predators are known to be far greater than what is usually met in other agricultural crops. About 22 predators have been reported to be closely associated with lac insects, of which three are major predators viz. Eublemma amabilis, Moore (Noctuidae); Pseudohypatopa pulverea Meyr. (Blastobasidae) and Chrysopa spp. (Chrysopidae). E. amabilis and P. pulverea alone are responsible for 30−40% of damage to the standing crop [27, 28] of which E. amabilis alone causes 20−25% damage [29]. These lepidopterous predators cut a hole in the lac and feed on the insect from inside by making a tunnel. Chrysopa, though a sporadic pest, sometimes causes havoc, particularly in kusmi strain.

Chemical communication between the lac insect-associated products and the lac predators have been evaluated under the laboratory condition and semio-chemicals identified for different stages of lac insect and its associated products [30]. Six major compounds viz. decane, dodecane, tetradecane, heptadecane, eicosane and octacosyl acetate constituted 78%, 79% and 85% in lac insect whole body, lac resin and lac wax extracts, respectively. Electroantennogram (EAG) revealed higher responses of adult male and female of lac insect predator, Eublemma amabilis to lac insect whole body extract than resin, wax, crawler and lac insect female extracts. Whereas, in Pseudohypatopa pulverea, EAG response was significantly higher in females towards lac insect whole body than resin, wax, adult female and crawler extracts than males. Both E. amabilis and P. pulverea exhibited high level of sensitivity to lac insect whole body extracts with different concentrations ranging from 1000 to 10,000 ppm than the identified semiochemicals viz., Decane, Hexadecane, Nonadecane, Eicosane and hexane.

4.2 Parasites

Thirty different parasites of lac insect have been reported by Varshney [31]. They lay eggs into the lac cell through the anal tubercle in/on the body of lac insect. The grub that hatches feeds only on lac insect.

4.2.1 Inimical parasites

Of all the parasites associated with lac insect, eight parasites namely, Coccophagus tschirchii, Erencyrtus dewitzi, Eupelmus tachardiae, Parechthrodryinus clavicornis, Tachardiaephagus tachardiae, Marietta javensis, T. somervillei and A. purpureus are of regular occurrence in the lac ecosystem. Among these, Tachardiaephagus tachardiae and Aprostocetus purpureus are the most abundant lac associated parasites. Extent of parasitisation varied between 15.5% in summer season (baisakhi) crop and 18.6% in rainy season (katki) crop of rangeeni strain. While for the kusmi strain it was 19.04% in winter season (aghani) crop and 22.8% in summer season (jethwi) crop [10]. Fecundity of lac insect is adversely affected by parasitisation. Parasitized cells adversely affect the resin production and brood value (fecundity) of the crop. Either, there is no emergence or very low emergence of young ones from the parasitized cells, which ultimately affects the inoculation of the next crop.

Parasitic losses: Percentage of parasitism recorded is much higher than the earlier reports of average 4.8 − 9.9 per cent parasitism based on seven years’ data [29]. Reinterpretation of the same data by Srivastava and Chauhan [32], however, revealed that average per cent parasitism for the crop on the basis of females alone worked out to be 20 to 37. In certain years and in some localities, it was as high as 50%. Jaiswal and Saha [33] have found a positive and significant correlation between density of lac insects and number of parasitoids. So it is highly likely that actual per cent parasitism would be higher than recorded since lac insects generally form a continuous encrustation and the present study was confined only to isolated female lac insects.

Rangeeni strain is more vulnerable to pest attack than kusmi and damage is more in rainy season crop. Parasites lay eggs into the lac cell through the anal tubercle in or on the body of the lac insect. The grub that hatches feeds only on lac insect and not on lac. As a result of parasitisation, fecundity and resin-producing capability Kerria lacca is adversely affected. Quality of the resin produced declined by 17.92% and 17.44% while fecundity decreased by 32.55% and 34.71% for kusmi and rangeeni strains, respectively [9, 34]. Damage caused by parasites varies depending upon the virulence of outbreak and stage of development of the lac insect at which damage is inflicted. Instances of super-parasitism (as many as 19 larvae have been reported from a single mature female lac insect) and multi-parasitism are not uncommon, which further aggravate the problem.

It was found that the decrease in fecundity of lac insect due to parasitisation ranged between 10−100%. Thus, proportionately more broodlac would be required as compared to healthy broodlac for inoculating the same number of trees. If parasitized at an early stage, the lac insect is practically eaten up by the developing parasitoid rendering the lac useless for broodlac purposes. Moreover, the broodlac harboring parasites if used for raising next crop would serve as a source of infection to new lac culture.

Lac insect is gregarious in nature and resin secreted by these coalesces to form a continuous encrustation. The thickness of lac encrustation is one of the criteria for assessing quality of broodlac. Size of parasitized as well as healthy cells did not differ much but comparison of weight of the resin secreted by the two revealed that the amount of the resin produced by the parasitized cells was significantly lower. Hence, visual assessment of broodlac quality on the basis of encrustation thickness alone may prove to be deceptive unless weight is also taken into account.

4.2.2 Beneficial parasites

Several types of insects are hyper-parasitic to the lac insect. Though their natural population constitutes only about 4−10% of the total fauna associated with lac insect, they act as a bio-control agent in controlling the damage done by inimical insects. Many ants and other insect species feed on the honey dew excreted by lac insect, and these prevent losses by fungus infection.

4.2.3 Effect of fungi on lac production

In addition to the damage caused by insect pests, lac crop yield suffers significant losses due to other biotic agents, particularly fungi. Few earlier reports suggested that the lac insects had mutualistic relationship with fungi. However, an association of fungi with lac insects is not beneficial always. Lac insects being phloem feeders excrete excess sugar in the form of honey dew, which invites sooty mold to grow over the lac encrustation. Besides, this rainy season crop is also prone to fungal infection when grown on ber and kusum due to their shady nature. Avoidable losses due to fungi alone were observed to be 40.9% to 59.85 in the kusmi strain of lac insect [35]. Similarly, Mishra et al. [36] have reported significant reduction (75.05%−88.41%) in mortality of second instar lac nymphs with application of different fungicides on the kusmi strain of lac insect.

4.2.3.1 Fungi associated with lac insects

The earliest record of honeydew that drips from colonies of lac insects on the twigs of host trees inviting black mold species of Capnodium and Fumago is that of Lindsay and Harlow [37]; the presence of pathogenic fungi, pythium sp. in female tests causes a heavy mortality in the larvae which fail to enclose satisafactorily and lie dead in clusters within the female resinous cell [38]; sooty mold fungi Conidiocarpus (Syn. Podoxyphium conidioxyphium) and Polychaeton spp. are obligate anaerobes capable of producing endospores and causing 30−40% damage to lac insect in Vietnam [39]; 11 species of saprophytic pathogenic fungi causing dark mildew on lac insect have been reported from China [40] and Three species of fungi belonging to family Eurotiaceae/Aspergillaceae causing severe damage to lac culture have been reported from India [41]; Aspergillus awamori Nakazawa forms black sheet-like covering on lac encrustation, Aspergillus terricola Marchal traverse the whole length of the anal tubercle and blocking it leading to disruption of mating and larval emergence and Penicillium citrinum Thom (Syn. Penicillium aurifluum biourage) blocks the breathing pores of lac insects. Fungal infection in lac cultures causes losses in lac yield by (i) inhibiting respiration, (ii) hindering mating process, (iii) blocking larval emergence and (iv) affecting lac host efficiency.

5. Interaction of lac insects with microbes

Insects and microbes’ interaction ranges from obligate mutualism to facultative parasitism. Insects harbor symbiotic bacteria on the integument, in the digestive tract and in some unique structures within their body [42, 43]. Interaction between insects and microbes is one of the important factors, which makes insects the most successful group of organisms on the earth. Insect microbiota plays significant role in growth, development, reproduction and adaptation of the insects to the environment. Endosymbionts assist insects in their survival by nutritional supplementation [44], aiding in digestion of recalcitrant food materials [45], protecting them from predators and parasitoids [46], detoxifying phytotoxins and pesticides [47], imparting resistance against insecticides [48], immune system stimulation [49], inter and intraspecific communication [50] etc.

Symbiosis with bacterial community is obligatory in insects whose diet is imbalanced such as vertebrate blood (by mosquitoes), phloem sap (by sap-sucking insects) and wood (by termites). Lac insect life stages are morphologically and physiologically highly diverse. Besides, their resin production potential also varies with different stages; crawlers and adult males do not secrete resin whereas, settlers and adult females after fertilization secrete resin albeit in different quantities. Lac insects are almost sedentary throughout their life and depend on nutritionally imbalanced sugar-rich phloem sap for their survival. It is postulated that lac insects must harbor myriad of endosymbionts for nutrition supplementation, host plant adaptability, defense etc. Earlier works have found that the presence of microbial flora in lac insects was beneficial during rainy season crops for higher lac yield. Some bacteria such as Micrococcus spp., Clostridium sp. and Bacillus subtilis have been reported from lac insects [51]. Micrococcus varians and Micrococcus conglomerates are associated with various stages of lac insect and are considered beneficial for good yield of lac production.

Till now, bacterial flora associated with lac insects has been identified either based on culturing method [52, 53] or PCR method for specific endosymbionts like Wolbachia [54]. Gender-specific bacterial flora has been identified from lac insects [53], and host plant-induced variation was observed in the bacterial composition of lac insects based on culturing method [52].

5.1 Association with Wolbachia

Wolbachia are members of the order Rickettsiales, a diverse group of intracellular bacteria that include species having parasitic, mutualistic and commensal relationships with their hosts. Wolbachia species are well known for their vast abundance, effect on hosts in terms of reproductive manipulation and mutualism and have potential applications in pest and disease vector control [55]. Wolbachia pipientis is the type species of Wolbachia genus. Based on the 16S ribosomal sequence and other sequence information, Wolbachia spp. have been divided into seventeen different supergroups (A-Q). Two supergroups (C and D) are commonly found in filarial nematodes, whereas other groups are found in arthropods, in which A and B are the most common. Wolbachia that participate in symbiotic relationships with arthropods have a range of phenotypic effects on their hosts and generally behave as reproductive parasites. Wolbachia manipulate host reproduction through cytoplasmic incompatibility, parthenogenesis, feminization and male-killing [56, 57, 58]. Mostly Wolbachia undergoes vertical transmission from mother to offspring. However, a horizontal transfer is also reported in nature [58].

In lac insect populations, there is a wide variation of male–female sex ratio, which ultimately affects the lac production as only females can produce lac. Vashishtha and co-workers [54] have found that lac insects are associated with Wolbachia based on 16S rDNA and wsp (Wolbachia cell surface protein) PCRs. Lac insect-associated Wolbachia was termed as wKerlac. Phylogenetic tree revealed it to be a subgroup “ori” of supergroup B, which is predominantly present in arthropods. Wolbachia of K. lacca was grouped with Wolbachia of Tagosodes orizicolus and Ephestia cautella. Wolbachia on both these hosts are responsible for cytoplasmic incompatibility. Further investigations are required on whether the identified Wolbachia would have any role in feminization. It is one of the most important factors in attributing lac yield because commercial lac is obtained solely from female lac insects.

5.2 Detection of Wolbachia phage (WO) in lac insects

Wolbachia species also harbor a bacteriophage called bacteriophage WO or phage WO [59]. Comparative sequence analyses of bacteriophage WO revealed the possibility of large-scale horizontal gene transfer between Wolbachia coinfections in the same host [60]. Molecular mechanism used by Wolbachia to manipulate its host in terms of cytoplasmic incompatibility, feminization, parthenogenesis, male killing etc. remain elusive and has been speculated due to genes on extrachromosomal factors such as plasmids or bacteriophages [61, 62]. Out of seventeen identified super-groups of Wolbachia, named A–Q, WO phage has been reported to infect Wolbachia belonging to super-group A, B, F and G [63, 64].

Screening and distribution of Wolbachia and WO phage sequences were studied by amplifying and sequencing the partial ftsZ, a cell cycle gene involved in cell division and a putative minor capsid protein, orf 7, respectively [65]. Two different lines kusmi and rangeeni each were found to be singly infected by Wolbachia belonging to Supergroup B. It was the first report on molecular detection of WO-phage infecting kusmi and rangeeni infrasubspecific forms of K. lacca. Further phylogenetic analysis revealed distinct differentiation of WO between kusmi and rangeeni infrasubspecific forms. In the phylogenetic tree made based on orf7 sequences, rangeeni and kusmi forms clustered with group III and group I, respectively. Although there is a differentiation of kusmi and rangeeni forms based on orf7 of WO sequences, the tripartite association of lac insect-Wolbachia-WO needs further investigation to implicate their role in such differentiation.

5.3 Association with yeast-like symbionts

Insects not only possess bacterial symbionts but also yeast-like fungal symbionts (YLS). Although microbiologists observed such yeast-like endosymbionts in 1960s, the identity was not known due to their fastidious nature and the lack of molecular tools at that time. Later, it was found that the phloem sap feeders harbor obligate intracellular yeast-like symbionts, YLS (subphylum Ascomycota, class Pyrenomycetes, family Clavicipitaceae) [66, 67] especially in the mycetocytes formed by fat body cells of abdomen. YLS have also been reported in Hemiptera (aphids, planthoppers, and scale insects) and Coleoptera (beetles) [68, 69]. They grow by budding and are vertically transmitted to the next generation by transovarial infection [70, 71]. YLS appears to play roles in nitrogen metabolism of the host through recycling of uric acid [72, 73], in insect metabolism by synthesizing sterols, the precursor molecule for many hormones (e.g., 20-OH ecdysone-a molting hormone), as insects, in general, are unable to synthesize them [74] and in detoxifying the toxic substances to the host [75]. Occurrence of YLS is highly essential for the survival and reproduction of the host insects because they play vital role in development, reproduction and embryonic development [76]. Transmission electron microscopy and PCR-based studies revealed the presence of YLS in lac insects. However, acquisition of YLS in lac insects seems to be different from that of aphids and plant hoppers and a horizontal transfer was also suggested for them [54].

5.4 Sex-specific endosymbionts

Male and female lac insects’ specific bacterial species were identified by Shamim and co-workers [53] based on 16S rDNA PCR and biochemical characterization. Eight different bacterial species were isolated and categorized as endosymbiont, gut bacterium or subsurface bacterium. Three of them were exclusive to males, three to females and two were common to both the sexes (Table 2). Bacillus megaterium, A. subterraneus and Pantoea ananatis were found to be the most abundant bacterial species. Among 13 bacterial isolates found in males, two were present as an internal gut bacterium which may get excreted out with honey dew. Single isolate of Paenibacillus barengoltzii was found at subsurface. P. ananatis was exclusively and majorly found in males as endosymbiont bacteria. P. fulva was also found in males as endosymbiont bacteria. Twelve bacterial species were isolated from the females and 50% populated with Bacillus sp. B. megaterium, Curtobacterium citreum and A. subterraneus were majorly reported in non-crushed samples, therefore, it was thought to be thriving at subsurface but B. cereus and Solibacillus silvestris were considered as endosymbiont. A. subterraneus, found in both the sexes, as endosymbiont in males and at subsurface in females.

Associated with	Bacterial spp.
Exclusively male lac insects	Paenibacillus barengoltzii, Pseudomonas fulva and Pantoea ananatis
Exclusively female lac insects	Bacillus cereus, Solibacillus silvestris and Curtobacterium citreum
Both male and female lac insects	Bacillus megaterium and Arthrobacter subterraneus

Table 2.

Bacterial species identified from lac insects.

Out of these bacterial species, B. cereus, B. megaterium and P. ananatis, are widespread in occurrence and have also been reported in other insect’s body. Some strains of P. ananatis, also referred as “ice nucleation-active” bacteria, are used in pest control because when present in the insect gut, they lower the cold resistance [77]. Owing to this property and bacterial abundance in the insect, absence of lac insects in colder regions could be attributed to the association of P. ananatis. Description about the bacterial species identified from both the sexes of lac insect [53] is given in Table 2.

5.5 Host plant-induced variation of endosymbionts

Since phloem sap constituents vary for host plants, variation in the endosymbionts of lac insects growing on different host plants is anticipated. Culture-based method was followed to isolate bacteria from lac insects grown on different host plants and 16S rDNA PCR based molecular method was followed [52] to identify them.

From 29 different bacterial isolates, 10 different bacteria were identified. Bacillus kochii, Bacillus oceanisediminis, Bacillus amyloliquefaciens, Bacillus nakamurai and Enterobacter cloacae were observed on kusmi lac insects collected from Kusum trees. Klebsiella quasipneumoniae subsp. similipneumoniae, Citrobacter amalonaticus, Providencia vermicola and B. nakamurai were found in bacteria isolated from lac insects collected from ber trees. Enterobacter ludwigii, Enterobacter cancerogenus and B. nakamurai were found in lac insects collected from semialata. In most of the cases, different species of Bacillus and Enterobacter were found. Bacillus is a very common genus found in different types of insects, which include B. subtilis, Bacillus thuringiensis, Bacillus cereus, Bacillus sphaericus, Bacillus popillae, Bacillus circulans, B. megaterium, Bacillus lentimorbus and Bacillus polymyxa [78]. Bacteria such as Enterobacter spp. K. quasipneumoniae, C. amalonaticus, P. vermicola belong to Enterobacteriaceae family. All these Enterobacters might have come from lac insect gut and belong to proteobacteria, primarily within the γ–subdivision.

B. nakamurai the most frequent bacteria identified from lac insects grown on three different hosts might be involved in some vital functions in lac insects. B. nakamurai was originally isolated from soil and known to produce black pigment [79]. B. amyloliquefaciens is known to control plant pathogens due to its antifungal activity [80]. Hence, antifungal activity may be anticipated for the B. amyloliquefaciens strain present in lac insects.

6. Conclusion

6.1 Impact on quantity and quality

Lac insects feed on host plant phloem sap by passive sucking mechanism through capillary action and turgor pressure of the sap [81]. Passive exudation of phloem sap through the intruded lac insect stylet as a function of phloem turgor pressure has a significant role in the supply of phloem sap to lac insects. Lac insects are sedentary soon after settlement on host plants. As a result, there is no escape mechanism for lac insects from the host plant’s defensive chemicals. Hence, lac insects should have ability to detoxify the host plant’s defensive chemicals and avoid them which is important for good lac insect − host plant interaction. Due to differences in the ability of lac insects to detoxify the defensive chemicals and the nature of defensive chemicals produced by the plants, there is difference in yield of lac on different lac host plant species and different genotypes of the same host plant. Lesser quantities of toxicant ingested when feeding on phloem sap of good host plants rich in nutrients would be detoxified quickly, whereas the larger quantities of toxicant ingested when feeding on poor quality host plant may not be detoxified by lac insects. Haque (1984) observed variations in the quality and quantity of amino acids present in honey dew (anal fluid) of K. lacca grown on different host plants such as Moghania (=Flemingia) macrophylla, Ficus glomerata, Ficus indica and Ficus religiosa [82], indicating the variations in nutritional composition of these host plants.

Due to sub-optimal nitrogen/carbon ratio in the phloem sap, phloem feeders need to ingest excess phloem sap along with excess organic carbon to obtain sufficient nitrogen for their growth. Non-optimal ratios of essentially required nutrients in the phloem sap warrant the insects to ingest supra optimal quantities of less required nutrients and also the defensive chemicals, which are toxic to them. These factors affect the resin-producing efficiency of lac insects. Resin production is found to be high on tree hosts in comparison with bushy hosts such as F. macrophylla and pumpkin fruit. Auclair [83] has reported that the phloem sap exudation rate through excised stylets in aphids is about an order of magnitude higher in woody plants compared to that in herbaceous plants. Since lac insect feeding habit is also similar to aphids and passive in nature, the higher turgor pressure of woody plants leads to greater food ingestion and higher resin production on tree hosts and vice versa in herbaceous hosts. Intraspecific variation in the host plant defense caused the deviation in the susceptibility of a host plant species in Nuculaspis californica [84]. Lac insects have the ability to manifest their biological parameters very specifically not only to lac host species but also to varieties, phenotypes of host plants [18, 85] locality and season of cultivation. Periodic host-plant resistance is considered to be a physiological response to meteorological and edaphic conditions, a response usually rendering the plant temporarily unsuitable to coccoid development.

It can be concluded that lac insects, as well as lac-host plants and associated flora and fauna, play significant roles in the quantity and quality of the lac production. The naturally existing high degree variability in lac insects and host plants can be successfully exploited for selection and evolution of high-yielding varieties and lac insect-host combinations. Quantity and quality of the lac resin can be significantly enhanced with better management of lac insects and their host plants.

6.2 Interaction with microbes

Crawlers i.e., early developmental stage of lac insect consists more of unknown and other bacterial types followed by Wolbachia and Mucilaginibacter. Wolbachia and Pantoea are the two important genera found in the adult female lac insects besides unknown bacteria. P. ananatis is already reported to be present exclusively in male lac insects based on the culture method [53]; Pantoea cypripedii and Pantoea dispersa are present in honeydew secreted by lac insects [86]. In insects, Pantoea is primary endosymbiont and its association with insects is mostly mutualistic and sometimes commensalistic. In the mutualistic association, insects provide habitat and nutrition to Pantoea, whereas Pantoea may help insects by hydrolysis of proteins, antagonism of pathogens, breakdown of toxic substances, nitrogen fixation, nutrition and digestion [87]. Since Pantoea carbekii genome encodes complete or near-complete canonical pathways for the production of several vitamins and cofactors such as folate, riboflavin, pyridoxal-5′-phosphate, glutathione, iron–sulfur clusters and lipoate, it is assumed that Pantoea supplements nutrition by providing essential vitamins and minerals to its host stink bug [88]. Taking these things into account, it is plausible to assume that the Pantoea spp. present in lac insects may be involved in nutrition supplementation because plant phloem sap on which lac insects feed is not a nutritionally balanced diet.

Wolbachia is an obligate endosymbiont of arthropods and nematodes and present in most of the insects wherein they play a major role in the reproductive manipulation of the host. They alter the reproduction of the host insects by the way of male killing, parthenogenesis, cytoplasmic incompatibility and feminization. Generally, the mode of transfer of Wolbachia in arthropods and nematodes from one generation to another is transovarial [89]. Due to such reproductive manipulation of the host by Wolbachia, the frequency of Wolbachia infected females increases in population sometimes at the expense of host fitness [90]. Vashishtha and co-workers [54] have reported the presence of Wolbachia in lac insects by 16S rDNA and wsp PCRs. Wolbachia infection is known to be biased based on sex or may increase as the development progress. Frequency of occurrence of Wolbachia was found to be more in female insects compared to crawlers in the current study. Similar results of higher Wolbachia incidence in the adult stage compared to an immature stage were obtained in several other insects. Besides manipulating host reproduction, Wolbachia may affect host fitness positively by nutrient supplementation. It has been demonstrated in bed bugs, Cimex lectularius that riboflavin provision ability of Wolbachia can positively impact the host’s growth, survival and reproduction [91]. In the scale insect, Dactylopius coccus, two species of Wolbachia were found to have metabolic capabilities for riboflavin and heme biosynthesis [92]. Besides, reproductive manipulation and nutrient supplementation, an additional function of protecting Drosophila from virus induced mortality was attributed by Wolbachia infection [93].

As far as lac insects are concerned, females are the productive gender as the commercial lac is obtained only from females but not from male insects. Since, Wolbachia can eliminate males, turn them into females, sterilize uninfected females or behave as a mutualistic symbiont [94], their role in lac production needs to be explored thoroughly. Whether the role of Wolbachia is restricted up to reproductive manipulation or it is extended to nutrient supplementation and virus protection in lac insects needs thorough investigation in future.

Lac insect endosymbionts are very diverse and supposed to carry out various vital functions in the insects. The available literature describe mainly cultivable bacteria and to certain extent uncultivable microbes. Much more uncultivable bacteria and also stage-dependent and strain-dependent endosymbionts may be anticipated to be present in lac insects. Culture-independent methods such as metagenomics would reveal more number of endosymbionts in lac insects. Different functions such as nutrition supplementation, sex differentiation, strain differentiation and protection from pests and predators are anticipated for the lac insect endosymbionts.